(en)The invention is based upon the discovery that small nucleic acids from non-viral pathogens are able to cross the kidney and are present in urine of a subject when the subject is infected with the non-viral pathogen. These transrenal DNAs are especially prevalent at smaller sizes under about 300 bp. Thus the invention provides compositions and methods for the diagnosis of infection of a subject with non-viral pathogens through the detection of transrenal nucleic acids from those pathogens in the urine of the subject.

1.ApplicationNumber: US-201113029308-A
1.PublishNumber: US-2012021412-A1
2.Date Publish: 20120126
3.Inventor: MELKONYAN HOVSEP
CANNAS ANGELA
TOMEI LOUIS DAVID
UMANSKY SAMUIL R.

4.Inventor Harmonized: MELKONYAN HOVSEP(US)
CANNAS ANGELA(IT)
TOMEI LOUIS DAVID(IT)
UMANSKY SAMUIL R(US)

5.Country: US
6.Claims:
(en)The invention is based upon the discovery that small nucleic acids from non-viral pathogens are able to cross the kidney and are present in urine of a subject when the subject is infected with the non-viral pathogen. These transrenal DNAs are especially prevalent at smaller sizes under about 300 bp. Thus the invention provides compositions and methods for the diagnosis of infection of a subject with non-viral pathogens through the detection of transrenal nucleic acids from those pathogens in the urine of the subject.

7.Description:
(en)RELATED APPLICATIONS
This application is a continuation application of U.S. Ser. No. 11/351,799, filed on Feb. 10, 2006, which is a continuation-in-part of U.S. Ser. No. 11/137,934, filed on May 25, 2005, now U.S. Pat. No. 7,803,929, which claims priority of Italian Patent Application No. RM2005A000068, filed on Feb. 17, 2005, and claims priority of U.S. Ser. No. 60/691,186, filed Jun. 16, 2005, which are incorporated herein by reference in their entireties.


BACKGROUND OF THE INVENTION
There are currently three types of in vitro diagnostic systems widely used for the detection of non-viral pathogens. These are direct culture of the pathogenic agent from the biological sample; immunological assays based on the detection of products or antigens of the infectious agent; and indirect immunological assays that can detect antibodies produced against the infectious agent during infection.
In the first system, the principal disadvantage is that the biological sample must be considered to be at risk for the transmission of the pathogenic agent. In the second and third systems, the disadvantages include sample retrieval that is often invasive and potentially infective sample when collected. In the third system, one major disadvantage is that there is often little possibility of discriminating between past and current infections.
More recently, molecular diagnostic methods have been developed based on the detection of the nucleic acids of the pathogenic agent in the blood or plasma samples, or in the cell cultures, taken from the patient. These assays are generally much more sensitive than the immunological assays. However, they may require the presence of special equipment and qualified personnel. Furthermore, the biological samples—in the case of plasma, blood, or cell cultures—are difficult to store unaltered, except under controlled temperature conditions, and are considered to be biohazardous to personnel who handle them.
Recently, molecular diagnostic methods based on transrenal DNA (Tr-DNA) have been described for monitoring the progress of allogeneic transplants, to diagnose the sex of a fetus, and to detect the presence of tumor markers. (Botezatu et al. Clinical Chemistry 46(8):1078-84 (2000); Su et al. Ann. NY Acad. Sci. 1022:81-89 (2004)) For example, U.S. Pat. No. 6,251,638 describes an analytical method for detecting male fetal DNA in the urine of pregnant women. U.S. Pat. No. 6,287,820 describes a system aimed at the diagnosis of tumors, particularly of adenocarcinomas of the colon and pancreas. U.S. Pat. No. 6,492,144 teaches that the Tr-DNA nucleic-acid analysis method may be used to monitor the progress of allogeneic transplants. The presence of transrenal DNA in urine, in the form of nucleic-acid fragments of fewer than 1000 base pairs was also described in Al-Yatama et al. (2001), Prenat Diagn, 21:399-402; and Utting, M., et al. (2002), Clin Cancer Res, 8:35-40. Keiko Koide, et al., Prenat Diagn, 2005; 25: 604-607; Mengjun Wang, et al., Clinical Chemistry, 2004, 50: 211-213; Y.-H. Su, et al., J. Mol. Diagn., 2004, 6: 101-107.
The presence of transrenal DNA has been explained through the apoptosis phenomenon. During cell death most of the nuclear DNA is converted into nucleosomes and oligomers (Umansky, S. R., et al. 1982 , Biochim. Biophys. Acta 655:9-17), which are finally digested by macrophages or neighboring cells. However, a portion of this degraded DNA escapes phagocytic metabolism, and can be found in the bloodstream (Lichtenstein, A. V., et al. 2001 , Ann NY Acad Sci, 945:239-249), and, as confirmed in the above-indicated patents, also in urine.
The application of this system to pathogenic microorganism infections has never been studied. Previously, it was only known that prokaryotic DNA could be isolated from urine sediment that contained bacteria (Frasier, et al. 1992, Acta Virol, 36:83-89). During a pathogenic infection, prokaryotes and parasites are generally ingested by the cells of the immune system, such as macrophages and dendritic cells. The prokaryotes are then dissolved by the phagolysosome vesicles. The prokaryotic DNA is then released by the cell and a portion of this DNA enters the bloodstream in either of two ways. Either the ingesting cell becomes apoptotic and breaks apart (Navarre, W. V. 2000 ; Cell Microbiol 2:265-273); or the phagolysosome vesicles release the fragments of the prokaryote (including the fragmented DNA) into the bloodstream (Friedlander, A. M. 1978 , Infect Immune 22:148-154). However, these fragmented nucleic acids have never been detected in the urine of the infected subject.
The instant invention describes a method of detecting the presence of non-viral pathogens in a subject through the detection of DNA sequences from those pathogens in the urine of the subject.
SUMMARY OF THE INVENTION
The invention provides compositions and methods for the diagnosis of an infection of a subject through the detection of the presence of pathogens by the presence of pathogen nucleic acids in a urine sample of the subject. The methods of the invention are also used to validate the diagnosis of an infection of a subject through the detection of the presence of pathogen nucleic acids in a urine sample of the subject, wherein a previous diagnosis has already been performed by a method of this invention or another diagnosis method. The compositions and methods of the invention are also used to determine the efficacy of a treatment of non-viral pathogen infection in a subject by detecting the presence or absence of a nucleic acid of the non-viral pathogen in the urine of the subject. More specifically, the compositions and methods of the invention are used to identify a drug resistant non-viral pathogenic infection in a subject by detecting the presence or absence of a nucleic acid of the non-viral pathogen in the urine of the subject after the subject has been treated for the infection with a drug. The compositions and methods of the invention are also used to determine the likelihood of pathology from the infection of the subject by a non-viral pathogen in a subject at risk therefrom. More specifically, the compositions and methods of the invention may be used to genotype a non-viral pathogen which has infected a subject by detecting and genotyping nucleic acids from the pathogen in the urine of the subject.
The invention provides a method of isolating a nucleic acid from urine, the method comprising providing urine from a subject; separating cells and cell debris from the urine by filtration or centrifugation; adding EDTA and Tris-HCl to the urine; adding a chaotropic salt to the urine; adding a resin, wherein the resin binds the nucleic acid in the presence of the chaotropic salt; removing the resin from the urine; and eluting the nucleic acid from the resin; thereby isolating the nucleic acid from urine. In one embodiment of the method of isolating a nucleic acid from urine, the concentration of EDTA and Tris-HCl after it is added to the urine is about 10 mM, and pH between about 8.0 and about 8.5.
In another embodiment of the method of isolating a nucleic acid from urine, the filtration is performed with a filter with a pore size between about 0.1 μm and about 5.0 μm.
In another embodiment of the method of isolating a nucleic acid from urine, the chaotropic salt is guanidine isothiocyanate. In one aspect of this embodiment, the guanidine isothiocyanate has a concentration after being added to the urine of at least about 3 M, and at most about 6 M.
In another embodiment of the method of isolating a nucleic acid from urine, the resin is constructed of silica.
In another embodiment of the method of isolating a nucleic acid from urine, the subject is a mammal. In one aspect of this embodiment, the mammal is a human.
The invention also provides a method of detecting a non-viral pathogen in a subject comprising detecting the presence of a nucleic acid from the non-viral pathogen in urine of the subject, wherein the non-viral pathogen is selected from Helicobacter pylori, Mycobacterium tuberculosis, Bacillus anthracia, Plasmodium species and Leishmania species. In one embodiment of the method of detecting a non-viral pathogen in a subject, the method further comprises the step of quantitating the nucleic acid.
In another embodiment of the method of detecting a non-viral pathogen in a subject, the detecting is performed by a method selected from polymerase chain reaction (PCR), nested PCR, semi-nested PCR, hybridization, Single-Strand Conformation Polymorphism analysis (SSCP), ligase chain reaction (LCR), strand displacement amplification (SDA), and pairing with molecular probes that are specific for the non-viral pathogen.
In another embodiment of the method of detecting a non-viral pathogen in a subject, the subject is a mammal. In one aspect of this embodiment, the mammal is a human.
The invention also provides a method for diagnosing a Helicobacter pylori infection in a subject, comprising detecting the presence of a Helicobacter pylori nucleic acid in a urine sample from the subject, thereby diagnosing the Helicobacter pylori infection. In one embodiment of the method for diagnosing a Helicobacter pylori infection in a subject, the method further comprises the step of quantitating the nucleic acid.
In another embodiment of the method for diagnosing a Helicobacter pylori infection in a subject, the detecting is performed by a method selected from PCR, nested PCR, semi-nested PCR, hybridization, SSCP, LCR, SDA, and pairing with molecular probes that are specific for the non-viral pathogen. In one aspect of this embodiment, a primer set is used in the method, and the primer set comprises a forward primer and a reverse primer. Optionally, the forward primer is selected from SEQ ID NOs: 5, 8, 10, 11, 12 and 15 and the reverse primer is selected from SEQ ID NOs: 6, 7, 9, 13, 14, 16 and 17.
In another embodiment of the method for diagnosing a Helicobacter pylori infection in a subject, the Helicobacter pylori nucleic acid comprises transrenal DNA.
In another embodiment of the method for diagnosing a Helicobacter pylori infection in a subject, the subject is a mammal. In one aspect of this embodiment, the mammal is a human.
In another embodiment of the method for diagnosing a Helicobacter pylori infection in a subject, the method further comprises the step of genotyping the Helicobacter pylori which infected the subject. In one aspect of this embodiment, the Helicobacter pylori is genotyped for the presence of a cag pathogenicity island. In another aspect of this embodiment, the Helicobacter pylori is genotyped for the presence of a vacA genotype selected from s1/m1, s1m2, s2 m1 and s2 m2.
The invention also provides a method for diagnosing a Mycobacterium tuberculosis infection in a subject, comprising detecting the presence of a Mycobacterium tuberculosis nucleic acid in a urine sample from the subject, wherein the Mycobacterium tuberculosis nucleic acid has crossed the kidney barrier and is from cells outside a urinary tract of the subject, thereby diagnosing the Mycobacterium tuberculosis infection. In one embodiment of the method for diagnosing a Mycobacterium tuberculosis infection in a subject, the method further comprises the step of quantitating the nucleic acid.
In another embodiment of the method for diagnosing a Mycobacterium tuberculosis infection in a subject, the detecting is performed by a method selected from PCR, nested PCR, semi-nested PCR, hybridization, SSCP, LCR, SDA, and pairing with molecular probes that are specific for the non-viral pathogen. In one aspect of this embodiment, a primer set is used for the method, wherein the primer set comprises a forward primer and a reverse primer. Optionally, the forward primer is selected from SEQ ID NOs: 34, 37 and 39 and the reverse primer is selected from SEQ ID NOs: 35, 36, 38 and 40.
In another embodiment of the method for diagnosing a Mycobacterium tuberculosis infection in a subject, the Mycobacterium tuberculosis nucleic acid comprises transrenal DNA.
In another embodiment of the method for diagnosing a Mycobacterium tuberculosis infection in a subject, the subject is a mammal. In one aspect of this embodiment, the mammal is a human.
The invention also provides a method for diagnosing a Bacillus anthracis infection in a subject, comprising detecting the presence of a Bacillus anthracis nucleic acid in a urine sample from the subject, thereby diagnosing the Bacillus anthracis infection. In one embodiment of the method for diagnosing a Bacillus anthracis infection in a subject, the method further comprises the step of quantitating the nucleic acid.
In another embodiment of the method for diagnosing a Bacillus anthracis infection in a subject, the detecting is performed by a method selected from PCR, nested PCR, semi-nested PCR, hybridization, SSCP, LCR, SDA, and pairing with molecular probes that are specific for the non-viral pathogen. In one aspect of this embodiment, a primer set is used for the method, wherein the primer set comprises a forward primer and a reverse primer. Optionally, the forward primer is selected from SEQ ID NOs: 41, 43, 45, 47, 48 and 50 and the reverse primer is selected from SEQ ID NOs: 42, 44, 46 and 49.
In another embodiment of the method for diagnosing a Bacillus anthracis infection in a subject, the Bacillus anthracis nucleic acid comprises transrenal DNA.
In another embodiment of the method for diagnosing a Bacillus anthracis infection in a subject, the subject is a mammal. In one aspect of this embodiment, the mammal is a human.
The invention also provides a method for diagnosing a Plasmodium species infection in a subject, comprising detecting the presence of a Plasmodium species nucleic acid in a urine sample from the subject, thereby diagnosing the Plasmodium species infection. In one embodiment of the method for diagnosing a Plasmodium species infection in a subject, the method further comprises the step of quantitating the nucleic acid.
In another embodiment of the method for diagnosing a Plasmodium species infection in a subject, the detecting is performed by a method selected from PCR, nested PCR, semi-nested PCR, hybridization, SSCP, LCR, SDA, and pairing with molecular probes that are specific for the non-viral pathogen. In one aspect of this embodiment, a primer set is used for the method, wherein the primer set comprises a forward primer and a reverse primer. Optionally, the forward primer is selected from SEQ ID NOs: 18, 21 and 23 and the reverse primer is selected from SEQ ID NOs: 19, 20 and 22.
In another embodiment of the method for diagnosing a Plasmodium species infection in a subject, the Plasmodium species nucleic acid comprises transrenal DNA.
In another embodiment of the method for diagnosing a Plasmodium species infection in a subject, the subject is a mammal. In one aspect of this embodiment, the mammal is a human.
The invention also includes a method for diagnosing a Leishmania species infection in a subject, comprising detecting the presence of a Leishmania species nucleic acid in a urine sample from the subject, thereby diagnosing the Leishmania species infection. In one embodiment of the method for diagnosing a Leishmania species infection in a subject, the method further comprises the step of quantitating the nucleic acid.
In another embodiment of the method for diagnosing a Leishmania species infection in a subject, the detecting is performed by a method selected from PCR, nested PCR, semi-nested PCR, hybridization, SSCP, LCR, SDA, and pairing with molecular probes that are specific for the non-viral pathogen. In one aspect of this embodiment, a primer set is used for the method, wherein the primer set comprises a forward primer and a reverse primer. Optionally, the forward primer is selected from SEQ ID NOs: 26, 28, 30, 32 and 51 and the reverse primer is selected from SEQ ID NOs: 24, 25, 27, 29, 31 and 33.
In another embodiment of the method for diagnosing a Leishmania species infection in a subject, the Leishmania species nucleic acid comprises transrenal DNA.
In another embodiment of the method for diagnosing a Leishmania species infection in a subject, the subject is a mammal. In one aspect of this embodiment, the mammal is a human.
The invention also provides a composition for use in the detection of Helicobacter pylori , wherein the composition comprises a nucleic acid sequence selected from SEQ ID NOs: 5-17. In one embodiment of the composition for use in the detection of Helicobacter pylori , the composition consists of a nucleic acid sequence selected from SEQ ID NOs: 5-17.
The invention also provides a composition for use in the detection of Plasmodium species, wherein the composition comprises a nucleic acid sequence selected from SEQ ID NOs: 18-23. In one embodiment of the composition for use in the detection of Plasmodium species, the composition consists of a nucleic acid selected from SEQ ID NOs: 18-23.
The invention also provides a composition for use in the detection of Leishmania species, wherein the composition comprises a nucleic acid sequence selected from SEQ ID NOs: 24-33 and 51. In one embodiment of the composition for use in the detection of Leishmania species, the composition consists of a nucleic acid selected from SEQ ID NOs: 24-33 and 51.
The invention also provides a composition for use in the detection of Mycobacterium tuberculosis , wherein the composition comprises a nucleic acid sequence selected from SEQ ID NOs: 34-40. In one embodiment of the composition for use in the detection of Mycobacterium tuberculosis , the composition consists of a nucleic acid selected from SEQ ID NOs: 34-40.
The invention also provides a composition for use in the detection of Bacillus anthracis , wherein the composition comprises a nucleic acid sequence selected from SEQ ID NOs: 41-50. In one embodiment of the composition for use in the detection of Bacillus anthracis , the composition consists of a nucleic acid selected from SEQ ID NOs: 41-50.
The invention also provides a kit for detecting Helicobacter pylori in a urine sample from a subject in need thereof, comprising at least one forward primer selected from SEQ ID NOs: 5, 8, 10, 11, 12 and 15 and at least one reverse primer selected from SEQ ID NOs: 6, 7, 9, 13, 14, 16 and 17, either in the same or separate packaging, and instructions for its use.
The invention also provides a kit for detecting Mycobacterium tuberculosis in a urine sample from a subject in need thereof, comprising at least one forward primer selected from SEQ ID NOs: 34, 37 and 39 and at least one reverse primer selected from SEQ ID NOs: 35, 36, 38 and 40, either in the same or separate packaging, and instructions for its use.
The invention also provides a kit for detecting Bacillus anthracis in a urine sample from a subject in need thereof, comprising at least one forward primer selected from SEQ ID NOs: 41, 43, 45, 47, 48 and 50 and at least one reverse primer selected from SEQ ID NOs: 42, 44, 46 and 49, either in the same or separate packaging, and instructions for its use.
The invention also provides a kit for detecting Plasmodium species in a urine sample from a subject in need thereof, comprising at least one forward primer selected from SEQ ID NOs: 18, 21 and 23 and at least one reverse primer selected from SEQ ID NOs: 19, 20 and 22, either in the same or separate packaging, and instructions for its use.
The invention also provides a kit for detecting Leishmania species in a urine sample from a subject in need thereof, comprising at least one forward primer selected from SEQ ID NOs: 26, 28, 30, 32 and 51 and at least one reverse primer selected from SEQ ID NOs: 24, 25, 27, 29, 31 and 33, either in the same or separate packaging, and instructions for its use.



BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 is a photograph of PCR reactions performed on human urine with primers specific for human b-actin resolved by electrophoresis.
FIG. 2 is a photograph of PCR reactions performed on rabbit urine with primers specific for rabbit b-globin resolved by electrophoresis.
FIG. 3A is a photograph of semi-nested PCR reactions performed on human urine with primers specific for H. pylori ureA sequences resolved by electrophoresis.
FIG. 3B is a photograph of semi-nested PCR reactions performed on human urine with primers specific for H. pylori cagA sequences resolved by electrophoresis.
FIG. 4 is a photograph of semi-nested PCR reactions performed on human urine with primers specific for Plasmodium falciparum sequences resolved by electrophoresis.
FIG. 5 is a photograph of semi-nested PCR reactions performed on human urine with primers specific for Leishmania infantum sequences resolved by electrophoresis.
FIG. 6 is a photograph of semi-nested PCR reactions performed on human urine from TB infected and healthy patients with primers specific for Mycobacterium tuberculosis sequences resolved by electrophoresis.
FIG. 7 is a photograph of semi-nested PCR reactions performed on the supernatants and pellets of centrifuged human urine from TB infected and healthy patients before and after treatment with primers specific for Mycobacterium tuberculosis sequences resolved by electrophoresis.
FIG. 8 shows photographs of semi-nested PCR reactions performed on human urine from TB infected patients with primers specific for Mycobacterium tuberculosis sequences of 129/67 by (left panel) and 330/69 by (right panel) resolved by electrophoresis.
FIG. 9 is a photograph of semi-nested PCR reactions performed on human urine from TB actively infected patients and patients two months after treatment for TB with primers specific for Mycobacterium tuberculosis sequences resolved by electrophoresis.
FIG. 10 is a photograph of semi-nested PCR reactions performed on rabbit urine from B. anthracis infected and uninfected subjects with primers specific for Bacillus anthracis sequences resolved by electrophoresis.



