Development and validation of heptaplex polymerase chain reaction assay for the discriminatory detection of selected meats in food products / Syed Muhammad Kamal Uddin

Syed Muhammad , Kamal Uddin (2022) Development and validation of heptaplex polymerase chain reaction assay for the discriminatory detection of selected meats in food products / Syed Muhammad Kamal Uddin. PhD thesis, Universiti Malaya.

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Abstract

Food fraud is one of the most prevalent problems of the present day. It is important for consumers to ascertain the authenticity of the declared ingredients in food products. Beef, buffalo, goat, sheep, chicken, duck, and pork are the heavily consumed meats having enormous importance from nutritional, economic, and cultural/religious viewpoints and are often found to be mutually adulterated in raw and processed states. Available DNA-based approaches for species authentication are commonly based on long DNA markers that can be damaged during food processing, rendering the methods less reliable. The objective of the project is to develop a heptaplex Polymerase chain reaction (PCR) assay targeting short length amplicons for the detection and differentiation of bovine, buffalo, goat, sheep, chickens, ducks, and porcine materials in food chain simultaneously. Both conventional and real-time PCR systems were developed, and authentic target detection was ensured through sequencing and Restriction Fragment Length Polymorphism (RFLP) analysis. Mitochondrial cytochrome b (cytb) and NADH dehydrogenase subunit 5 (ND5) genes were targeted and seven different targets (73-263 bp length) each for pig (73 bp), cow (106 bp), buffalo (138 bp), chicken (161 bp), duck (203 bp), goat (236 bp) and sheep (263 bp) were amplified from raw as well as boiled, microwaved, and autoclaved meats under pure and admixed states. The specificity of the PCR assays was tested against seven target species and other related 19 non-targets. Specific PCR products were obtained only from the targeted cow, buffalo, goat, sheep, chicken, duck, and pig without any cross-species amplification. The use of universal eukaryotic primers eliminated any chance of false-negative detection. The assay was sensitive enough to detect 0.01-0.005 ng of DNA from raw meat and 0.5% (w/w) adulterated meat under mixed and commercial matrices. The amplified PCR products from the targets showed more than 98% (98.5-100%) sequence similarity with specific target sequences in GenBank. The PCR products were digested by the restriction enzymes namely FatI, BfaI and HPY188I that confirmed the authentic molecular fingerprints from the seven target species. The novel methods were applied to screen various commercially processed foods, namely meatballs, frankfurters, burgers and meat curries. A market survey revealed that 100% of beef meatballs were adulterated with buffalo along with total beef replacement in 20% of cases. Beef frankfurters and burgers were adulterated with buffalo in 84% cases. Beef curry samples contained 90% buffalo contamination. In contrast, porcine products were found to be 100% authentic and no porcine was detected in the halal branded foods. Finally, the developed TaqMan probe-based multiplex real-time PCR (mqPCR) systems successfully detected 0.006 ng DNA in raw state and 1% adulterated meat in mixed and commercial matrices. Screening of commercial products by mqPCR assays showed that 85% of beef burgers and 100% of beef frankfurters, meatballs and cocktails were adulterated with buffalo. Lamb products were also buffalo contaminated. Pork products contained chicken (50%). Given some advantageous features, including stringent specificity, exceptional stability and sensitivity, the developed approach could discriminatorily detect and quantify the target species even in severely processed specimens.

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