Genomic identification of pathogenic and non-pathogenic Leptospires by multilocus variable-number tandem-repeat analysis
Abstract number: P1720
Khaki P., Abdollahpour Alittapeh M., Moradi Bidhendi S., Esmaelizad M., Tadayon K.
Background: Leptospirosis is an emerging zoonotic diseases which caused by pathogenic Leptospires. Detection and identification of Leptospires has conventionally been performed by cultural and serological methods. These methods are tedious, time consuming, and potentially biohazardous. Recently several PCR based techniques such as VNTR have been demonstrated to provide useful tools in detection and identification of leptospiral serovars but most these techniques have drawbacks. VNTR can also provide information relating to both the evolutionary and functional areas of bacterial diversity.
Objective: The present study was carried out to set up of VNTR technique for the genotyping of pathogenic and nonpathogenic Leptospiral serovars.
Methods: The pathogenic and non-pathogenic reference serovars of Leptospira spp. were obtained from the Microbial Culture Collection at the National Reference Laboratory for Leptospira at the Microbiology Department of Razi Vaccine & Serum Research Institute, karaj, Iran. Leptospiral strains were subcultured into the liquid EMJH medium and incubated at 28 for 7 days.
Genomic DNA of Leptospiral serovars were extracted using the phenol-chloroform method. PCR was performed with the three selected VNTR loci (VNTR4, 10, 36). The amplified products were analyzed by agarose gel electrophoresis. The sizes of the amplified products were estimated by comparison with a 100-bp ladder.
Result: All loci successfully amplified in all pathogenic leptospiral serovars with the primers. Although VNTR-4 was unable to discriminate between Grippotyphosa and Hardjo, Icterohemoragaea and Pomona, this locus was thoroughly differentiated between pathogenic and non-pathogenic. VNTR-10 had an appropriate discrimination to differentiate among these strains but was unable to differentiate between pomana and semaranga. VNTR-36 successfully amplified in all of the strains. Although This locus aptly separated serovar pomona from other strains, but could not separated Grippotyphosa and semaranga.
Conclusion: The sizes of the amplified products displayed a wide range of polymorphisms, suggesting variation tandem-repeat copy numbers in the VNTR loci. The combination of the VNTR-4, VNTR-10, and VNTR-36 loci was useful for typing L. interrogans. Although VNTR-4 was poorly discriminatory, was the only marker for separation between pathogenic and non-pathogenic. The VNTR method provides rapid typing as well as a highly discriminant assay to identify L. interrogans serovars.
|Session name:||Abstracts 20th European Congress of Clinical Microbiology and Infectious Diseases|
|Location:||Vienna, Austria, 10 - 13 April 2010|
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