Differentiation of Leptospira species and serovars by PCR-restriction endonuclease analysis, arbitrarily primed PCR and low-stringency PCR

Abstract

Reference strains from 30 serovars representing seven species of Leptospira and 48 recent isolates from human patients, dogs and rats, were characterised by polymerase chain reaction-restriction endonuclease analysis (PCR-REA), arbitrarily primed PCR (AP-PCR) and low stringency PCR (LS-PCR). PCR-REA analysis yielded seven groups among 29 serovars of pathogenic Leptospira; the non-pathogenic L. bzjiexa serovar patoc was not amplified with the primer pairs studied. AP-PCR and LS-PCR fingerprinting resulted in 25 and 21 distinct profiles, respectively, among the 30 reference strains. The results of the three PCR-based techniques were highly concordant and were in general agreement with those from previous DNA studies, confirming the high level of polymorphism among Leptospira species and serovars, and supported the concept of the serovar as the basic taxonomic unit of leptospiral classification. Results of the PCRbased typing methods for 11 randomised leptospiral strains, 36 clinical isolates from human patients and dogs and 12 survey isolates from trapped rats agreed with those from serological identification. With one exception, isolates of the same serovar gave identical profiles irrespective of the source. AP-PCR and LS-PCR are simple to perform and interpret, and appear to be useful for characterising isolates of Leptospira spp. for diagnostic and epidemiological purposes.

Identification of New Repetitive Element in Leptospira interrogans Serovar Copenhageni and Its Application to PCR-Based Differentiation of Leptospira Serogroups

“…These spirochetes are taxonomically classified by genetic techniques such as DNA-DNA hybridization (2,3,6,21,35), but in many laboratories, diagnosis and classification rely on serologic methods based on the microscopic agglutination test. In reference laboratories, methods such as the cross-absorption agglutinin test and the use of monoclonal antibodies allow serovar identification (6,7).…”

“…First, the use of a single oligonucleotide primer eliminates the need for difficult and time-consuming techniques such as maintenance of reference serum batteries, dark-field microscopy (3,6,7,16,17,23), and preparation of homologous rabbit antiserum used for the serologic assays. Second, iRep1 PCR was specific enough to detect leptospiral DNA from cultures that became contaminated with other bacteria, a frequent problem in the tropical environment.…”

“…Batteries of monoclonal antibodies raised against each isolated strain or reference serovar are also used for rapid diagnosis and identification, but are limited by the availability or access to these antibodies, which are usually confined to few reference laboratories (6). These tests are often complicated by the extensive cross-reactivity of the antisera to shared Leptospira serovar antigenic epitopes (3,4,6,7,9,13). Because of this complexity of serologic identification, several laboratories have developed new classification techniques based on genetic heterogeneity among members of the genus Leptospira (3,4,7,8,9,12,19,24).…”

Multilocus Sequence Analysis for Typing Leptospira interrogans and Leptospira kirschneri

“…A wide range of molecular methods to type leptospiral isolates, including PCR-restriction endonuclease analysis (4,19), pulsed-field gel electrophoresis (7), and random amplification (6,17), have been applied with more or less success. The genomic DNA-DNA hybridization method has been widely used for the determination of phylogenetic relationships between closely related strains (3,5,14).…”

SEROLOGIC SURVEY AND RESULTS OF URINARY PCR TESTING FOR LEPTOSPIROSIS IN CAPTIVE BLACK-TAILED PRAIRIE DOGS (CYNOMYS LUDOVICIANUS)

“…6. Arbitrarily primed PCR (AP-PCR), random amplification of polymorphic DNA (RAPD), or low stringency PCR (LS-PCR) (Brown & Levett, 1997;de Caballero et al, 1994;Perolat et al, 1994).…”