PCR-BASED MOLECULAR IDENTIFICATION OF SPECIES AND SEX IN DOMESTIC RUMINANTS USING CYTOCHROME B, SRY AND GAPDH MARKERS

Abstract

Accurate identification of animal species and sex is important in veterinary forensic investigations, wildlife-related cases, meat authentication and the examination of biological evidence. Conventional identification based on morphological characteristics may become difficult when samples are fragmented, degraded or putrefied. The present study evaluated polymerase chain reaction (PCR)-based molecular markers for identification of species and sex from blood samples of domestic ruminants. Blood samples were collected from 48 animals comprising six males and six females each of buffalo (Bubalus bubalis), cattle (Bos indicus), goat (Capra hircus) and sheep (Ovis aries). Genomic DNA was isolated using a silica membrane-based commercial extraction kit and assessed for concentration and purity using a spectrophotometer. Species identification was performed using species specific forward primers targeting the mitochondrial cytochrome b gene in combination with a common reverse primer. Sex identification was performed by amplification of the male-specific SRY gene, while GAPDH was used as an autosomal amplification control. A duplex PCR combining SRY and GAPDH was subsequently evaluated. Species-specific PCR generated distinct amplicons of 106 bp in buffalo, 163 bp in cattle, 232 bp in goat and 308 bp in sheep. The SRY assay produced a 122-bp fragment in male animals, whereas no SRY-specific amplification was observed in females. GAPDH produced a 218-bp product in both sexes and was therefore useful as an internal amplification control. Duplex PCR generated two products of 122 and 218 bp in males and only the 218-bp product in females. The findings demonstrate that species-specific cytochrome b PCR can distinguish the four domestic ruminant species examined, while the combined SRY-GAPDH assay provides a practical molecular approach for sex determination. These assays may be useful in veterinary forensic investigations and other applications requiring molecular identification of domestic ruminant biological samples.