Espacenet - Bibliographic data
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`https://worldwide.espacenet.com/publicationDetails/biblio?CC
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`6/28/2016 12:53 AM
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`(12) INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCT)
`
`(19) World Intellectual Property Organization
`International Bureau
`
`(43) International Publication Date
`24 August 2006 (24.08.2006)
`
`
`
`(51) International Patent Classification:
`
`Not classified
`
`(21) International Application Number:
`PCT/US2006/005225
`
`(22) International Filing Date:
`14 February 2006 (14.02.2006)
`
`(25) Filing Language:
`
`(26) Publication Language:
`
`(30) Priority Data:
`RM2005 A000067
`11/137,934
`60/69 1,186
`11/351,799
`
`English
`
`English
`
`(74)
`
`(81)
`
`17 February 2005 (17.02.2005)
`25 May 2005 (25.05.2005)
`16 June 2005 (16.06.2005)
`10 February 2006 (10.02.2006)
`
`IT
`US
`US
`US
`
`(63) Related by continuation (CON) or continuation-in-part
`(CIP) to earlier applications:
`US
`Filed on
`US
`Filed on
`
`Not furnished (CON)
`Not furnished
`RM2005A000068 (CIP)
`17 February 2005 (17.02.2005)
`
`(71) Applicant (for all designated States except US): ISTI-
`TUTO NAZIONALE PER LE MALATTIE INFET-
`
`TIVE IRCCS LAZZARO SPALLANZANI [IT/IT]; Via
`Portucnsc 292, I—00149 Roma (IT).
`
`(72) Inventors; and
`(75) Inventors/Applicants
`
`(for US only):
`
`lVLILKMAN,
`
`(84)
`
`(10) International Publication Number
`
`WO 2006/088895 A2
`
`Hooves [RU/US]; 546 Ewing Street, Princeton, New Jer—
`sey 08540 (US). CANNAS, Angela [IT/IT]; Via Genne’s
`Frongia 11.29, I—09031 Arbus (IT). TOMEI, Louis, David
`[US/IT]; Piazzale Clementi #5, I—00030 Genazzano, Rome
`(IT). UMANSKY, Samuil, R. [US/US]; 3 Orchid Center,
`Princeton, New Jersey 08540 (US).
`Agent: ELRIFI, Ivor, R.; MINTZ, LEVIN, COHN, FER—
`RIS, GLOVSKY AND POPEO PC, One Financial Center,
`Boston, Massachusetts 02111 (US).
`Designated States (unless otherwise indicated, for every
`kind of national protection available): AE, AG, AL, AM,
`AT, AU, AZ, BA, BB, BG, BR, BW, BY, BZ, CA, CH, CN,
`CO, CR, CU, CZ, DE, DK, DM, DZ, EC, EE, EG, ES, FI,
`GB, GD, GE, GH, GM, HR, HU, ID, IL, IN, IS, JP, KE,
`KG, KM, KN, KP, KR, KZ, LC, LK, LR, LS, LT, LU, LV,
`LY, MA, MD, MG, MK, MN, MW, MX, MZ, NA, NG, NI,
`NO, NZ, OM, PG, PH, PL, PT, RO, RU, SC, SD, SE, SG,
`SK. SL, SM, SY, TJ, TM, TN, TR, TT, TZ, UA, UG, US
`(patent), UZ, VC, VN, YU, ZA, ZM, ZW.
`Designated States (unless otherwise indicated, for every
`kind of regional protection available): ARIPO (BW, GH,
`GM, KE, LS, MW, NIZ, NA, SD, SL, SZ, TZ, UG, ZM,
`ZW), Eurasian (AM, AZ, BY, KG, KZ, MD, RU, '1], TM),
`European (AT, BE, BG, CII, CY, CZ, DE, DK, EE, ES, FI,
`FR, GB, GR, HU, IE, IS, IT, LT, LU, LV, MC, NL, PL, PT,
`RO, SE, SI, SK, TR), OAPI (BF, Bl, CF, CG, CI, CM, GA,
`GN, GQ, GW, ML, MR, NE, SN, TD, TG).