DETAILED DESCRIPTION OF THE INVENTION
The invention is based, in part, upon the discovery described herein that pathogenic nucleic acids are detectable in the urine of a subject infected with the pathogen. In some instances, nucleic acids from pathogenic organisms cross the kidney barrier and pass into the urine of mammals. The nucleic acids which cross the renal barrier, termed herein as transrenal nucleic acids, and more specifically transrenal DNA tend to be shorter than 1,000 by in length, but are preferably less than 500 by in length, and more preferably shorter than 250-300 by in length or shorter than 250 by in length. While it had been shown in the past that DNA from mammalian cells, cancerous and non-cancerous which were hypothesized to die by apoptosis in the body of a subject may be able to cross the renal barrier, this is the first time it has been shown that the DNA of pathogens also crosses this barrier. Further, nucleic acids from pathogens found in the kidney would shed pathogen specific nucleic acids into the urine without crossing the kidney barrier. There may be other mechanisms by which pathogen specific nucleic acids pass into the urine of a subject infected with the pathogen.
Based on this discovery, the invention provides compositions and methods for the detection of non-viral pathogens in the urine of a subject. The compositions and methods of the invention are used to detect transrenal-nucleic acids derived from pathogenic microorganisms which traverse the renal barrier and are present in the urine of a subject. The invention includes methods for the isolation, amplification and detection of these transrenal nucleic acids. The invention also includes compositions which may be used to isolate, amplify these transrenal nucleic acids.
One method of the invention for the isolation of urinary DNA from a subject includes the following steps. Urine is first centrifuged or filtered to separate cells and cell debris from the urine. EDTA/Tris HCl is then added to the urine to bind bivalent ions and to adjust pH to approximately pH 8. To this solution is then added a chaotropic salt, for example guanidine isothiocyanate to a final concentration of at least 3 M. Then a silica resin is added to the urine to which DNA binds. This resin is then washed, and the DNA eluted from it, thereby isolating the transrenal DNA.
DNA isolated from the urine of a subject may then be amplified in order to be detected. Amplification methods include polymerase chain reaction (PCR), nested PCR, semi-nested PCR, Single-Strand Conformation Polymorphism analysis (SSCP), ligase chain reaction (LCR) and strand displacement amplification (SDA). Detection of transrenal DNAs is also performed through hybridization of at least one labeled probe.
The amplification and detection of urinary nucleic acids from non-viral pathogens may be used to detect the presence of these pathogens, and thus diagnose the infection of a subject with these pathogens and to diagnose pathologies associated with the infection of a subject by these pathogens. Further, the detection of these pathogens may be used to monitor treatment of the infections and pathologies associated with these pathogens. Also, detection of these pathogens may be used to suggest treatments for a subject in which the pathogens are detected. These treatments may be used to relieve symptoms which are associated with the detection of pathogens or diagnosis with infection of the pathogens.
Non-viral pathogens which are able to be detected by the presence of their nucleic acids in mammalian urine include bacteria and parasites. Bacterial species which are detectable using the compositions and methods of the invention include Helicobacter pylori ( H. pylori ), Mycobacterium tuberculosis ( M. tuberculosis , MTB or TB), and Bacillus anthracis ( B. anthracis ). Parasite species which are detectable using the compositions and methods of the invention include Plasmodium species including Plasmodium falciparum and Leishmania species.
Detection of the Presence of Pathogenic Microorganisms
Bacteria

Helicobacter pylori

Most cases of peptic ulcer disease, gastric mucosa associated lymphoid tissue (MALT) lymphoma and cancer of the distal stomach are complications of Helicobacter pylori infection. (Axon A T. Gut. 1999 July; 45 Suppl 1:11-4). However, most H. pylori -positive individuals remain symptom free throughout their life. Symptoms associated with H. pylori infection include abdominal discomfort, weight loss, poor appetite, bloating, burping, nausea and vomiting. A widely accepted explanation of this phenomenon is that the outcome of H. pylori colonization is determined by combination of several factors including the genotypes of the bacteria and the host and the environmental cofactors such as diet and smoking. For the successful medical management of the infection it is very important the development of diagnostics tools to direct the decision concerning the treatment of the carriers. The test should give information about the genotype of the bacteria in the carrier.
Colonizing human stomach H. pylori strains are genetically very diverse. Observed diversity is attributed to both horizontal transfer of genetic material between coexisting strains as well as to the instability of the bacterial genome. Polymorphism occurs due to point mutations, substitutions, insertions, deletions that may involve one or more genes (Akopyanz, N. S., et al. 1992. Nucleic Acids Res. 20:5137-5142; Achtman, M., et al. 1999. Mol. Microbiol. 32:459-470; Tomb, J.-F., et al. 1997. Nature. 388:539-547; Alm, R. A., et al. 1999. Nature. 397:176-180; Kleanthous, H., et al. 1991. Mol. Microbiol. 5:2377-2389; Kersulyte, D., et al. 1998. Gene. 223:175-186.) Therefore, important initial step for the development of genetic test is the selection of specific genetic markers of H. pylori associated with clinical symptoms. There are numerous publications concerning the genetic markers associated with elevated virulence of the bacteria and the disease.
A major genetic determinant of H. pylori virulence is the cag pathogenicity island (cag PAI) (Blaser, M. J., et al. 1995. Cancer Res. 55:2111-2115; Cover, T. L., et al. 2001. Helicobacter pylori pathogenesis. Academic Press, San Diego, Calif.; Mobley, H. L. 1996. Am. J. Med. 100:2 S-11S.), a 40-kb region of chromosomal DNA that is present in some H. pylori strains but absent from others. The cag PAI encodes a type IV secretion system and an immunodominant antigen, CagA, which is translocated into gastric epithelial cells. In comparison to infection with cag PAI-negative H. pylori strains, infection with cag PAI-positive strains is associated with an increased severity of gastric mucosal inflammation, an increased risk for development of peptic ulceration, and an increased risk of gastric cancer (Ho, S. A., et al. 1991. J. Clin. Microbiol. 29:2543-2549). cagA is present in only 60-70% of H pylori isolates in Western countries. Over 80% of patients with ulcers harbor cagA positive strains (Atherton J C. Gut. 1997 June; 40(6):701-3; Tummuru M K R, et al. Infect Immun 1993; 61:1799-809; Atherton J C, et al. Curr Microbiol. 1999 October; 39(4):211-8.).
In contrast to cagA, the gene encoding the vacuolating cytotoxin (vacA) is present in all H. pylori strains analyzed so far. But only 50% of them produce an active vacuolating cytotoxin. Production and cytotoxicity of the vacA protein is determined by its structure. There are two polymorphic regions in the vacA gene: the region encoding the signal sequence with two distinct types of s1 and s2, and midregion marked as m1 and m2. Existence of combination of all s and m types was registered except s2/m1. Most active genotype of vacA gene is shown to be s1/m1 followed by s1/m2. Very little activity or no activity was seen for the type s2/m2. Studies in US demonstrated that over 90% of patients with duodenal ulcer disease had vacA s1 strains and those with s2 strains were at the level of uninfected patients. There is very close genetic linkage between cagA and vacA s1/m1 genotypes. In this pair of genetic markers vacA is considered to be surrogate for the cagA (Cover L, T et al. J Biol Chem 1994; 269:10566-73; Graham D Y, and Yamaoka Y. Disease-specific Helicobacter pylori virulence factors: the unfulfilled promise. Helicobacter. 2000; 5 Suppl 1:S3-9; discussion S27-31).
There are many other H. pylori genetic markers studied such as: 16S rRNA gene, the random chromosome sequence, the 26-kDa species-specific antigen (SSA) gene, the urease A (ureA) gene, and the urease C (ureC) gene (Valentine, J. L., et al. 1991. J. Clin. Microbiol. 29:689-695; Hammer, M., T. et al. 1992. J. Clin. Microbiol. 30:54-58; Clayton, C. L., et al. 1992. J. Clin. Microbiol. 30:192-200; Bickley, J., et al. 1993. J. Med. Microbiol. 39:338-344; Lu J J, et al. J Clin Microbiol. 1999 March; 37(3):772-4.).
Thus, according to the methods and compositions of the invention, and explained in more detail in the Examples below, H. pylori presence and virulence can be detected through the isolation and detection of H. pylori transrenal-DNA. Methods of detecting the presence and genotype of H. pylori in urine have not been shown before.

Mycobacterium tuberculosis

Tuberculosis remains the second most common cause of death from an infectious disease in the world in spite of advances in the implementation of control strategies during the last decade (Elzing a G, et al. Lancet 2004; 363:814-9). Symptoms of tuberculosis include cough that is worse in the morning (sometimes with hemoptysis, blood in the sputum), chest pain, breathlessness, night sweats, and signs of pneumonia. In advanced disease, there may be extreme weight loss. However, no highly sensitive molecular-based test for the detection of M. tuberculosis has yet been discovered. Effective treatment of active tuberculosis is the cornerstone of tuberculosis control, and it has been shown that a mere 1% increase in the rate of detection of active cases can save thousands of lives and avert thousand of new tuberculosis cases in high incidence countries (Currie C S, et al. AIDS. 2003; 17:2501-8). Consequently, the availability of new diagnostic tools that are more accurate and accessible will greatly benefit individual patients and significantly contribute to the control of the disease.
Advances in knowledge of the genetic structure of tubercle bacillus have recently contributed to the development of several new molecular methods for detection and identification of Mycobacterium tuberculosis from cultures or directly from biological specimens (Drobniewski F A, et al. Lancet Infect Dis. 2003; 3:141-7). Molecular diagnostic tests which involve the detection of specific nucleic acid sequences could be used for rapid diagnosis of both pulmonary and extrapulmonary tuberculosis. In pulmonary tuberculosis specificity of diagnostic tests based on direct identification of M. tuberculosis in the sputum using PCR generally exceeds 98% and sensitivity is also high in patients whose sputum smear is positive for acid-fast bacilli on microscopic examination. However, the sensitivity of such tests may be less than 50% for patients with negative sputum smear. (Drobniewski F A, et al., Piersimoni C and Scarparo C. J Clin Microbiol. 2003; 41:5355-65; Sarmiento O L, et al. J Clin Microbiol. 2003; 41:3233-40). The presence of DNA fragments derived from M. tuberculosis have been shown before, but only when the pathogen itself was present in the urine. (Del Portillo et al. J. Clin. Microbiol., 29(10):2163-2168 (1991); Kolk et al. J. Clin. Microbiol., 30(10):2567-2575 (1992); Kox et al. Neurology, 45(12):2228-2232 (1995) and Zambardi et al. Mol. Cell. Probes, 9:91-99 (1995)).
The present invention allows for the detection of M. tuberculosis through identifying its transrenal DNA in the urine of patients with pulmonary tuberculosis. This approach is more attractive than tests using sputum samples because urine specimens are easier and safer to collect than sputum which can generate infectious aerosols and prove to be difficult to obtain, especially in children.
Despite a previous study suggesting the potential utility of a urine test for M. tuberculosis DNA by PCR in HIV infected patients with pulmonary tuberculosis (Aceti A, et al. Thorax. 1999; 54:145-6), subsequent prospective studies conducted on HIV infected and uninfected patients with pulmonary tuberculosis, resulted in only a 40 to 60% sensitivity. This is too low a sensitivity to support the use of a diagnostic test in clinical practice. In these and other such studies, target urinary DNA was of relatively high molecular weight, amplicon sizes of >500 by having been employed in the PCR reactions. (Kafwabulula M, et al. Int J Tuberc Lung Dis. 2002; 6:732-7; Torrea G, et al. J Med. Microbiol. 2005; 54:39-44).
Previously reported PCR based tests have been used successfully to detect M. tuberculosis in the urine of patients with genitourinary tuberculosis (van Vollenhoven P, et al. Urol Res. 1996; 24(2):107-11; Hemal A K, et al. Urology. 2000 Oct. 1; 56(4):570-4; Moussa O M, et al. J Urol 2000; 164:584-8) in which cases the infectious bacteria are present in the urinary tract. Among these patients, high sensitivity and specificity has been achieved by amplifying large (245 to 1000 bp) fragments of mycobacterial DNA purified from urine sediment and the results of molecular test are strongly correlated with the presence of viable mycobacteria which can be cultured from the urine. Thus, presence of specific DNA fragments in these patients appears to reflect urinary excretion of mycobacteria actively replicating in the urogenital tract.
In contrast, we have discovered that small cell-free M. tuberculosis DNA fragments originating from sites of infection outside the urogenital tract are present in the urine in this previously unrecognized range of molecular sizes of less than approximately 200 bp. M. tuberculosis specific DNA sequences can be easily detected as short fragments of less than 200 by in the soluble fraction of urine specimens from patients with pulmonary tuberculosis. These specific DNA fragments disappear following successful tuberculosis treatment. This is demonstrated in more detail in the Examples below.
The data on M. tuberculosis Tr-DNA disappearance during the course of treatment supports the potential utility of this test in monitoring the clinical course of the disease. It is also clear that small urine specimens can be obtained in the field more easily and safely which is important when broad screening of large populations is of concern to public health. It is also likely that Tr-DNA testing for diagnosis of tuberculosis would have particular value for clinics located in regions having limited resources. Although PCR is currently the preferred method for detecting specific nucleic acid sequences, new emerging technologies can be expected to eventually provide the tools to perform the relatively simple Tr-DNA test in the field (Storhoff J. J., et al. (2004) Biosensors & Bioelectronics 19:875-883; Nam J. M., et al. (2002) J. Am. Chem. Soc 124:3820-3821; Ho H A, et al. (2005) J Am Chem. Soc. 127:12673-12676) offering practical advantages over those of PCR can be readily applied to the Tr-DNA test. All steps of the diagnostic process, from Tr-DNA purification to biomarker detection, can be automated and brought to the point of care in small hospitals or even in the field with greater facility than can be done with conventional microbiological testing.

