• We are sorry, but NCBI web applications do not support your browser and may not function properly. More information
Logo of jcmPermissionsJournals.ASM.orgJournalJCM ArticleJournal InfoAuthorsReviewers
J Clin Microbiol. Dec 1997; 35(12): 3140–3146.
PMCID: PMC230137

Identification of pathogenic Leptospira genospecies by continuous monitoring of fluorogenic hybridization probes during rapid-cycle PCR.


Partial sequences of 23S rRNA gene PCR products from 23 strains of 6 pathogenic Leptospira genospecies and from 8 strains of the saprophytic Leptospira biflexa were determined. Sequence analyses enabled Leptospira genus-specific amplification primers and pathogen-specific fluorogenic adjacent hybridization probes to be designed and synthesized. A PCR protocol was developed in which changes in fluorescence emission resulting from specific annealing of fluorogenic adjacent hybridization probes to the target DNA were continuously monitored. Nine strains of the pathogenic Leptospira genospecies could be differentiated from Leptonema illini, Escherichia coli, and eight strains of Leptospira biflexa. The PCR method was rapid, requiring 18 min for the completion of 45 cycles. It was also simple and flexible, as DNA templates prepared by four different methods, including the simple boiling method, could be used without adverse effects. Two hundred copies of target, equivalent to 100 cells, could be detected.

