Short targeting multiplex PCR assay to detect and discriminate beef, buffalo, chicken, duck, goat, sheep and pork DNA in food products

ABSTRACT

Food fraud is a global problem raising increased concerns during the past decades and food authenticity is now a burning issue. Beef, buffalo, chicken, duck, goat, sheep, and pork are heavily consumed meats bearing nutritional, economic and cultural/religious importance and are often found to be adulterated in raw and processed states. To authenticate these species, we developed and validated a highly specific multiplex (heptaplex) PCR assay targeting short length amplicons (73–263 bp) using seven pairs of species-specific primer sets targeting mitochondrial cytochrome b (cytb) and NADH dehydrogenase subunit 5 (ND5) genes. Specificity checking (in silico and in vitro) against 25 non-target species revealed no cross-species amplification. The developed multiplex assay was validated with various adulterated and heat-treated (boiled, microwaved and autoclaved) meatball products and were found to show high sensitivity and stability under all processing conditions. The assay was sensitive enough to detect 0.01–0.005 ng of DNA from raw meat and 0.5% (w/w) adulterated meat in mixed matrices. A market survey revealed mislabelling of 95% beef and 15% chicken products while pork products were found pure. Given some advantageous features including short sizes of amplicons, exceptional stability and superior sensitivity, the developed assay could be conveniently used for discriminatory detection of target species with a variety of raw meat as well as processed meat products undergoing extreme processing treatments.

GRAPHICAL ABSTRACT

KEYWORDS:

• Multiplex PCR
• adulteration
• short amplicon
• authentication
• meatballs
• food products

Acknowledgments

This work was supported by University of Malaya Research Grant No. ST017-2020 and RU001-2020. The authors acknowledge Wildlife Malaysia and Dewan Bandaraya, Kuala Lumpur (DBKL) for providing monkey, dog and cat meat samples.

Disclosure statement

No potential conflict of interest was reported by the author(s).

Ethical compliance

The ethical permission was obtained from the University of Malaya Ethical Committee and all the animals and meats used in this study were handled following the institutional and national guideline for animal care.

Supplementary material

Supplemental data for this article can be accessed on the publisher’s website.

Additional information

Funding

This work was supported by the Universiti Malaya [ST017-2020 and RU001-2020].