Bacillus anthracis

Bacillus anthracis is a spore-forming gram-positive bacterium well known for its recent use as a bioterrorist agent. Bacillus anthracis is a large, Gram-positive, spore forming rod shaped bacterium, 1-1.2 μm in width×3-5 μm in length. The bacterium grows in most media under aerobic or anaerobic conditions. Bacillus anthracis infection can cause anthrax. Until recently, anthrax was primarily associated with contact with domestic animals. However, more recently Bacillus anthracis has been associated with weaponized forms used in terrorism. In the United States, the incidence of naturally-acquired anthrax is extremely rare (1-2 cases of cutaneous disease per year). However, in fall 2001, 22 cases of anthrax (11 inhalation cases, 11 cutaneous cases) were identified in the United States following intentional contamination of the mail.
The most common form of the disease in humans is cutaneous anthrax, which is usually acquired via injured skin or mucous membranes. A minor scratch or abrasion of the skin is contacted by Bacillus anthracis spores. The spores germinate, vegetative cells replicate, and an edema develops at the site. This develops into papule within 12-36 hours which changes rapidly to a vesicle, then a pustule (malignant pustule), and finally into a necrotic ulcer from which infection may disseminate, giving rise to septicemia. Lymphatic swelling also occurs and in severe cases, where the blood stream is eventually invaded, the disease is frequently fatal.
Another form of the disease, inhalation anthrax (woolsorters' disease), results from the inhalation of spore-containing dust. The disease begins with a high fever and chest pain and progresses to a systemic hemorrhagic pathology. It is often fatal if treatment cannot stop the invasive aspect of the infection.
The virulence of B. anthracis has been linked to two plasmids, pXO1 and pXO2 (Turnbull, P. C. B., and J. M. Kramer. 1995. Bacillus , p. 349-356. In P. R. Murray, E. J. Baron, et al. (ed.), Manual of clinical microbiology, 6th ed. ASM Press, Washington, D.C.). Isolated Strains lacking pX01 and/or pX02 plasmid were shown to be non-virulent (Mikesell, P., et al. 2002. Plasmids, Pasteur, and anthrax. ASM News 49:320-322. Welkos, S. L. 1991. Microb. Pathog. 10:183-198.).
The plasmid pXO1 (110-MDa) carries three anthrax toxin proteins coding genes, edema factor (cya), lethal factor (lef), and protective antigen (pag) (Uchida, I., K. et al. 1986. J. Gen. Microbiol. 132(Pt. 2):557-559.). Products of these genes act in paired combinations to produce the two anthrax toxins: edema toxin (pag and cya) and lethal toxin (pag and lef) (Leppla, S. H. 1995. Bacterial toxins and virulence factors in disease, p. 543-572. Marcel Dekker, New York, N.Y.). These toxicity loci on pXO1 are commonly used as specific genetic markers for PCR detection of B. anthracis . Chromosomal genetic markers such as rpoB, gyrA, rRNA were also successfully utilized for the same purposes.(Fasanella, A., S. et al. 2001. Vaccine 19:4214-4218., Qi, Y., et al. 2001. Appl. Environ. Microbiol. 67:3720-3727., Ramisse, V., et al. 1996. FEMS Microbiol. Lett. 145:9-16. Oggioni M R, et al. 2002 J Clin Microbiol. 40(11):3956-63.) Blackwood K S, et al. 2004 J Clin Microbiol. April; 42(4):1626-30; Drago, L., et al. 2002. J. Clin. Microbiol. 40:4399. Hurtle, W., et al. 2004. J. Clin. Microbiol. 42:179-185.)
Identification of B. anthracis can be done clinically by Gram stain, colony morphology, and various biochemical tests (Logan, N. A., and P. C. B. Turnbull. 2004. Bacillus and other aerobic endospore-forming bacteria, p. 445-460. In P. R. Murray, et al. (ed.), Manual of clinical microbiology, 8th ed. American Society for Microbiology, Washington, D.C.). However, these methods are time-consuming, therefore more rapid tests, such as PCR, have been developed to detect B. anthracis in clinical samples (Oggioni, M. R., et al. 2002. J. Clin. Microbiol. 40:3956-3963.). PCR assays have been widely used to identify B. anthracis on the basis of genes associated with pXO1 and pXO2 plasmids. (Higgins, J. A., et al. 2003. Appl. Environ. Microbiol. 69:593-599. Oggioni, M. R., F. et al. 2002. J. Clin. Microbiol. 40:3956-3963., Patra, G., et al. 2002. Ann. N.Y. Acad. Sci. 969:106-111.)
However, previous tests used samples isolated from nasal passages. These samples may potentially contain infective pathogen, while urine samples do not. The methods and compositions of the invention are used to detect the presence of B. anthracis in subjects through identification of transrenal DNA from B. anthracis in the urine of the subjects. These compositions and methods are described in more detail in the Examples below.
Parasites
Plasmodium Species
It is estimated that there are at least 300 million acute cases of malaria each year globally, resulting in 1.5 to 2.7 million deaths. About 90% of these deaths occur in Africa, mostly in young children. According to the U.S. Centers for Disease Control and Prevention, more than 1000 new cases are reported annually in U.S. travelers returning from malaria-endemic areas. Symptoms of malaria include chills, fever, anemia and jaundice.
Four species of malaria parasites can infect humans under natural conditions: Plasmodium falciparum, P. vivax, P. ovale and P. malariae . The first two species cause the most infections worldwide. Plasmodium falciparum is the agent of severe, potentially fatal malaria, causing an estimated 700,000-2.7 million deaths annually, most of them in young children in Africa. Plasmodium vivax and P. ovale have dormant liver stage parasites (“hypnozoites”) which can reactivate (“relapse”) and cause malaria several months or years after the infecting mosquito bite. Plasmodium malariae produces long-lasting infections and if left untreated can persist asymptomatically in the human host for years, even a lifetime.
Molecular diagnostic methods have been developed based on the detection of the nucleic acids of the pathogenic agent in the blood or plasma samples, or in the cell cultures, taken from the patient. These assays are generally much more sensitive than the immunological assays. However, they may require the presence of special equipment and qualified personnel. Furthermore, the biological samples—in the case of plasma, blood, or cell cultures—are difficult to store unaltered, except under controlled temperature conditions. For example, methods for the detection of Plasmodium in blood have been reported by Gal S., et al. (2001), Ann N Y Acad. Sci. 945: 234-238.
The present invention provides compositions and methods for the detection of Plasmodium species, potentially very soon after infection, by isolating and detecting Plasmodium species transrenal DNA in subjects. A more detailed description of these compositions and methods are found below in the Examples.
Leishmania Species
About 12 million people throughout the world suffer from leishmaniasis. The disease threatens populations of 30 million people in 88 countries. Over the last 10 years endemic regions have been growing, with a sharp increase in the number of recorded cases of the disease. In addition, as a result of epidemiological changes, leishmaniasis is sharply increasing in populations which are also threatened by overlapping AIDS infection. Molecular diagnostic methods have been developed based on the detection of the nucleic acids of the pathogenic agent in the blood or plasma samples, or in the cell cultures, taken from the patient. These assays are generally much more sensitive than the immunological assays. However, they may require the presence of special equipment and qualified personnel. Furthermore, the biological samples—in the case of plasma, blood, or cell cultures—are difficult to store unaltered, except under controlled temperature conditions. For example, methods for the detection of Leishmania in blood have been reported by Disch J, et al. Acta Trop. 2004 November-December; 92(3):279-83; Disch J, et al., Trans R Soc Trop Med. Hyg. 2003 July-August; 97(4):391-5; and Lee N, et al., Cell Death Differ. 2002 January; 9(1):53-64. The symptoms of leishmaniasis include fever, fatigue, weakness, appetite loss, weight loss, abdominal discomfort, vomiting, diarrhea, cough, scaly skin, thinning hair, macule or papule forming on the skin, skin ulcer, nasal stuffiness, nosebleed, difficulty swallowing and difficulty breathing.
The present invention provides compositions and methods for the detection of Leishmania species, potentially very soon after infection, by isolating and detecting Leishmania species transrenal DNA in subjects. A more detailed description of these compositions and methods are found below in the Examples.
Urinary Nucleic Acids in Non-Viral Pathogen Infections
The present invention describes a method for diagnosing and/or monitoring of a non-viral pathogen infection by detecting and quantification of the nucleic acids of pathogenic agents in urine. It has been discovered that the nucleic acids of these pathogenic agents are detectable in urine. Many of these pathogen specific nucleic acids cross the transrenal barrier (Tr-NA) and can be detected in urine as low-molecular-weight fragments (which tend to be shorter than 1,000 by in length, but are preferably less than 500 by in length, and more preferably shorter than 250-300 by in length or shorter than 250 by in length) through molecular methods known in the art. Other pathogen specific nucleic acids may be shed by cells that are within the kidney, and thus do not have to cross the transrenal barrier in order to be detected in the urine. Further, some pathogen specific nucleic acids may be found in the urine through other mechanisms besides crossing the transrenal barrier or being generated by cells in the kidney.
The presence of transrenal nucleic acids in the case of infections caused by pathogenic agents according to the present invention also relates to infections that do not directly involve the urinary tract, even in the absence of hematuria or of pathologies that lead to the rupture, or that alter the normal integrity, of the renal barrier. Transrenal nucleic acids are nucleic acids that have crossed the kidney barrier. Generally, the transrenal nucleic acids (Tr-NA) according to the invention are not associated with, and are not derived from, the DNA of cells that are lost or released in the urinary tract. Instead, the transrenal nucleic acids that are detected in the present invention generally have crossed the kidney barrier as a cell-free material. Thus, their lengths are smaller than about 1000 base pairs, e.g., smaller than about 500, smaller than about 300, smaller than about 250, or between about 100 and about 200 bases or base pairs, as opposed to other cases in which DNA usually has a longer length, greater than 1000 bases or base pairs. In embodiments, the Tr-NAs are DNA fragments.
The discovery confirms the presence of urinary nucleic acids or transrenal nucleic acids derived from pathogenic bacteria and parasites in urine, and therefore is applicable to the diagnosis of all infectious diseases caused by non-viral pathogens.
In one embodiment, the invention relates to a method for the diagnosis a non-viral pathogenic infection in a subject by detection of pathogen-related transrenal nucleic acids in a urine sample from the subject. In some embodiments, the invention further includes the step of quantifying the pathogen-related transrenal nucleic acids. In other embodiments, the invention provides for methods of monitoring a parasite infection by repeated detection of pathogen-related transrenal nucleic acids in a subject over a period of time.
In one embodiment, the invention relates to a method for diagnosis of an infection in a subject, including the step of detection of the presence pathogen-related transrenal nucleic-acid sequences in a urine sample of the subject. In a further embodiment, the invention relates to the above method in which the transrenal nucleic acids are isolated from the urine sample prior to detecting the nucleic acids.
In another embodiment, the method according to the invention may include an initial treatment of the urine sample prior to detection. In a specific embodiment, the invention includes the pretreatment of the urine sample with agents that inhibit the degradation of the nucleic acids. Included are the enzymatic inhibitors, such as chelating agents, detergents, or denaturing agents, and preferably DNase or RNase inhibitors, which include EDTA, guanidine HCl, guanidine isothiocyanate, N-lauryl sarcosine, and sodium dodecyl sulfate.
According to another embodiment, the urine sample may be centrifuged (at a speed between 3000 g and 5000 g, such as between 3500 g and 4500 g) or filtered in order to separate the fraction consisting of cells and their fragments from the supernatant containing the soluble DNA or RNA and their proteinous complexes. However, the urine sample may also be subjected to nucleic acid detection procedures without this fractionation.
The optional isolation or purification and quantification of the transrenal nucleic acids are achieved through the use of chemical or physical methods that are already known in the art. It includes at least one purification step, using methods selected from among extraction with organic solvents, filtration, precipitation, absorption on solid matrices (e.g., via ion exchange), affinity chromatography or else molecular exclusion chromatography or combinations of these methods.
However, the purification method must be appropriate for the isolation of DNA (single- or double-helix) that are less than 1000 nucleotides in length, with a corresponding molecular weight, assuming, as the average molecular weight, that of a nucleotide having a value of 330 Daltons. In some embodiments, the purification is specific for fragments that are smaller than 500 nucleotides (nt) in length, with a corresponding molecular weight, such as fragments whose lengths are less than 300 nt, fragments less than 250 nt in length, or fragments whose lengths are between 100 and 200 base pairs of nucleic acids (nt).
In an embodiment, the DNA isolation method is implemented by pretreating the urine sample with a denaturing agent, such as urea, guanidine HCl, or guanidine isothiocyanate. The sample is then caused to pass through a solid phase, such as a matrix consisting of a silica-based resin which, in the presence of chaotropic salts, such as guanidine isothiocyanate, binds the nucleic acids. Even more preferably, a resin such as Wizard Purification Resin® (Promega®) is utilized. The sample is then collected by elution in a buffer, such as Tris-EDTA (Tris 1-10 mM, EDTA 1-10 mM), or in water.
In a preferred embodiment, the characterization and the determination of the presence of DNA of the pathogenic agent is performed through a technique selected from the group consisting of: hybridization of the nucleic acids, a cycling probe reaction (F. Bekkaoui et al., in BioTechniques 20:240-248 [1996]), a polymerase chain reaction ( PCR Protocols: A Guide to Methods and Applications , by M. Innis et al.; Elsevier Publications, 1990), a nested polymerase chain reaction, single-strand conformation polymorphism, or ligase chain reaction (LCR) (F. Barany, in PNAS USA, 88:189-93 [1991]), strand displacement amplification (SDA) (G. K. Terrance Walker, et al., in Nucleic Acid Res, 22:2670-77 [1994], and restriction fragments length polymorphism. Polymerase chain reaction (PCR) is the preferred method for the detection or analysis of nucleic acids. More preferred is dual-amplification PCR, with the use of a nested primer in nested or semi-nested PCR (reference: bio.davidson.edu/courses/genomics/method/NestedPCR.html).
Methods for the Amplification and Detection of Urinary Nucleic Acids
The term nucleic acid refers to an oligonucleotide, nucleotide, polynucleotide, or fragments/parts thereof and to DNA or RNA of natural (e.g., genomic) or synthetic origin. It may have a double or single helix, and may also represent the sense or antisense direction of a sequence. Parallel helix (5′→3′); antiparallel helix (3′→5′). The terms oligonucleotide, polynucleotide and nucleic-acid polymer are equivalent, and are understood as referring to a molecule consisting of more than two deoxyribonucleic or ribonucleic acid bases. The number of nucleotides (bases) and the length of the oligonucleotide fragment may vary. They may be synthesized in different ways. The sequences are traditionally defined as starting with 5′ and ending with a 3′. These numbers indicate the direction of the sequence.
