A rapid and reliable multiplex PCR assay for simultaneous detection of fourteen animal species in two tubes

Highly convenient and highly specific-and-sensitive PCR using Se-atom modified dNTPs

“…Encouraged by the high specificity on simplex PCR with dNTPaSe, we explored multiplex PCR (MP-PCR), a powerful tool for simultaneously detecting many target sequences in one tube. [23][24][25] Our results indicated that using canonical dNTPs, the conventional MP-PCRs still formed many by-products even after the time-consuming optimization ( Fig. 5), while in the presence of dCTPaSe, MP-PCRs conveniently offered clean reactions without significant by-products, indicating the suppression of nonspecific amplifications.…”

A Simple and Reliable Single Tube Septuple PCR Assay for Simultaneous Identification of Seven Meat Species

“…Nowadays, PCR-based techniques are the effective methods for species authentication. Real-time PCR techniques and microchip electrophoresis-dependent multiplex PCR methods require special infrastructures [ 11 , 29 , 30 ]; multiplex PCR assays through simple agarose gel analysis minimizes the cost drastically on a large scale and can be easily carried out to verify the identity of ingredients in foodstuffs [ 6 , 31 , 32 ]. As seen in Table 3 , much is known about multiplex PCR assays that simultaneously verify two to six meat ingredients in one reaction.…”

“…Although one study authenticated 10 animal species (beef, sheep, pork, chicken, turkey, cat, dog, mouse, rat, human), it was achieved by two tube multiplex assays, where every five animal species were verified by a pentaplex PCR assay in one reaction [ 33 ]. Similarly, using two tube independent pentaplex PCR assays with ten pairs of primers, 14 animal species including cattle, donkey, Canidae (dog, fox, raccoon-dog), deer and horse, pig, Ovis (sheep, goat), poultry (chicken, duck), cat and mouse were detected through chip electrophoresis; however, the multiplex PCR failed to accurately distinguish sheep and goat within Ovis, dog, fox and raccoon-dog within Canidae, and chicken and duck within poultry [ 11 ]. Therefore, there is still a lack of more efficient detection methods with low cost for supervising more meat content.…”

“…Notably, there is increasing evidence supporting that meat may trigger allergic reactions, especially for sensitized patients, which may cause a severe health risk of infectious diseases, metabolic disorders and allergies [ 9 ]. In addition, meat adulteration could also violate religious concerns; as is known, meat products containing pork ingredients are not permitted by Kosher and Halal food laws [ 10 , 11 ]. Therefore, it remains a pressing need for identifying meat species with 100% accuracy in real-world foodstuffs.…”

Species substitution in the meat value chain by high-resolution melt analysis of mitochondrial PCR products

“…Adulteration of meat with products from multiple species is increasingly being reported (Ali et al, 2015; Di Pinto et al, 2015; Izadpanah et al, 2018; Kitpipit et al, 2014; O’Mahony, 2013), raising the demand for affordable and faster techniques for their detection. Many studies describing multi-species analysis of vertebrates in meat have utilized multiplex PCR (Ali et al, 2015; Balakrishna et al, 2019; Izadpanah et al, 2018; Kitpipit et al, 2014; Li et al, 2019; Liu et al, 2019; Qin et al, 2019; Wang et al, 2020). While useful, multiplex PCR requires use of expensive probes and post-PCR procedures such as agarose gel electrophoresis for size separation of amplicons and/or DNA sequencing, thereby increasing analysis time, cost, and risk of cross-contamination.…”