Compositions and Methods for Detecting Pathogen Specific Nucleic Acids in Urine

Abstract

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 . (canceled) 
     
     
         2 . A method for diagnosing  Helicobacter pylori, Bacillus anthracis, Plasmodium  species or  Leishmania  species infection in a subject, comprising:
 a) obtaining a urine sample from a subject;   b) separating a soluble fraction of said urine sample; and   c) detecting the presence of a cell-free  Helicobacter pylori, Bacillus anthracis, Plasmodium  species or  Leishmania  species nucleic acid in said soluble fraction of said urine sample, wherein said nucleic acid is less than about 300 by in length, wherein said detecting step is performed by a method selected from the group consisting of PCR, nested PCR, semi-nested PCR, LCR, and SDA, using a primer set comprising at least one forward primer selected from the group consisting of SEQ ID NOs: 5, 8, 10, 11, 12, 15, 18, 21, 23, 26, 28, 30, 32, 41, 43, 45, 47, 48, 50, and 51 and at least one reverse primer selected from the group consisting of SEQ ID NOs: 6, 7, 9, 13, 14, 16, 17, 19, 20, 22, 24, 25, 27, 29, 31, 33, 42, 44, 46 and 49, wherein the presence of said nucleic acid diagnoses  Helicobacter pylori, Bacillus anthracis, Plasmodium  species or  Leishmania  species infection.   
     
     
         3 . The method of  claim 2 , further comprising the step of quantitating the nucleic acid. 
     
     
         4 . The method of  claim 2 , wherein the subject is a mammal. 
     
     
         5 . The method of  claim 4 , wherein the mammal is a human. 
     
     
         6 . A kit, comprising: a) one or more reagents or materials for the isolation and purification or for the detection and quantitation of cell-free pathogen-related transrenal nucleic acids from a urine sample of a subject, wherein said reagents or materials comprise a matrix including a silica-based resin, wherein said silica-based resin isolates cell-free nucleic acids that are less than about 300 nucleotides; and wherein said reagents or materials comprise an agent that inhibits the degradation of the cell-free nucleic acids; and b) two or more primers for detecting said cell-free pathogen-related nucleic acids in said urine sample, wherein said two or more primers are selected from the group consisting of SEQ ID NOs: 5-33 and 41-51. 
     
     
         7 . The kit according to  claim 6 , further comprising reagents for the performance of a technique selected from the group consisting of a hybridization of said cell-free pathogen-related nucleic acids, a cycling probe reaction, a polymerase chain reaction, a nested polymerase chain reaction, a ligase chain reaction, and a strand displacement amplification. 
     
     
         8 . The kit according to  claim 6 , further comprising a chaotropic agent. 
     
     
         9 . The kit according to  claim 6 , wherein said agent that inhibits the degradation of the nucleic acids is selected from the group consisting of a ion-chelating agent, a denaturing agent, and an ionic detergent. 
     
     
         10 . The kit according to  claim 9 , wherein said ion-chelating agent is EDTA; said denaturing agent is guanidine HCL or guanidine isothiocyanate; and said ionic detergent is N-lauryl sarcosine or sodium dodecyl sulfate.