DNA isolated from the urine of a subject may then be amplified in order to be detected. Amplification methods include polymerase chain reaction (PCR), nested PCR, semi-nested PCR, Single-Strand Conformation Polymorphism analysis (SSCP), ligase chain reaction (LCR) and strand displacement amplification (SDA). Detection of transrenal DNAs is also performed through hybridization of at least one labeled primer.
Hybridization is a method that allows two nucleic-acid sequences to recognize each other as complementary and to join together (annealing). Complementarity/Complementary sequences are sequences of polynucleotides that interact with each other, depending on the interaction between the bases. For example, the AGTC sequence is complementary to TCAG according to standard Watson Crick base pairing. However, other combinations such as Hoogstein base pairing are well known to those having ordinary skill in the art. It is possible to have a fully or partially complementary sequence, and this is what determines the efficiency or attractive force between the two sequences. Average complementarity would prevent a strong complementarity from hybridizing, under conditions that would allow it to remain attached.
The ability of nucleic sequences to hybridize is a well-known phenomenon. The first hybridization method was described in Marmur & Lane, PNAS USA, 46:453 (1960) and 461 (1960), but since then has been perfected as a technique in molecular biology. Today, the term “hybridization” includes, among others, slot/dot and blot hybridization. The conditions that allow nucleotide sequences to recognize each other (hybridization) can be modified in such a way as to produce complete hybridization (complementarity with high specificity) or partial hybridization (complementarity with average specificity). In the present application, whenever the term “hybridization” is used, the conditions should be understood as referring to those that allow average or high complementarity. The technician in the field can calculate how many artificial sequences are needed to encourage hybridization between two complementary sequences in the opposite direction, known as antiparallel association.
A probe is an oligonucleotide that can be produced artificially or naturally, and that forms a combination with another nucleic-acid sequence. The probes are useful in discovering specific sequences in a sample containing unknown DNA. In this patent, all of the probes can be bound to a signaling molecule (or reporter). The reporter molecule makes it possible to detect the probe (for example, through enzymatic reactions (e.g., ELISA (Enzyme-Linked Immunosorbent Assay)), radioactivity, fluorescence, or other systems).
Polymerase chain reaction (PCR) is a method of amplification of a DNA sequence using complementary primers and a heat sensitive polymerase. One class of enzymes utilized in the amplification of specific nucleic acids are DNA polymerases referred to as Taq ( Thermus aquaticus ) polymerases. Primers are oligonucleotides from which, under proper conditions, the synthesis of a polynucleotide sequence can be initiated. A primer may exist naturally (for example, in an enzymatic digestion of a polynucleotide), or may be obtained through chemical synthesis. The product amplified in PCR is often referred to as an amplicon.
Nested PCR is a second PCR which is performed on the product of an earlier PCR using a second set of primers which are internal to the first set of primers, referred to as nested primers. This significantly improves the sensitivity and specificity of the PCR. Nested primers are primers internal to an amplicon obtained with a first PCR cycle. The amplification process that uses at least one nested primer improves specificity, because the non-specific products of the first cycle are not amplified in the second cycle, because they lack the sequence that corresponds to the nested primer. Semi-nested PCR is a second PCR which uses one new primer and one of the original primers. This process also improves specificity.
Ligase Chain Reaction (LCR) is a method of DNA amplification similar to PCR. LCR differs from PCR because it amplifies the probe molecule rather than producing an amplicon through polymerization of nucleotides. Two probes are used per each DNA strand and are ligated together to form a single probe. LCR uses both a DNA polymerase enzyme and a DNA ligase enzyme to drive the reaction. Like PCR, LCR requires a thermal cycler to drive the reaction and each cycle results in a doubling of the target nucleic acid molecule. LCR can have greater specificity than PCR.
In Single-Strand Conformation Polymorphism (SSCP) analysis a small PCR product (amplicon) is denatured and electrophoresed through a non-denaturing polyacrylamide gel. Thus, as the PCR product moves into and through the gel (and away from the denaturant), it will regain secondary structure that is sequence dependent (similar to RNA secondary structure). The mobility of the single-stranded PCR products will depend upon their secondary structure. Therefore, PCR products that contain substitutional sequence differences as well as insertions and deletions will have different mobilities.
Strand displacement amplification (SDA) is an isothermal nucleic acid amplification method based on the primer-directed nicking activity of a restriction enzyme and the strand displacement activity of an exonuclease-deficient polymerase.
The terms purification or isolation refers to a process for removing contaminants from a sample, where the result is a sample containing 50%, 60%, 75%, 90% or over 90% of the material toward which the purification procedure is directed.
For stringent temperature conditions in the case of nucleic-acid hybridization, these terms usually refer to a variable temperature between a maximum, for a nucleic acid, represented by Tm less 5° C., and a minimum represented by Tm less 25° C. The technique used in the field utilizes stringent temperature conditions, in combination with other parameters (e.g., saline concentration), to distinguish sequences with a quasi-exact homology.
Stringent conditions are known to those skilled in the art and can be found in Ausubel, et al., (eds.), C URRENT P ROTOCOLS IN M OLECULAR B IOLOGY , John Wiley & Sons, N.Y. (1989), 6.3.1-6.3.6. Preferably, the conditions are such that sequences at least about 65%, 70%, 75%, 85%, 90%, 95%, 98%, or 99% homologous to each other typically remain hybridized to each other. A non-limiting example of stringent hybridization conditions are hybridization in a high salt buffer comprising 6×SSC, 50 mM Tris-HCl (pH 7.5), 1 mM EDTA, 0.02% PVP, 0.02% Ficoll, 0.02% BSA, and 500 mg/ml denatured salmon sperm DNA at 65° C., followed by one or more washes in 0.2×SSC, 0.01% BSA at 50° C.
In a second embodiment, a nucleic acid sequence that is hybridizable to the nucleic acid molecule comprising the nucleotide sequence, or fragments, analogs or derivatives thereof, under conditions of moderate stringency is provided. A non-limiting example of moderate stringency hybridization conditions are hybridization in 6×SSC, 5×Reinhardt's solution, 0.5% SDS and 100 mg/ml denatured salmon sperm DNA at 55° C., followed by one or more washes in 1×SSC, 0.1% SDS at 37° C. Other conditions of moderate stringency that may be used are well-known within the art. See, e.g., Ausubel, et al. (eds.), 1993, C URRENT P ROTOCOLS IN M OLECULAR B IOLOGY , John Wiley & Sons, NY, and Krieger, 1990; G ENE T RANSFER AND E XPRESSION , A L ABORATORY M ANUAL , Stockton Press, NY.
In a third embodiment, a nucleic acid that is hybridizable to the nucleic acid molecule, or fragments, analogs or derivatives thereof, under conditions of low stringency, is provided. A non-limiting example of low stringency hybridization conditions are hybridization in 35% formamide, 5×SSC, 50 mM Tris-HCl (pH 7.5), 5 mM EDTA, 0.02% PVP, 0.02% Ficoll, 0.2% BSA, 100 mg/ml denatured salmon sperm DNA, 10% (wt/vol) dextran sulfate at 40° C., followed by one or more washes in 2×SSC, 25 mM Tris-HCl (pH 7.4), 5 mM EDTA, and 0.1% SDS at 50° C. Other conditions of low stringency that may be used are well known in the art (e.g., as employed for cross-species hybridizations). See, e.g., Ausubel, et al. (eds.), 1993, C URRENT P ROTOCOLS IN M OLECULAR B IOLOGY , John Wiley & Sons, NY, and Kriegler, 1990, G ENE T RANSFER AND E XPRESSION , A L ABORATORY M ANUAL , Stockton Press, NY; Shilo and Weinberg, 1981 . Proc Natl Acad Sci USA 78: 6789-6792.
The invention also provides a kit for detecting and/or genotyping Helicobacter pylori in a urine sample from a subject in need thereof, comprising at least one forward primer selected from SEQ ID NOs: 5, 8, 10, 11, 12 and 15 and at least one reverse primer selected from SEQ ID NOs: 6, 7, 9, 13, 14, 16 and 17, either in the same or separate packaging, and instructions for its use.
The invention also provides a kit for detecting and/or genotyping Mycobacterium tuberculosis in a urine sample from a subject in need thereof; comprising at least one forward primer selected from SEQ ID NOs: 34, 37 and 39 and at least one reverse primer selected from SEQ ID NOs: 35, 36, 38 and 40, either in the same or separate packaging, and instructions for its use.
The invention also provides a kit for detecting and/or genotyping Bacillus anthracis in a urine sample from a subject in need thereof; comprising at least one forward primer selected from SEQ ID NOs: 41, 43, 45, 47, 48 and 50 and at least one reverse primer selected from SEQ ID NOs: 42, 44, 46 and 49, either in the same or separate packaging, and instructions for its use.
The invention also provides a kit for detecting and/or genotyping Plasmodium species in a urine sample from a subject in need thereof, comprising at least one forward primer selected from SEQ ID NOs: 18, 21 and 23 and at least one reverse primer selected from SEQ ID NOs: 19, 20 and 22, either in the same or separate packaging, and instructions for its use.
The invention also provides a kit for detecting and/or genotyping Leishmania species in a urine sample from a subject in need thereof, comprising at least one forward primer selected from SEQ ID NOs: 26, 28, 30, 32 and 51 and at least one reverse primer selected from SEQ ID NOs: 24, 25, 27, 29, 31 and 33, either in the same or separate packaging, and instructions for its use.
The invention will be further described in the following examples, which do not limit the scope of the invention described in the claims.
EXAMPLES
All of the methodology described herein may be modified by the technician in the field with no change in the basic principal idea.
Example 1
Stabilization and Preparation of the Samples
All of the steps of the preparation of the urine samples and of the analysis of the transrenal DNA were performed at room temperature. Briefly, approximately 50-60 ml of urine samples were collected from each patient participating in the study. Within 30 minutes after collection, a solution consisting of 0.5M EDTA and 0.5M Tris-HCl, at a pH ranging from 8.0 to 8.5, was added at a final concentration of 10 mM in order to inhibit the nucleases that might be present in urine samples. At least three nucleolytic enzymes were identified in urine, DNase I, DNase II and phosphodiesterase. EDTA inhibits the activity of DNase I and phosphodiesterase by chelating divalent metals such as Mg 2+ , Ca 2+ and/or others that are required for their activity. DNase II is maximally active in acidic environment. Supplementing the urine with Tris-HCl pH 8.0 buffer increases the pH of urine thereby inhibiting the activity of DNase II.
The stabilized urine samples can be stored, in aliquots of 5 ml, at −80° C. Optionally, 25 mL of saline solution is frozen to act as a control.
Other methods for the preparation of urine samples and for the extraction of the DNA are described in PCT patent No. WO 98/54364, which is incorporated by reference in its entirety.
Example 2
Urine Fractionation
For Tr-DNA based applications, that involve quantitative Tr-DNA analysis, urine fractionation is often required. It reduces the impact of nucleoproteins from cells present in urine on accurate quantitation of Tr-DNA/protein complex, total Tr-DNA, or specific genetic markers. In our experiments we have employed two procedures for urine fractionation, centrifugation and filtration. Both were carried out immediately after urine collection and, before supplementing the specimen with EDTA-Tris stabilizing mixture. Urine may be kept in this condition as long as urine cells are intact and their constituents do not leak into urine and contaminate Tr-DNA by cellular DNA.
For filtration one can use filters with pore size ranging from 0.1 to 5 μm with reduced nucleic acid and protein binding capacity. The choice of the filter depends on target under analysis. For bacterial pathogens smaller pores are required. In our experiments we used luer-lock 0.45 μm Filter Units (Cat. No. SLHV033RS) or 0.45 μm 150 ml Filter Unit STERICAP (Cat. No. SCHVU01RE) both from Millipore. After filtration the samples were supplemented with EDTA-Tris conservation solution and taken into downstream processes of DNA extraction or aliquoted and stored frozen at −80° C. Urine cellular fraction can be harvested by extracting it from the filter by applying a solution (high concentrations of salts or chaotropic agents) that is known to dissolve cells.
Urine fractionation by centrifugation in our experiments was carried immediately after sample collection, before addition of preservation mixture. Centrifugation was performed at room temperature at RCF ranging between 3500 and 4000×g for approximately 15 min. Supernatant was carefully collected and manipulated per the procedure described above for urine filtration. The pellet consisting mainly of cells and cellular debris was resuspended in physiological solution and stored at −80° C.
Example 3
Extraction and Purification of the Nucleic Acids from Urine
In our experiment we used silica based DNA extraction protocol in two different formats. One was vacuum driven and the second, centrifugation.
Vacuum Based Procedure
To the urine sample 6 M Guanidine Thiocyanate (GITC) (AMRESCO, Cat. No. 0380) was added to final concentration not less than 3 M. This solution was mixed vigorously and supplemented with 0.25 ml of Wizard silica suspension (PROMEGA, Cat. No. A7141) per 10 ml of initial urine volume. To capture DNA the mixture was continuously rotated for 1 hour at room temperature. Resin with captured DNA was collected by loading the mixture on a minicolumn (PROMEGA, Cat. No. A7211) syringe assembly attached to a vacuum line. Resin was collected by applying vacuum, washed twice with 5 ml of 3M GITC solution. The resin was further washed twice by a solution consisting of 80% ethanol and 50 mM NaCl. Then the minicolumn was detached and the resin was additionally washed twice with 96% ethanol by centrifugation on a benchtop microcentrifuge in 1.5 ml tubes. DNA was eluted by brief centrifugation with hot water in a volume of not more than 1/20 of urine initial volume.
Centrifugation Based Procedure
To the urine sample 6 M Guanidine Thiocyanate (GITC) (AMRESCO, Cat. No. 0380) was added to final concentration not less than 3 M. This solution was mixed vigorously and supplemented with 0.25 ml of Wizard silica suspension (PROMEGA, Cat. No. A7141) per 10 ml of initial urine volume. To capture the DNA, the mixture was continuously rotated for 1 hour at room temperature. Resin with captured DNA was collected by centrifugation at approximately 4000×g for about 5 min at room temperature. Pelleted resin was washed once with GITC solution and once with wash buffer, then was transferred into a mini-column and washed one additional time with 96% ethanol. DNA was eluted with hot water into a microcentrifuge tube by brief centrifugation. DNA was eluted by brief centrifugation with hot water in a volume of not more than 1/20 of urine initial volume.
Example 4
Monitoring of the Integrity of DNA Extraction Procedure
The DNA extraction procedure was routinely monitored by testing the purified DNA for the presence of selected control DNA sequences. For the purposes of detection of Tr-DNA of infectious agents the integrity of DNA purification procedure was monitored using beta-actin and beta-globin as markers for human and rabbit urine, respectively. PCR amplification of above targets is a surrogate for the estimation of DNA yield and quality. Primer pairs used in these studies are presented in Table 1.










