title: Evaluation of lig-based conventional and real time PCR for the detection of pathogenic leptospires.
abstract: Leptospirosis is globally important infectious disease affecting almost all mammals. Pathogenic Leptospira encodes immunoglobulin-like protein (Lig) that is found to express only during infection. We report the development of conventional and real time PCR assays targeting lig genes of leptospires for the early diagnosis of leptospirosis. Sensitivity of the newly designed Lig1/Lig2 primers for conventional PCR was compared with previously published primers LP1/LP2 and G1/G2. G1/G2 primers amplified the target DNA from all the serovars including non-pathogenic Leptospira biflexa whereas LP1/LP2 and Lig1/Lig2 primers amplified only pathogenic leptospires. Diagnostic PCR assay was also developed for the detection of pathogenic Leptospira interrogans in urine samples. We obtained the highest sensitivity in PCR using our Lig1/Lig2 primers with a detection of 6 leptospires. A rapid and sensitive lig-based real time PCR assay was also developed with a detection range of 10-10(7) gene copies. To evaluate the early diagnosis for leptospirosis, we compared the culture with conventional and real time PCR for the detection of spirochetes in experimentally infected hamsters during a time-course study. Culture of infected hamster tissues detected the presence of leptospires from Day 2 of infection but not on the day of infection or Day 1, whereas conventional PCR and real time PCR detected the leptospires from the day of infection. Hence, conventional and real time PCR with lig primers would be a sensitive and rapid tool for early diagnosis of leptospirosis.
authors.affiliation: College of Veterinary Medicine, Cornell University, Ithaca, NY 14853, USA.
authors.forename: Raghavan U M
authors.initials: RU
authors.lastname: Palaniappan
authors.forename: Yung-Fu
authors.initials: YF
authors.lastname: Chang
authors.forename: Chao-Fu
authors.initials: CF
authors.lastname: Chang
authors.forename: M J
authors.initials: MJ
authors.lastname: Pan
authors.forename: C W
authors.initials: CW
authors.lastname: Yang
authors.forename: Peter
authors.initials: P
authors.lastname: Harpending
authors.forename: Sean P
authors.initials: SP
authors.lastname: McDonough
authors.forename: Edward
authors.initials: E
authors.lastname: Dubovi
authors.forename: Thomas
authors.initials: T
authors.lastname: Divers
authors.forename: Jiaxin
authors.initials: J
authors.lastname: Qu
authors.forename: Bruce
authors.initials: B
authors.lastname: Roe
mesh_terms: D000818:Animals; D000942:Antigens, Bacterial / Q000235:genetics; D006224:Cricetinae; D017931:DNA Primers; D018628:Gene Dosage; D005798:Genes, Bacterial; D007919:Leptospira / Q000235:genetics* / Q000302:isolation & purification; D007921:Leptospira interrogans / Q000235:genetics / Q000302:isolation & purification; D007922:Leptospirosis / Q000235:genetics* / Q000382:microbiology / Q000652:urine; D016133:Polymerase Chain Reaction
chemical_list: D000942:Antigens, Bacterial; D017931:DNA Primers