Authentication of beef, carabeef, chevon, mutton and pork by a PCR-RFLP assay of mitochondrial cytb gene

Abstract

Authentication of meat assumes significance in view of religious, quality assurance, food safety, public health, conservation and legal concerns. Here, we describe a PCR-RFLP (Polymerase Chain Reaction- Restriction Fragment Length Polymorphism) assay targeting mitochondrial cytochrome-b gene for the identification of meats of five most common food animals namely cattle, buffalo, goat, sheep and pig. A pair of forward and reverse primers (VPH-F & VPH-R) amplifying a conserved region (168-776 bp) of mitochondrial cytochrome-b (cytb) gene for targeted species was designed which yielded a 609 bp PCR amplicon. Further, restriction enzyme digestion of the amplicons with Alu1 and Taq1 restriction enzymes resulted in a distinctive digestion pattern that was able to discriminate each species. The repeatability of the PCR-RFLP assay was validated ten times with consistent results observed. The developed assay can be used in routine diagnostic laboratories to differentiate the meats of closely related domestic livestock species namely cattle from buffalo and sheep from goat.

Molecular detection of adulteration in commercial buffalo meat products by multiplex PCR assay

“…Meat vendors often use cheaper meats to adulterate buffalo meat to meet the consumer demand and achieve monetary gain. On past researches, several investigators have used PCR-based approaches to identify buffalo meat from cattle meat and other animal meats targeting mitochondrial D-loop region (Karabasanavar et al, 2011;Mane et al, 2012;Girish et al, 2013), 12S rRNA gene (Patil et al, 2015), cytb gene (Gupta et al, 2011;Kumar et al, 2014;Bhat et al, 2016) and ND5 gene (Hossain et al, 2017). However, no multiplex PCR assay has been documented for the detection of commercial buffalo meat products.…”

Self‐signal‐on fluorescent colorimetric protocol for rapid authentication of horsemeat adulterated beef samples with functional designed probes

“…Compared to traditional and classic PCR with agarose gel electrophoresis required, at least additional 30–60 min is needed for both the qualitative and quantitative analysis. For the reported PCR‐ELISA or PCR‐RFLP‐based protocol (Asensio et al ., ; Kumar et al ., ; Santaclara et al ., ), additional 2 h or overnight is required to achieve the final results of the amplifications respectively. Even compared to our previous results of mimicking DNAzyme‐included primer‐based PCR protocol (Cheng et al ., ), the hairpin‐based fluorescent signal‐on protocol in this research is about 25–30 min faster.…”

PCR-RFLP identification of meat from red deer, sika deer, roe deer, fallow deer, mouflon, wild boar, hare and cattle

“…Thanks to the point mutations in mtDNA, even closely related species can be accurately discriminated after amplification of a section of mtDNA by an appropriate set of primers. Specific mtDNA sequences fitting for species identification have been published for many species (Kumar et al 2014). The main advantage of mtDNA is its abundance in the samples -unlike nDNA, mtDNA is present in multiple copies in each cell and can be isolated from samples with very low quantity and/or quality of DNA due to its degradation.…”

“…The common targets in mtDNA for species identification are represented by the D-loop region and genes like CytB and mt rRNA (5 s, 12 s, 16 s, 18 s etc). The CytB gene is a preferred target not only for taxonomic and phylogenetic studies, but also for meat speciation (Kumar et al 2014).…”