TABLE 1





Pr. Length

Amp. Size
SEQ ID

ID
Sequence
(nt)
Tm
(bp)
NO:



ES0007
AAGACAGTGTTGTGGGTGTAGG
22
59.0
77
1



ES0008
TCCAAGGCCGCTTTACAC
18
59.8

2



LEBG_F
GGCACAGGTTTCATCCATTC
20
54.1
70
3



LEBG_R
GATAGGTCTCTCTTTATGTCTTCAG
25
54.4

4
























PCR conditions for both targets were identical. Amplification of isolated DNA equivalent to 300 μl of urine was carried out in 25 μl mixture containing 0.2 μmol/liter of primers ES0007 and ES0008 or LEBG_F and LEBG_R for human b-actin or rabbit b-globin, respectively, 200 μmol/liter dNTP each, and 1 U GoTaq Polymerase (Promega). Thermal profile for both types of reactions was: 94° C. for 5 minutes followed by 35 cycles of 94° C. for 30 seconds, 60° C. for 30 seconds, 72° C. for 1 minute, and 1 cycle of 72° C. for 5 minutes. Ten μl the products were resolved by electrophoresis in 7% polyacrylamide gel, and visualized by ethidium bromide staining. FIG. 1 and FIG. 2 depict the results of PCR of DNA purified from human urine and rabbit urine, respectively.
FIG. 1 shows the results of PCR performed to control the integrity of DNA extraction from human urine. Urine samples are from patients attending doctor's office for endogastroscopy for a test for Helicobacter pylori . M—DNA molecular weight standards, 50 by ladder; NTC—no template control; 1-13 urine from patients.
FIG. 2 . shows the results of PCR performed to control the integrity of DNA extraction from rabbit urine. Urine samples were harvested from rabbits infected with Bacillus anthracis spores. M—DNA molecular weight standards, 50 by ladder; NTC—no template control; 1—DNA from urine of uninfected rabbit; 2 and 3—urine of infected rabbit collected 6 an 14 hours after infection, respectively; +c—rabbit genomics DNA
Example 5
Design of PCR Primers
As a general rule, primers for the PCR based analysis of Tr-DNA were designed for target amplicons not more than 200 by in length.
All primers were screened against the complete sequence of human genome.
For primer design FastPCR software was used (http://www.biocenter.helsinki.fi/bi/bare-1_html/oligos.htm). The critical parameters for primer design were as follows:
Allow 1 mismatch in 5 nucleotides at 3′ end.
At least 2 G or C residues at 3′ end.
Only one A or T residue at 3′ end.
Maximum Tm of 23° C. for 6 nucleotides at 3′ end.
Nested primers were designed using Primer 3 package [see Steve Rozen and Helen J. Skaletsky (2000) Primer3 on the WWW for general users and for biologist programmers. In: Krawetz S, Misener S (eds) Bioinformatics Methods and Protocols: Methods in Molecular Biology. Humana Press, Totowa, N.J., pp 365-386] and is available online (http://frodo.wi.mit.edu/cgi-bin/primer3/primer3_www.cgi). The only parameter specified for the nested primers was the Tm in order to have primers with annealing temperature not less than external primers.
Example 6
Selection of PCR Primer Sets for the Detection of H. pylori Gene Sequences for PCR
PCR primers were designed based on consensus DNA sequences created by alignment of corresponding genetic markers nucleotide sequences. DNA sequences of target genes were extracted from GeneBank at NCBI and aligned using BioEdit package (IBIS Therapeutics, Carlsbad, Calif.). Primers are listed in Table 2. For the purposes of present invention two sets of genetic markers were selected: ureA as specie specific marker and cagA as a marker of virulence.
TABLE 2 Nested SEQ ID ID Primer Sequence Amplicon Amplicon NO: cagA-f TCRGAAATTTGGGRMTCAGCGTTAC 102 bp 65 bp 5 cagA-r TCCATAAAATTTYGGATBBDTYGGGTGTTG 6 cagA-rn ACGGATCDTTTTGAWGGGACAC 7 vacSl-f AGCDYCAYACCRCAAVAAAGTCATGCYGC 104 bp 71 bp 8 vacS-r CTGADACCGTTCCTACAGC 9 vacS-fn YTTYACRACCGTGATYATTCCAGC 10 vacS2-f CKCCAAAYGATCCCATACACAGCGAGAG 109 bp 71 bp 11 ureA-f TGATGGGACYAAACTCGTAACCG 86 bp 50 bp 12 ureA-r CTTCATTTTTTAAGAACAACTCACCAGGA 13 ureA-nr ACCATTRGCCTCAATAGGGGTATGC 14 HG16sf CACTGGGCGTAAAGAGYGCGTAG 113 bp 63 bp 15 HG16sr CCACCTRCCTCTCCCAYACTC 16 HG16snr GGTTAAGCCATAGGATTTCACAYCTGAC 17
Set 1. cagA Gene primers for Semi-Nested-PCR