`
`[Continued on next page]
`
`(54) Title: COMPOSITIONS AND METI IODS FOR DETECTING PATI IOGEN SPECIFIC NUCLEIC ACIDS IN URINE
`
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`06/088895A2|||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
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`c (57) Abstract: The invention is based upon the discovery that small nucleic acids from non—Viral pathogens are able to cross the
`N 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
`
`W0 of the subject.
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`

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`WO 2006/088895 A2
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`|||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
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`Published:
`— without international search report and to be republished
`upon receipt of that report
`
`For two—letter codes and other abbreviations, refer to the ”Guid—
`ance Notes on Codes andAbbreviations” appearing at the begin—
`ning of each regular issue of the PCT Gazette.
`
`

`

`WO 2006/088895
`
`PCT/U52006/005225
`
`COMPOSITIONS AND METHODS FOR DETECTING PATHOGEN SPECIFIC
`
`NUCLEIC ACIDS IN URINE
`
`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): 1 078-84 (2000); Su et al. Ann. NY Acad. Sci. 1022:81-89 (2004)) For example, US.
`
`Patent No. 6,251,638 describes an analytical method for detecting male fetal DNA in the
`
`urine of pregnant women. US. Patent No. 6,2 87,820 describes a system aimed at the
`
`diagnosis of tumors, particularly of adenocarcinomas of the colon and pancreas. U.S.
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`Patent 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 :3 99-402; 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., er
`
`al. 2001, Ann NYAcad 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, AM. 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.
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`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
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`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
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`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—H01 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
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`filtration is performed with a filter with a pore size between about 0.1 um and about 5.0 am.
`
`In another embodiment ofthe method of isolating a nucleic acid from urine, the
`
`chaotropic salt is guanidine isothiocyanate. In one aspect ofthis 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.
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`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 anthracis, Plasmodz'um 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
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`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
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`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
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`which infected the subject. In one aspect of this embodiment, the Helicobacter pylori is
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`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 sl/ml, slmZ, 32ml and sZmZ.
`
`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 filycobacterium 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 N03: 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
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`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.
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`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
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`human.
`
`The invention also provides a method for diagnosing a Plasmodium species infection
`
`in a subject, comprising detecting the presence of a Plasmodz'um species nucleic acid in a
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`urine sample from the subject, thereby diagnosing the Plasmodium species infection. In one
`
`embodiment of the method for diagnosing a Plasmoalz'um species infection in a subject, the
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`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 N05: 18, 21 and 23 and the reverse
`
`primer is selected from SEQ ID N03: 19, 20 and 22.
`
`In another embodiment of the method for diagnosing a Plasmodz'um species infection
`
`in a subject, the Plasmoa’ium species nucleic acid comprises transrenal DNA.
`
`In another embodiment of the method for diagnosing a Plasmodz'um 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 Leishmam'a species infection in
`
`a subject, comprising detecting the presence of a Leishmam'a species nucleic acid in a urine
`
`sample from the subject, thereby diagnosing the Leishmanz'a 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.
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`Optionally, the forward primer is selected from SEQ ID N03: 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 Leishmam‘a species infection
`
`in a subject, the Leishmania species nucleic acid comprises transrenal DNA.
`
`In another embodiment of the method for diagnosing a Leislzmam‘a species infection
`
`in a subject, the subject is a mammal. In one aspect of this embodiment, the mammal is a
`human.
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`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 Plasmodz'um
`
`species, wherein the composition comprises a nucleic acid sequence selected from SEQ ID
`
`NOs: 18-23. In one embodiment of the cemposition 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.
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`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.
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`The invention also provides a composition for use in the detection of Bacillus
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`anthracis, wherein the composition comprises a nucleic acid sequence selected fiom SEQ ID
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`NOs: 41-50. In one embodiment of the composition for use in the detection of Bacillus
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`anthracis, the composition consists of a nucleic acid selected from SEQ ID NOS: 41-50.
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`The invention also provides a kit for detecting Helicobacter pylori in a urine sample
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`from a subject in need thereof, comprising at least one forward primer selected from SEQ ID
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`NOS: 5, 8, 10, 11, 12 and 15 and at least one reverse primer selected from SEQ ID NOS: 6, 7,
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`9, l3, 14, 16 and 17, either in the same or separate packaging, and instructions for its use.
