Application of real-time polymerase chain reaction using species specific primer targeting on mitochondrial cytochrome-b gene for analysis of pork in meatball products

Abstract

Objective: This study aimed to design specific primers derived from mitochondrial cytb of Sus Scrofa (1F1R primer) used in the pork meatball analysis using real time polymerase chain reaction (RT-PCR) method. Materials and Methods: Such designed primers were validated and these included specificity of primer, linearity, and sensitivity of the method as well as the repeatability test. The primers were specifically affirmed in the fresh tissue of chickens, cows, pigs, and goats. The linearity and sensitivity of the method was conducted by measuring the amplification curve from a series of dilution (0, 1, 1, 10, 100, 1,000, and 10,000 pg/μl of DNA) extracted from 100% pork meatball formulation. The repeatability test was conducted by determining the cycle threshold ( Ct ) values of RT-PCR amplification from 100% pork meatball formulation as many as six times. Results: Primer of 1F1R (forward: 5′-ACG CGA TAT AAG CAG GTA AA-3′; reverse: 5′-CTG CTT TCG TAG CAC GTA TT-3′) was specific in analyzing the presence of pork in meatball formulation at 47.1°C, which was optimum annealing temperature. The DNA identification was able to use the primers by RT-PCR with 1 pg as the limit of detection, efficiency value was 242.58%, and the coefficient of determination value ( R 2 ) was 0.956. The coefficient of variance was 4.13%. The developed method was also fruitfully applied to analyze commercial meatballs. Conclusion: RT-PCR method using specific primers targeting on mitochondrial gene (1F1R primer) could be used as the standard method for identification of pork in food samples intended for halal authentication studies.

Real-Time Polymerase Chain Reaction: Current Techniques, Applications, and Role in COVID-19 Diagnosis

“…Furthermore, the technique has been employed for the detection of pork in meat-based food products by using specific primers targeting the mitochondrial cytochrome-b gene. Notably, pork is considered non-halal (prohibited from eating according to Islamic law) for Muslim communities, and therefore accurate labeling of meat-based products is essential [ 35 ].…”

Analytical approaches of meat authentication in food

“…Mitochondrial cytB primers for porcine species have been designed for detection of porcine DNA in beef adulterated pork meatball utilising SYBR as detection chemistry in qPCR. Detection limit was recorded as low as 1 pg with R 2 of 0.956 (Orbayinah et al ., 2019). In addition, the use of highly sensitive and specific EvaGreen dye permits detection as low as 0.1 pg DNA in raw and heat treated in beef adulterated horse meat model (Meira et al ., 2017).…”

Combination of Loop-Mediated Isothermal Amplification and AuNP-Oligoprobe Colourimetric Assay for Pork Authentication in Processed Meat Products

“…The LAMP-AuNPprobe assay, however, has its limitations as it delivers qualitative information and is less sensitive when compared with the quantitative PCR techniques (Table 2). Real-time PCR (RT-PCR) and digital PCR (dPCR) are the two PCR-based methods that can provide quantitative data with high specificity and sensitivity (Ballin et al 2009;Cai et al 2017;Farrokhi and Joozani 2011;Floren et al 2015;Orbayinah et al 2019;Rodríguez et al 2005;Shehata et al 2017). Nevertheless, both RT-PCR and dPCR require relatively expensive reagents as well as special devices to perform the PCR reactions, which may not be affordable for laboratories with limited budgets (Ali et al 2012;Basu 2017).…”