External primers:


cagA-f (SEQ ID NO: 5):


TCRGAAATTTGGGRMTCAGCGTTAC





cagA-r (SEQ ID NO: 6):


TCCATAAAATTTYGGATBBDTYGGGTGTTG


Product: 102 bp.





Internal primers:


cagA-f (SEQ ID NO: 5):


TCRGAAATTTGGGRMTCAGCGTTAC





cagA-rn (SEQ ID NO: 7):


ACGGATCDTTTTGAWGGGACAC


Product: 65 bp.






















VacA Region Primers for Semi-Nested-PCR,
Set 2. S1 Specific








External primers


vacS1-f (SEQ ID NO: 8)


AGCDYCAYACCRCAAVAAAGTCATGCYGC





vacS-r (SEQ ID NO: 9)


CTGADACCGTTCCTACAGC


Product 104 bp





Internal primers


vacS-fn (SEQ ID NO: 10)


YTTYACRACCGTGATYATTCCAGC





vacS-r (SEQ ID NO: 9)


CTGADACCGTTCCTACAGC


Product 71 bp






















Set 3. S2 Specific

External Primers vacS2-f (SEQ ID NO: 11) CKCCAAAYGATCCCATACACAGCGAGAG vacS-r (SEQ ID NO: 9) CTGADACCGTTCCTACAGC Product 109 bp Internal Primers vacS-fn (SEQ ID NO: 10) YTTYACRACCGTGATYATTCCAGC vacS-r (SEQ ID NO: 9) CTGADACCGTTCCTACAGC Product 71 bp
Set 4. H. pylori Specific








External Primers


ureA-f (SEQ ID NO: 12)


TGATGGGACYAAACTCGTAACCG





ureA-r (SEQ ID NO: 13)


CTTCATTTTTTAAGAACAACTCACCAGGA


Product 86 bp





Internal Primers


ureA-f (SEQ ID NO: 12)


TGATGGGACYAAACTCGTAACCG





ureA-nr (SEQ ID NO: 14)


ACCATTRGCCTCAATAGGGGTATGC


Product 50 bp






















Set 5. Helicobacter Genus-Specific








External Primers


HG16s-f (SEQ ID NO: 15)


CACTGGGCGTAAAGAGYGCGTAG





HG16s-r (SEQ ID NO: 16)


CCACCTRCCTCTCCCAYACTC


Product 113 bp





Internal Primers (i)


HG16s-f (SEQ ID NO: 15)


CACTGGGCGTAAAGAGYGCGTAG





HG16s-nr (SEQ ID NO: 17)


GGTTAAGCCATAGGATTTCACAYCTGAC


Product 63 bp






















Example 7
Detection of H. pylori in Urine of Patients
Study participants were randomly selected from the patients of the S. Orsola-Malpighi Hospital visiting the Department of Internal Medicine (Bologna, Italy) for routine upper gastrointestinal endoscopy. Infection of these patients was diagnosed by two assay: fast urease test and histological analysis of the biopsy for the presence of bacteria.
Urine specimens were obtained before the endoscopy, and the DNA from whole urine was purified following the protocol described in Examples 1, 2 and 3.
Detection of ureA and cagA specific sequences in urine was carried out by semi-nested PCR. Initial twenty cycles of PCR amplification were performed as following: isolated DNA equivalent to that contained in 5 ml of urine was added to a 25 μl mixture containing 0.2 μmol/liter of outer primers specific for ureA or cagA (SEQ ID NO 12 and 13, or SEQ ID NO 5 and 6, respectively), 5 μl 5×PCR buffer (Promega), 200 μmol/liter dNTP each, and 1 U GoTaq Polymerase (Promega), denatured at 94° C. for 5 minutes followed by 20 cycles of 94° C. for 30 seconds, 62° C. for 30 seconds, 72° C. for 1 minute, and 1 cycle of 72° C. for 5 minutes. One μl of the product from this amplification was diluted 1:10 and 1 μl of the dilution was re-amplified 35 cycles using internal ureA and cagA primers (SEQ ID NO 12 and 13, or SEQ ID NO 5 and 6, respectively), under the same conditions as in the first reaction. The products from the second amplification were resolved by electrophoresis in 7% polyacrylamide gel, and visualized by ethidium bromide staining.
FIG. 3 shows semi-nested PCR detection of ureA (A) and cagA (B) genes specific DNA sequences in urine of patients (lanes 1-13). NTC—no template control; −c—DNA from the urine of a healthy volunteer; +c—positive control.
Example 8
Selection of PCR Primer Sets for the Detection of Plasmodium Sp.
Primers were designed to detect 18S rRNA genes of all four human pathogenic subtypes Plasmodium sp: P. falciparum; P. vivax; P. malariae , and P. ovale (accession numbers: M19172; X13926; M54897 and L48987, respectively).
PCR primers were designed based on consensus DNA sequences created by alignment of corresponding genes' nucleotide sequences. Nucleotide sequences were aligned using BioEdit package (IBIS Therapeutics, Carlsbad, Calif.). Primers are presented in the Table 3.





TABLE 3



PCR primer pairs for the detection of Plasmodium sp .









Seq

Seq



Pair
Name

Name

Product

No.
<ID>
Forward sequence
<ID>
Reverse sequence
(bp)



1
F-358
gggtattggcctaacatggctatgac
R-405
aggctccctctccggaatcg
67 bp


<18>

<19>





2
F-358
gggtattggcctaacatggctatgac
R-456
agaattgggtaatttacgcgcct
123 bp


<18>

<20>





3
F-1458
cttgatttcttggatggtgatgc
R-1526
ggttaagatctcgttcgttatcgg
92 bp


<21>

<22>





4
F-1385
cggcttaatttgactcaacacg
R-1526
ggttaagatctcgttcgttatcgg
164 bp


<23>

<22>

































Example 9
Selection of PCR Primer Sets for the Detection of Leishmania Sp.
For the detection of pathogenic Leishmania strains that cause visceral leishmaniasis two sets of primers were designed: first, a set specific for kinetoplast minicircle DNA conserved sequences common for the donovani complex, and a second set specific for 18S rRNA gene common for all Leishmania sp.





TABLE 4



PCR primer pairs designed for the detection of Leishmania sp .









Seq

Seq



Pair
Name

Name

Product

No.
<ID>
Forward sequence
<ID>
Reverse sequence
(bp)










1
F-81
rggtaggggcgttctgc
R-160
agtttyccgccccggag
101 bp


<51>

<24>





2
F-81
rggtaggggcgttctgc
R-156
cccagtttbccgccycgga
97 bp


<23>

<25>





3
F-340
ctattggagattatggagctgtgc
R-382
cacacaccgaaccgargttgc
64 bp


<26>

<27>





4
F-505
gggagaacgtactggggcgtc
R-547
tcgctgtagttcgtcttggtg
64 bp


<28>

<29>





5
F-37
ctgttgctgttaaagggttcgtag
R-91
tccgacatggacgggacgacc
76 bp


<30>

<31>





6
13A*
gtgggggaggggcgttct
13B*
attttacaccaacccccagtt
120 bp


<32>

<33>



*BELLI A et al. 1998, SIMPLIFIED POLYMERASE CHAIN REACTION DETECTION OF NEW WORLD LEISHMANIA IN CLINICAL SPECIMENS OF CUTANEOUS LEISHMANIASIS, Am. J. Trop. Med. Hyg., 58(1), pp. 102-109









































Example 10
Detection of Plasmodium species
Urine sample was taken from a malaria patient infected with Plasmodium falciparum . DNA isolated from the whole urine of non-infected patients or from blood of malaria patients was used as negative and positive controls, respectively. For the detection of Plasmodium sp specific DNA sequences in urine of a patient semi-nested PCR was performed using two sets of primers targeting two conserved regions of S18 ribosomal RNA gene.
FIG. 4 illustrates the electrophoresis of the DNA fragments that were amplified via two amplification cycles (semi-nested PCR). Two sets of reactions were carried out:
1. Primer pairs F-358/R-456 for the first PCR and F-358/R-405 for subsequent semi-nested reaction, lanes 1; 2 and 3 are negative control; Tr-DNA from infected patient and DNA extracted from the whole blood of an infected patient, respectively. 2. Primer pairs F-1385/R-1526 for the first PCR and F-1458/R-1526 for subsequent semi-nested reaction, lanes 4; 5 and 6 are negative control; Tr-DNA from infected patient and DNA extracted from the whole blood of an infected patient, respectively.
Lane M—molecular weight standards. For primer pairs and PCR product sizes see Table 3.


Example 11
Detection of Leishmaniasis Species
Urine sample was taken from a leishmaniasis patient infected with Leishmania infantum . DNA isolated from the whole urine of non-infected patients or from blood of leishmaniasis patients was used as negative and positive controls, respectively. For the detection of Leishmania sp specific DNA sequences in urine of a patient PCR amplification reaction was carried out using a set of primers specific for kinetoplast minicircle DNA conserved domain (primers 13A/13B).
FIG. 5 illustrates the electrophoresis of the DNA fragments that were amplified using primers specific for pathogenic Leishmania sp
Lane M—DNA molecular weight standards; Lane 2—Tr-DNA from non-infected subject; Lane 3—Tr-DNA from an infected patient Lanes 4 and 5—DNA extracted from blood of infected patients was used as a positive control.