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`The invention also provides a kit for detecting Mycobacterium tuberculosis in a urine
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`sample from a subject in need thereof, comprising at least one forward primer selected from
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`SEQ ID N05: 34, 37 and 39 and at least one reverse primer selected from SEQ ID N05: 35,
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`36, 38 and 40, either in the same or separate packaging, and instructions for its use.
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`The invention also provides a kit for detecting Bacillus anthracis in a urine sample
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`from a subject in need thereof, comprising at least one forward primer selected from SEQ ID
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`NOS: 41, 43, 45, 47, 48 and 50 and at least one reverse primer selected from SEQ ID NOS:
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`42, 44, 46 and 49, either in the same or separate packaging, and instructions for its use.
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`The invention also provides a kit for detecting Plasmodz'um species in a urine sample
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`from a subject in need thereof, comprising at least one forward primer selected from SEQ ID
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`NOS: 18, 21 and 23 and at least one reverse primer selected from SEQ ID N03: 19, 20 and
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`22, either in the same or separate packaging, and instructions for its use.
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`The invention also provides a kit for detecting Leishmam‘a species in a urine sample
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`from a subject in need thereof, comprising at least one forward primer selected from SEQ ID
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`NOS: 26, 28, 30, 32 and 51 and at least one reverse primer selected from SEQ ID N05: 24,
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`25, 27, 29, 31 and 33, either in the same or separate packaging, and instructions for its use.
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`BRIEF DESCRIPTION OF THE DRAWINGS
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`Figure 1 is a photograph of PCR reactions performed on human urine with primers
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`specifc for human b-actin resolved by electrophoresis.
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`Figure 2 is a photograph of PCR reactions performed on rabbit urine With primers
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`specifc for rabbit b-globin resolved by electrophoresis.
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`Figure 3A is a photograph of semi-nested PCR reactions performed on human urine
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`with primers specifc for H. pylori ureA sequences resolved by electrophoresis.
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`Figure 3B is a photograph of semi-nested PCR reactions performed on human urine
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`with primers specifc for H. pylori cagA sequences resolved by electrophoresis.
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`Figure 4 is a photograph of semi-nested PCR reactions performed on human urine
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`with primers specifc for Plasmodz'umfalciparum sequences resolved by electrophoresis.
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`Figure 5 is a photograph of semi-nested PCR reactions performed on human urine
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`with primers specifc for Leishmania infanl'um sequences resolved by electrophoresis.
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`Figure 6 is a photograph of semi-nested PCR reactions performed on human urine
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`from TB infected and healthy patients with primers specifc for Mycobacz‘erz’um tuberculosis
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`sequences resolved by electrophoresis.
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`Figure 7 is a photograph of semi-nested PCR reactions performed on the supernatants
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`and pellets of centrifuged human urine from TB infected and healthy patients before and after
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`treatment with primers specifc for Mycobacterium tuberculosis sequences resolved by
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`electrophoresis.
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`Figure 8 shows photographs of semi—nested PCR reactions performed on human urine
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`from TB infected patients with primers specific for Mycobacierium tuberculosis sequences of
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`129/67 bp (left panel) and 330/69 bp (right panel) resolved by electrophoresis.
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`Figure 9 is a photograph of semi-nested PCR reactions performed on human urine
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`from TB actively infected patients and patients two months after treatment for TB with
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`primers specifc for Mycobacterium tuberculosis sequences resolved by electrophoresis.
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`Figure 10 is a photograph of semi-nested PCR reactions performed on rabbit urine
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`from B. anthracis infected and uninfected subjects with primers specifc for Bacillus anthI-‘acis
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`sequences resolved by electrophoresis.
