Development of novel heminested PCR assays based on mitochondrial 16s rRNA gene for identification of seven pecora species -: art. no. 42

Background: Characterization of molecular markers and the development of better assays for precise and rapid detection of wildlife species are always in demand. This study describes a set of seven novel heminested PCR assays using specific primers designed based on species-specific polymorphism at the mitochondrial 16S rRNA gene for identification of Blackbuck, Goral, Nilgai, Hog deer, Chital, Sambar and Thamin deer. Results: The designed heminested PCR assays are two consecutive amplifications of the mitochondrial 16S rRNA gene. In the first stage, similar to 550 bp region of the 16S rRNA gene was amplified by PCR using template DNA and universal primers. In the second stage, a species-specific internal region of the 16S rRNA gene was amplified by PCR using the amplicon of the first PCR along with one universal primer and another species-specific primer as the reverse or forward primer. The amplicon generated after two consecutive amplifications was highly unique to target species. These assays were successfully validated for sensitivity, specificity, and ruggedness under a wide range of conditions. Conclusion: The validation experiments confirm that the designed heminested PCR assays for identification of the seven species are highly specific, sensitive, reliable and provide a reproducible method allowing analysis of low copy number DNA recovered from decomposed or highly processed tissues. The assays for identification of other species could be devised by extrapolating the principle of designed heminested PCR.