Multiplex PCR assay discriminates rabbit, rat and squirrel meat in food chain

ABSTRACT

Rabbit meat is receiving increasing attention because it contains a high level of proteins with relatively little fat. On the other hand, squirrel meat is served in upper-class meals in certain countries, so is sold at higher prices. The other side of the coin is rat meat, which has family ties with rabbit and squirrel but poses substantial threats to public health because it is a potential carrier of several zoonotic organisms. Recently, rat meat was mislabelled and sold as lamb after chemical modification. Thus, the chances of rabbit and squirrel meat substitution by rat meat cannot be ruled out. For the first time, a multiplex PCR assay was developed in Malaysia for the discriminatory identification of rat, rabbit and squirrel in the food chain. Rabbit (123 bp), rat (108 bp) and squirrel (243 bp) targets were amplified from ATP6 and cytb genes, along with a eukaryotic internal control (141bp). The products were sequenced and cross-tested against 22 species. A total of 81 reference samples and 72 meatball specimens were screened to validate the assay. Analyte stability was evaluated through boiling, autoclaving and micro-oven cooking. The tested lower limits of detection were 0.01 ng DNA for pure meat and 0.1% for meatballs.

Graphical Abstract

KEYWORDS:

• Multiplex PCR
• internal control
• analyte stability
• species identification
• meat substitution

Acknowledgements

The authors would like to thank to Dewan Bandaraya, Kuala Lumpur and Wildlife and National Parks, Malaysia for providing cat, dog, rat and monkey meat samples.

Disclosure statement

No potential conflict of interest was reported by the authors.

Compliance with Ethics requirements

Ethical clearance of ref. No: NANOCAT/23/07/2013/A(R) was obtained from the Institutional Animal Care and Use Committee, University of Malaya (UM IACUC) and all experiments were conducted following the national and institutional guideline while handling animal meats used in this study.

Supplemental data

Supplemental data for this article can be accessed here.

Additional information

Funding

This study was supported by the University of Malaya Grant No. GC001A-14SBS and PG288-2016A to M. E. Ali.