Heptaplex Polymerase Chain Reaction Assay for the Simultaneous Detection of Beef, Buffalo, Chicken, Cat, Dog, Pork, and Fish in Raw and Heat-Treated Food Products

Abstract

Species authentication of meat and fish products is crucial to safeguard public health, economic investment, and religious sanctity. We developed a heptaplex polymerase chain reaction assay targeting short amplicon length (73−198 bp) for the simultaneous detection and differentiation of cow, buffalo, chicken, cat, dog, pig, and fish species in raw and processed food using species-specific primers targeting mitochondrial cytb, ND5, and 16s rRNA genes. Assay validation of adulterated and various heat-treated meatball matrices showed excellent stability and sensitivity under all processing conditions. The detection limit was 0.01−0.001 ng of DNA under pure states and 0.5% meat in meatball products. Buffalo was detected in 86.7% (13 out of 15) of tested commercial beef products, while chicken, pork, and fish products were found to be pure. The developed assay was efficient enough to detect target species simultaneously, even in highly degraded and processed food products at reduced time.

One‐step triplex high‐resolution melting (HRM) analysis for rapid identification of Atlantic cod (Gadus morhua), Alaska pollock (Gadus chalcogrammus) and haddock (Melanogrammus aeglefinus)

“…Despite the limitation in quantitative analysis (Mohamad et al ., 2013), mitochondrial sequences are still preferentially chosen as the target in lots of qualitative analysis (Fernandes et al ., 2018; Shi et al ., 2020), since they are maternally inherited, evolve faster than other DNA and exist in multiple copies per cell. Additionally, the candidate gene marker should display either low or no intra‐specific variations and have a high inter‐specific variability (Lo & Shaw, 2018; Hossain et al ., 2019). In the present study, after detailed sequence alignment of the target species with different species of various families, the mitochondrial COI, cytb and ND2 gene fragments were selected as the molecular marker for designing PCR primers specific for G. morhua , G. chalcogrammus and M. aeglefinus , respectively (Fig.…”

“…3c,d), which are relatively poorer than the results of simplex PCR (Fig. S1), and previous studies (Hossain et al ., 2019; Shi et al ., 2020; Xiong et al ., 2020b). Although the high sensitivity with PCR‐based techniques has been widely reported, many factors should be considered regarding sensitivity evaluation, such as the used extraction method, DNA degradation and also primer interactions (Trotta et al ., 2005; Böhme et al ., 2019).…”

Standardization of Polymerase Chain Reaction Assay for the Authentication of Arapaima gigas fish

“…The detection levels obtained were 0.01% for the DNAs of rabbit, cow and hare and 0.1% for the DNAS of dear, pork, and red meat. Hossain (2019) researched the presence of cow, buffalo, chicken, cat, dog, swine, and fish in raw and processed foods, obtained a sensitivity threshold of 0.001% for raw food and 0.5% for Heat-Treated Food Products.…”

Detection of meat from horse, donkey and their hybrids (mule/hinny) by duplex real-time fluorescent PCR

“…Additionally, real-time PCR reduces the potential of contamination of the PCR mixture as the reaction tubes remain closed throughout the assay. Research papers from different countries, including the USA [10], Germany [11], Switzerland [12,13], Portugal [14], and Malaysia [15], for the detection of meat origin and adulteration with this method have been published.…”