Robust detection of cow and female buffalo DNA through Real-Time PCR assay

Abstract

Cow, Bos taurus, and female buffalo, Bubalus bubalis, are considered sacred animals that are a part of rural livelihood in India. The purity of products from these bovine species has significant sentimental implications in the dairy and meat industry. Therefore, the mitochondrial DNA and the sex origin, targeting the X and Y chromosomes from these bovine species, were selected to design three multiplex real-time probe PCR assays: Hi-PCR® Cow Detection Kit (MBPCR184), Hi-PCR® Buffalo Detection Kit (MBPCR185) and Hi-PCR® Cattle Sex Determination Kit (MBPCR186). Scientific Working Group on DNA Analysis Methods (SWGDAM) guidelines were followed to perform different studies using reference control DNAs. An Internal Reagent Control (IRC) was part of every assay, thus ensuring a successful reaction. The assays were 100% specific, with no cross-amplification of the two bovine species. The amplification of the X chromosomal target was observed for male and female DNAs, whereas Y chromosome amplification was observed only for the male DNA. The assays were 100% specific to the target genes in these organisms with no non-specificity towards any other targets or organisms. The limit of detection for sex determination was 0.01 ng/µl, whereas the differential capability of the assay was 3 copies/µl and 30 copies/µl for Bos taurus and Bubalus bubalis, respectively. The assays were reproducible at 1 ng/µl genomic DNA with 95% CI. The assays are open and compatible with other brands of Real-Time PCR systems used in forensic labs. The experiments presented here verify that the developed real-time PCR assays are robust, produce reliable and reproducible results for detection and differentiation of Bos taurus and Bubalus bubalis and their sex even at low DNA concentrations.