The size of the PCR product is 120 by (see Table 4).
Example 12
Selection of PCR Primer Sets for the Detection of Mycobacterium tuberculosis
PCR primers for the detection of M. tuberculosis complex species DNA were selected for the region of IS6110 shown to be highly specific (Hellyer). Primers used in this study are presented in Table 5.







TABLE 5





Product size,

ID
Sequence
bp









F-785
ACCAGCACCTAACCGGCTGTGG (SEQ ID NO: 34)
129



R-913
CATCGTGGAAGCGACCCGCCAG (SEQ ID NO: 35)




Rn-851
GTAGGCGAACCCTGCCCAGGTC (SEQ ID NO: 36)
67



F-489
GCCCCATCGACCTACTACG (SEQ ID NO: 37)
330



R-819
TGAGGTCTGCTACCCACAGC (SEQ ID NO: 38)




Fn-627
CCCTGAACCGTGAGGGCATCG (SEQ ID NO: 39)
69



Rn-690
ACAGGCCGAGTTTGGTCATCAGC (SEQ ID NO: 40)


































Example 13
Detection of Mycobacterium tuberculosis Bacteria DNA Sequences in Urine
All study participants were enrolled at the National Institute for Infectious Diseases (INMI) “L. Spallanzani” in Rome, Italy. In this study we included patients who had been clinically diagnosed with pulmonary tuberculosis as confirmed by isolation of M. tuberculosis from sputum culture. Nineteen of the 20 patients had a sputum smear positive for acid-fast bacilli, whereas, only one was HIV positive. No patient exhibited clinical evidence of extra-pulmonary involvement, and all urine cultures for M. tuberculosis performed were negative. Urine specimens were obtained either before or within one week of initiation of anti-tuberculosis therapy. Information regarding age, sex, ethnicity, M. bovis BCG vaccination, and results of laboratory examinations for tuberculosis were collected. In order to determine whether subsequent therapy had an influence on the ability to detect M. tuberculosis DNA, 8 of the patients with pulmonary tuberculosis were asked to return to donate urine specimens two months after initiating anti-tuberculosis therapy. In addition, ten healthy individuals were included as controls in the study.
Urine specimens were processed as described in Examples 1-3 above.
Twenty cycles of PCR amplification were performed as following: isolated DNA equivalent to that contained in 300 μl of urine was added to a 25 μl mixture containing 0.2 μmol/liter of outer primers F-785 and R-913, 5 μl 5×PCR buffer (Promega), 200 μmol/liter dNTP each, and 1 U GoTaq Polymerase (Promega), denatured at 94° C. for 5 minutes followed by 20 cycles of 94° C. for 30 seconds, 62° C. for 30 seconds, 72° C. for 1 minute, and 1 cycle of 72° C. for 5 minutes. 1 μl of the product from this amplification was diluted 1:10 and 1 μl of the dilution was re-amplified 35 cycles using primers F-785 and Rn-851 under the same conditions as in the first reaction. Identical setting was used for the amplification of large, 330 by fragment. The products from the second amplification were resolved by electrophoresis in 7% polyacrylamide gel, and visualized by ethidium bromide staining.
Results from semi-nested amplification are shown in the right panel of FIG. 6 . The M. tuberculosis -specific product of 67 by was found in urine specimens from all 20 patients analyzed. Eight healthy individuals used as controls were all found to be negative for the presence of M. tuberculosis -specific sequences. The last lane in the gels is the genomic DNA of Mycobacterium tuberculosis strain H37RV, as a positive control.
Example 14
Demonstration that TB Specific DNA Sequences Found in Urine are Cell Free
Previous reports on detection of M. tuberculosis DNA in urine employed isolation methods designed to extract high molecular weight DNA from whole urine or urinary sediments. Furthermore, in these reports larger amplicon sizes were used (>200 bp) (Torrea G. J., et al. Med Microbiol. 2005; 54:39-44; Piersimoni C. J., et al. Clin. Microbiol. 40:4138-4142 (2002) Kafwabulula M., et al. Int J Tuberc Lung Dis. 2002; 6:732-7; Marei A. M., et al. J Med Microbiol. 2003 April; 52(Pt 4):331-5. Del Portillo P. et al. J Clin Microbiol. 1991 October; 29(10):2163-8). Therefore, it was important to further characterize the nature of the M. tuberculosis Tr-DNA found in urine. In order to address this question, two experimental designs were employed. In the first, specimens were fractionated by centrifuged and DNA was isolated separately from urine sediment and supernatant (see Examples 2 and 3).
Primer pairs F-785/R-913 and semi-nested primers F-785/Rn-851 specific for short amplicon were used (see Table 5). As shown in FIG. 7 , 6 of 8 of the supernatant samples display the presence of M. tuberculosis DNA, whereas only 2 of 8 of the pellet samples were positive.
The second experiment was aimed on comparison of PCR products obtained with primers designed for larger amplicon size, in the range used by several other investigators. Results shown in FIG. 8 demonstrate that MTB DNA sequences again were detected in all 7 samples of DNA that had been isolated from urine of pulmonary TB patients when using primers for 129/67 amplicons. However, no PCR products were detected with primers for 330/69 amplicons. In Panel A semi-nested PCR for the short amplicon (target 129 bp). In Panel B semi-nested PCR for the long amplicon with primers F489/R819 (target 330 bp). Respective final semi-nested PCR products for short and long amplicons are 67 by and 69 bp. Lanes 3-9 represent products of PCR amplification of DNA purified from patients urine. Lanes 1 in both gels show the DNA standards, Lanes 2 in both gels are negative controls, and Lanes 10 in both gels are positive genomic DNA controls. These data confirmed that bacterial DNA fragments extracted from urine are relatively short fragment. Furthermore, these results strongly suggest that it is unlikely that MTB DNA can be reproducibly detected in urine specimens of pulmonary TB patients when using urine sediment PCR analysis combined with large amplicon sizes.
Example 15
Analysis of TB Patients Before and After Treatment
One of the critical problems faced in the management and eradication of pulmonary tuberculosis worldwide is the accurate and early detection of the efficacy of therapeutic treatment of patients. Currently general practice of monitoring of the success of a treatment is based on clinical symptoms, which are relatively late indicators and therefore do not provide information for a “real time” adjustment of the regiment of therapy.
In this regard the impact of Tr-DNA platform technology is twofold. First, it gives a safe, easy and inexpensive means for detection of mutations known to be associated with increased drug resistance, and second, it ideally fits to the early monitoring tasks owing to its inherent capabilities of detection and quantification of non-host origin genetic markers in urine.
In order to determine whether MTB-specific DNA sequences in urine can be used for evaluation of therapeutic treatment, 8 of the patients with TB who had been found to be MTB Tr-DNA positive at enrolment were asked to return to donate urine samples approximately two months following initiation of chemotherapy. At the time of the second test clinical symptoms have resolved in these patients and all were sputum smear negative. Tr-DNA extracted from these samples was analyzed and no product corresponding to 67 by specific TB sequence was observed, as shown in FIG. 9 .
Example 16
Selection of PCR Primer Sets for the Detection of Bacillus anthracis and Diagnosis of Anthrax
A virulent strain of B. anthracis , A0843, was used for spore production. This strain, isolated from an outbreak of anthrax, which occurred in Italy, was microbiologically characterized by Turnbull et al. (Bacillus, p. 187-210. In M. T. Parker and L. H. Collier (ed.), Topley and Wilson's principles of bacteriology, virology and immunity, VII ed. Edward Arnold, London, United Kingdom 1990), and the presence of plasmids pXO1 and pXO2 has been confirmed by PCR (Fasanella A., et al. (2001) Vaccine 19: 4214-4218; Green B. D., et al. (1985) Infect. Immun. 49:291-297; Ramisse V., et al. (1996). FEMS Microbiol. Lett. 145: 9-16). Genotyping analysis performed by multiple-locus variable-number tandem repeat analysis assigned this strain to the cluster Ala, genotype 3 (Keim, P., et al. (2000). J. Bacteriol. 182: 2928-2936.).
B. anthracis infection in rabbits was used as a model system for validation of applicability of Tr-DNA technology for detection of bacterial pathogens and for developing a Tr-DNA assay to diagnose Anthrax infection in human patients. Rabbit represents a more advanced animal model with a disease progression similar to human.
Rabbits were randomly clustered in several groups of 4 animals. Each rabbit was infected by subcutaneous injection of 100,000 spores of the pathogen strain A0843. Animals from each group were sacrificed post-infection at time intervals 6 and 14 hr. After euthanasia urine was harvested from the bladder by syringe aspiration. The volume of collected urine specimens varied from 2 to 10 ml. Four uninfected rabbits were used as controls.
DNA from the rabbit urine specimens were extracted according to the established for the human urine protocol summarized in Examples 1-3 above. Tr-DNA isolated from the urine of 4 non-infected rabbits were used as negative controls.
As targets we have selected pXO1 derived toxicity genetic markers. PCR primers were designed using Beacon Designer software (Premier Biosoft, Palo Alto, Calif.).










TABLE 6



SEQ






ID


Length

Amplicon

NO
ID
Sequence
(nt)
Tm
(bp)












41
LEF_F
GGTTATATGTTCCAGAATCC
20
47.7
125



42
LEF_R
ATATCCAGCATAATCATCCA
20
48




43
LEF_FN
AAGGTGTAGAATTAAGGAATGATAGTGAG
29
57.8
74



44
LEF_RN
TCCAGCATAATCATCCACAGCAT
23
57.8




45
CYA_F
AACAACGAAGTACAATACAAGAC
23
53
122



46
CYA_R
GCTGTTAACGGCTTCAAGA
19
52.6




47
CYA_FSN
AATTGGAGAAATATAGAAGTGATG
24
50.5
62



48
PAG_F
AAGTACAAGTGCTGGACCTAC
21
54.5
83



49
PAG_R
CAACCGTATATCCTTCTACCTCTAA
25
55.3




50
PAG_FSN
TTCCAGACCGTGACAATGATG
21
55.5
60









































Detection of pag specific sequences in urine was carried out by semi-nested PCR using the primer set specific for the pag loci (see Table 6). PCR amplification and electrophoresis analysis was performed per the protocol used in previous Examples. Briefly, twenty cycles of PCR amplification were performed in a 25 μl mixture with primers PAG_F and PAG_R. One μl of the product from this amplification was diluted 1:10 and 1 μl of the dilution was re-amplified 35 cycles using primers PAG_R and PAG_FSN under the same conditions as in the first reaction. The products from the second amplification were resolved by electrophoresis in 7% polyacrylamide gel, and visualized by ethidium bromide staining.
FIG. 10 shows the detection of B. anthracis A0843 specific DNA sequences in the urine of experimentally infected rabbits. Lanes: M—50 by ladder DNA molecular weight markers; 1—no template control; 2—urine from uninfected rabbit; 3 and 4—urine collected from infected rabbits 6 and 14 hr post-infection, respectively; 5— B. anthracis A0843 genomic DNA, positive control.