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`DETAILED DESCRIPTION OF THE INVENTION
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`The invention is based, in part, upon the discovery described herein that pathogenic
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`nucleic acids are detectable in the urine of a subject infected with the pathogen. In some
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`instances, nucleic acids from pathogenic organisms cross the kidney barrier and pass into the
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`urine of mammals. The nucleic acids which cross the renal barrier, termed herein as
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`transrenal nucleic acids, and more specifically transrenal DNA tend to be shorter than 1,000
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`bp in length, but are preferably less than 500 bp in length, and more preferably shorter than
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`250—3 00 bp in length or shorter than 250 bp in length. While it had been shown in the past
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`that DNA from mammalian cells, cancerous and non-cancerous which were hypothesized to
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`die by apoptosis in the body of a subject may be able to cross the renal barrier, this is the first
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`time it has been shown that the DNA of pathogens also crosses this barrier. Further, nucleic
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`acids from pathogens found in the kidney would shed pathogen specific nucleic acids into the
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`urine Without crossing the kidney barrier. There may be other mechanisms by which
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`pathogen specific nucleic acids pass into the urine of a subject infected with the pathogen.
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`Based on this discovery, the invention provides compositions and methods for the
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`detection of non—viral pathogens in the urine of a subject. The compositions and methods of
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`the invention are used to detect transrenal—nucleic acids derived from pathogenic
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`microorganisms which traverse the renal barrier and are present in the urine of a subject. The
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`invention includes methods for the isolation, amplification and detection of these transrenal
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`nucleic acids. The invention also includes compositions which may be used to isolate,
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`amplify these transrenal nucleic acids.
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`One method of the invention for the isolation of urinary DNA from a subject includes
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`the following steps. Urine is first centrifuged or filtered to separate cells and cell debris from
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`the urine. EDTA/Tris HCl is then added to the urine to bind bivalent ions and to adjust pH to
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`approximately pH 8. To this solution is then added a chaotropic salt, for example guanidine
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`isothiocyanate to a final concentration of at least 3 M. Then a silica resin is added to the
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`urine to which DNA binds. This resin is then washed, and the DNA eluted from it, thereby
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`isolating the transrenal DNA.
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`DNA isolated from the urine of a subject may then be amplified in order to be
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`detected. Amplification methods include polymerase chain reaction (PCR), nested PCR,
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`semi-nested PCR, Single-Strand Conformation Polymorphism analysis (SSCP), ligase chain
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`reaction (LCR) and strand displacement amplification (SDA). Detection of transrenal DNAs
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`is also performed through hybridization of at least one labeled probe.
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`The amplification and detection of urinary nucleic acids from non-viral pathogens
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`may be used to detect the presence of these pathogens, and thus diagnose the infection of a
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`subject with these pathogens and to diagnose pathologies associated with the infection of a
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`subject by these pathogens. Further, the detection of these pathogens may be used to monitor
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`treatment of the infections and pathologies associated with these pathogens. Also, detection
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`of these pathogens may be used to suggest treatments for a subject in which the pathogens are
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`detected. These treatments may be used to relieve symptoms which are associated with the
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`detection of pathogens or diagnosis with infection of the pathogens.
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`Non-viral pathogens which are able to be detected by the presence of their nucleic
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`acids in mammalian urine include bacteria and parasites. Bacterial species which are
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`detectable using the compositions and methods of the invention include Helicobacter pylori
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`(H. pylori), Mycobacterium tuberculosis (M tuberculosis, MTB or TB), and Bacillus
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`anthracis (B. anthracis). Parasite species which are detectable using the compositions and
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`methods of the invention include Plasmodium species including Plasmodium falciparum and
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`Leishmania species.
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`Detection of the Presence of Pathogenic Microorganisms
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`Bacteria
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`Helicobacter pylori
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`Most cases of peptic ulcer disease, gastric mucosa associated lymphoid tissue
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`(MALT) lymphoma and cancer of the distal stomach are complications of Helicobacter
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`pylori infection. (Axon AT. Gut. 1999 Jul; 45 Suppl 1:11-4). However, most H. pylori—
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`positive individuals remain symptom free throughout their life. Symptoms associated with H.
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`pylori infection include abdominal discomfort, weight loss, poor appetite, bloating, burping,
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`nausea and vomiting. A widely accepted explanation of this phenomenon is that the outcome
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`of H. pylori colonization is determined by combination of several factors including the
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`genotypes of the bacteria and the host and the environmental cofactors such as diet and
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`smoking. For the successful medical management of the infection it is very important the
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`development of diagnostics tools to direct the decision concerning the treatment of the
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`carriers. The test should give information about the genotype of