FlaB PCR-based identification of pathogenic leptospiral isolates.
OBJECTIVE
The genus Leptospira comprises pathogenic and saprophytic strains. Conventional methods for the identification of pathogenic leptospiral isolates are cumbersome and laborious. In view of these limitations, the search for alternative methods have been focused on DNA based techniques. In this study, we have developed an effective method for the rapid identification of pathogenic and saprophytic leptospiral isolates based on DNA-based techniques.


METHODS
A polymerase chain reaction(PCR)-based approach was developed using specific primer sets (flaB, G1-G2, B64I-II, and A-B) to differentiate pathogenic and saprophytic leptospiral strains. Fifty-five leptospiral isolates were used for this study. The pathogenic status of the isolates was compared with the results obtained using conventional techniques, which included growth in the presence of 8-azaguanine and growth at 13 degrees C.


RESULTS
In this analysis, 46 leptospiral isolates were confirmed as pathogenic and nine were confirmed as saprophytic. PCR with the A-B primer set yielded an amplified product of 331 bp in all of the pathogenic and saprophytic isolates. The other primer sets, G1-G2, B64I-II and flaB, yielded products of 258 bp, 568 bp, and 793 bp, respectively, exclusively for the pathogenic leptospiral strains. None of the saprophytic strains yielded products with these primer sets.


CONCLUSIONS
The flaB-specific primers consistently yielded an amplification product for all of the pathogenic leptospiral isolates, indicating the presence of the flaB gene only among pathogenic leptospires, and making this a useful tool for distinguishing between pathogenic and saprophytic leptospires. The efficiency of PCR-based identification corroborates the implementation of these techniques for the identification of pathogenic and saprophytic leptospiral strains.
Natarajaseenivasan
Kalimuthusamy
K
Division of Medical Microbiology, Department of Microbiology, School of Life Sciences, Bharathidasan University, Tiruchirappalli, Tamilnadu, India. natarajaseenivasan@rediffmail.com
Vijayachari
Paluru
P
Sharma
Sameer
S
Sugunan
Attayoor Purushothaman
AP
Vedhagiri
Kumaresan
K
Selvin
Joseph
J
Sehgal
Subhash Chandra
SC
D000818:Animals; D001426:Bacterial Proteins / Q000235:genetics*; D001431:Bacteriological Techniques / Q000379:methods*; D017931:DNA Primers / Q000235:genetics; D005408:Flagellin / Q000235:genetics*; D006801:Humans; D007919:Leptospira / Q000145:classification* / Q000235:genetics / Q000302:isolation & purification*; D007922:Leptospirosis / Q000175:diagnosis* / Q000382:microbiology; D016133:Polymerase Chain Reaction / Q000379:methods*; D051381:Rats; D012680:Sensitivity and Specificity; D014871:Water Microbiology
D001426:Bacterial Proteins; D017931:DNA Primers; D005408:Flagellin; C416532:flaB flagellin