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Volume 43, Number 1, February 2010

FlaB PCR-based Identification of Pathogenic Leptospiral Isolates


Kalimuthusamy Natarajaseenivasana, Paluru Vijayachari, Sameer Sharma, Attayoor Purushothaman Sugunan, Kumaresan Vedhagiri, Joseph Selvin, Subhash Chandra Sehgal


Received: September 30, 2008    Revised: January 20, 2009    Accepted: March 25, 2009   

 

Corresponding author:

Division of Medical Microbiology, Department of Microbiology, School of Life Sciences, Bharathidasan University, Tiruchirappalli 620 024, India.
E-mail: natarajaseenivasan@rediffmail.com



 

Background and purpose: 

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°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.



 

Conclusion:

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.



 

Key words:

8-azaguanine, G1-G2, B64I-II, flaB, PCR, pathogenic leptospires