Full Text

The Full Text of this article is available as a PDF (449K).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.
  • Bonfield JK, Smith K f, Staden R. A new DNA sequence assembly program. Nucleic Acids Res. 1995 Dec 25;23(24):4992–4999. [PMC free article] [PubMed]
  • Boom R, Sol CJ, Salimans MM, Jansen CL, Wertheim-van Dillen PM, van der Noordaa J. Rapid and simple method for purification of nucleic acids. J Clin Microbiol. 1990 Mar;28(3):495–503. [PMC free article] [PubMed]
  • Breslauer KJ, Frank R, Blöcker H, Marky LA. Predicting DNA duplex stability from the base sequence. Proc Natl Acad Sci U S A. 1986 Jun;83(11):3746–3750. [PMC free article] [PubMed]
  • Cardullo RA, Agrawal S, Flores C, Zamecnik PC, Wolf DE. Detection of nucleic acid hybridization by nonradiative fluorescence resonance energy transfer. Proc Natl Acad Sci U S A. 1988 Dec;85(23):8790–8794. [PMC free article] [PubMed]
  • Corney BG, Colley J, Djordjevic SP, Whittington R, Graham GC. Rapid identification of some Leptospira isolates from cattle by random amplified polymorphic DNA fingerprinting. J Clin Microbiol. 1993 Nov;31(11):2927–2932. [PMC free article] [PubMed]
  • de Caballero OL, Dias Neto E, Koury MC, Romanha AJ, Simpson AJ. Low-stringency PCR with diagnostically useful primers for identification of Leptospira serovars. J Clin Microbiol. 1994 May;32(5):1369–1372. [PMC free article] [PubMed]
  • Fukunaga M, Masuzawa T, Okuzako N, Mifuchi I, Yanagihara Y. Linkage of ribosomal RNA genes in Leptospira. Microbiol Immunol. 1990;34(7):565–573. [PubMed]
  • Gerritsen MA, Smits MA, Olyhoek T. Random amplified polymorphic DNA fingerprinting for rapid identification of leptospiras of serogroup Sejroe. J Med Microbiol. 1995 May;42(5):336–339. [PubMed]
  • Gerritsen MJ, Olyhoek T, Smits MA, Bokhout BA. Sample preparation method for polymerase chain reaction-based semiquantitative detection of Leptospira interrogans serovar hardjo subtype hardjobovis in bovine urine. J Clin Microbiol. 1991 Dec;29(12):2805–2808. [PMC free article] [PubMed]
  • Gravekamp C, Van de Kemp H, Franzen M, Carrington D, Schoone GJ, Van Eys GJ, Everard CO, Hartskeerl RA, Terpstra WJ. Detection of seven species of pathogenic leptospires by PCR using two sets of primers. J Gen Microbiol. 1993 Aug;139(8):1691–1700. [PubMed]
  • Hillier L, Green P. OSP: a computer program for choosing PCR and DNA sequencing primers. PCR Methods Appl. 1991 Nov;1(2):124–128. [PubMed]
  • Hookey JV. Detection of Leptospiraceae by amplification of 16S ribosomal DNA. FEMS Microbiol Lett. 1992 Jan 15;69(3):267–274. [PubMed]
  • Kee SH, Kim IS, Choi MS, Chang WH. Detection of leptospiral DNA by PCR. J Clin Microbiol. 1994 Apr;32(4):1035–1039. [PMC free article] [PubMed]
  • Mérien F, Amouriaux P, Perolat P, Baranton G, Saint Girons I. Polymerase chain reaction for detection of Leptospira spp. in clinical samples. J Clin Microbiol. 1992 Sep;30(9):2219–2224. [PMC free article] [PubMed]
  • Oliveira MA, Caballero OL, Dias Neto E, Koury MC, Romanha AJ, Carvalho J, Hartskeerl RA, Simpson AJ. Use of nondenaturing silver-stained polyacrylamide gel analysis of polymerase chain reaction amplification products for the differential diagnosis of Leptospira interrogans infection. Diagn Microbiol Infect Dis. 1995 Aug;22(4):343–348. [PubMed]
  • Pearson WR, Lipman DJ. Improved tools for biological sequence comparison. Proc Natl Acad Sci U S A. 1988 Apr;85(8):2444–2448. [PMC free article] [PubMed]
  • Savio ML, Rossi C, Fusi P, Tagliabue S, Pacciarini ML. Detection and identification of Leptospira interrogans serovars by PCR coupled with restriction endonuclease analysis of amplified DNA. J Clin Microbiol. 1994 Apr;32(4):935–941. [PMC free article] [PubMed]
  • Wagenaar JA, Segers RP, Van der Zeijst BA. Rapid and specific detection of pathogenic Leptospira species by amplification of ribosomal sequences. Mol Biotechnol. 1994 Aug;2(1):1–14. [PubMed]
  • Wittwer CT, Herrmann MG, Moss AA, Rasmussen RP. Continuous fluorescence monitoring of rapid cycle DNA amplification. Biotechniques. 1997 Jan;22(1):130–138. [PubMed]
  • Woo TH, Smythe LD, Symonds ML, Norris MA, Dohnt MF, Patel BK. Rapid distinction between Leptospira interrogans and Leptospira biflexa by PCR amplification of 23S ribosomal DNA. FEMS Microbiol Lett. 1997 May 1;150(1):9–18. [PubMed]
  • Woodward MJ, Sullivan GJ, Palmer NM, Woolley JC, Redstone JS. Development of a PCR test specific for Leptospira hardjo genotype bovis. Vet Rec. 1991 Mar 23;128(12):282–283. [PubMed]
  • Zhang Y, Li S, Dai B. [Amplified 23S rRNA gene of 52 strains of Leptospira and detection of leptospiral DNA in 55 patients by PCR]. Hua Xi Yi Ke Da Xue Xue Bao. 1993 Sep;24(3):262–267. [PubMed]
  • Zuerner RL, Alt D, Bolin CA. IS1533-based PCR assay for identification of Leptospira interrogans sensu lato serovars. J Clin Microbiol. 1995 Dec;33(12):3284–3289. [PMC free article] [PubMed]

Articles from Journal of Clinical Microbiology are provided here courtesy of American Society for Microbiology (ASM)


Related citations in PubMed

See reviews...See all...

Cited by other articles in PMC

See all...


Recent Activity

Your browsing activity is empty.

Activity recording is turned off.

Turn recording back on

See more...