Development and validation of short-amplicon length PCR assay for macaques meat detection under complex matrices

ABSTRACT

The macaque (Macaca fascicularis) monkeys are the third largest primate population in tropical forests. Being the potential carrier of Simian Immunodeficiency and Ebola and Corona viruses, as well as being a religiously-protected and wildlife-protected species could not save the macaques from being over-hunted and consumed. Despite having needs, methods to detect monkey species in foods are rarely documented. To fill this gap, this article describes a monkey-specific polymerase chain reaction assay targeting a short-site (120 bp) of mitochondrial d-loop gene because the short-length targets are thermodynamically more stable than the longer ones under degrading states. Specificity was tested against 17 terrestrial and aquatic meat and fish species and no cross-species amplification was detected under raw, processed, and admixed states. The sensitivity of the assay was 0.0001 ng DNA under pure states and 0.1% monkey meats in binary meat mixtures. Finally, the assay was validated by digesting the polymerase chain reaction products with AluI and CViKI-1 and distinctive restriction fingerprints for macaque species were demonstrated.

KEYWORDS:

• Macaca fascicularis
• Short-amplicon-length PCR assay
• Mitochondrial d-loop gene
• Religious and wildlife restriction
• Restriction fingerprints

Acknowledgment

The authors are thankful to the Department of Wildlife and National Park Malaysia for the donation of macaque meat-tissue samples for conducting this study.

Funding

This research was supported by the University of Malaya fund PPP fund no. PG0004-2013B and GC001A-14SBS to M. E. Ali.

Additional information

Funding

This research was supported by the University of Malaya fund PPP fund no. PG0004-2013B and GC001A-14SBS to M. E. Ali.

Notes on contributors

Md. Eaqub Ali

All authors declare no conflict of interest in publishing this article. Ethical clearance of ref. No: NANOCAT /17/07/3013/NRAR (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.

Nur Raifana Abdul Rashid

All authors declare no conflict of interest in publishing this article. Ethical clearance of ref. No: NANOCAT /17/07/3013/NRAR (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.

Sharifah Bee Abd Hamid

All authors declare no conflict of interest in publishing this article. Ethical clearance of ref. No: NANOCAT /17/07/3013/NRAR (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.

S. M. Azad Hossain

All authors declare no conflict of interest in publishing this article. Ethical clearance of ref. No: NANOCAT /17/07/3013/NRAR (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.

Asing Asing

All authors declare no conflict of interest in publishing this article. Ethical clearance of ref. No: NANOCAT /17/07/3013/NRAR (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.

M. A. Motalib Hossain

All authors declare no conflict of interest in publishing this article. Ethical clearance of ref. No: NANOCAT /17/07/3013/NRAR (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.

I. S. M. Zaidul

All authors declare no conflict of interest in publishing this article. Ethical clearance of ref. No: NANOCAT /17/07/3013/NRAR (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.