A suitable method to detect potential fraud of bringing Malayan box turtle ( Cuora amboinensis ) meat into the food chainby Md. Eaqub Ali, Asing, Sharifah Bee Abd Hamid, Md. Abdur Razzak, Nur Raifana Abd Rashid, Md. Al Amin, Shuhaimi Mustafa

Food Additives & Contaminants: Part A

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A suitable method to detect potential fraud of bringing

Malayan box turtle (Cuora amboinensis) meat into the food chain

Md. Eaqub Aliab, Asinga, Sharifah Bee Abd Hamida, Md. Abdur Razzaka, Nur Raifana Abd

Rashida, Md. Al Amina & Shuhaimi Mustafac a Nanotechnology and Catalysis Research Center (NANOCAT), University of Malaya, Kuala

Lumpur 50603, Malaysia b Centre for Research in Biotechnology for Agriculture (CEBAR), University of Malaya, Kuala

Lumpur 50603, Malaysia c Institute of Halal Products Research, University Putra Malaysia, 43400 UPM Serdang,

Selangor, Malaysia

Accepted author version posted online: 11 Jun 2015.Published online: 10 Jul 2015.

To cite this article: Md. Eaqub Ali, Asing, Sharifah Bee Abd Hamid, Md. Abdur Razzak, Nur Raifana Abd Rashid, Md. Al Amin & Shuhaimi Mustafa (2015) A suitable method to detect potential fraud of bringing Malayan box turtle (Cuora amboinensis) meat into the food chain, Food Additives & Contaminants: Part A, 32:8, 1223-1233, DOI: 10.1080/19440049.2015.1058535

To link to this article: http://dx.doi.org/10.1080/19440049.2015.1058535

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A suitable method to detect potential fraud of bringing Malayan box turtle (Cuora amboinensis) meat into the food chain

Md. Eaqub Alia,b*, Asinga, Sharifah Bee Abd Hamida, Md. Abdur Razzaka, Nur Raifana Abd Rashida, Md. Al Amina and Shuhaimi Mustafac aNanotechnology and Catalysis Research Center (NANOCAT), University of Malaya, Kuala Lumpur 50603, Malaysia; bCentre for

Research in Biotechnology for Agriculture (CEBAR), University of Malaya, Kuala Lumpur 50603, Malaysia; cInstitute of Halal Products

Research, University Putra Malaysia, 43400 UPM Serdang, Selangor, Malaysia (Received 31 March 2015; accepted 31 May 2015)

Malayan box turtle (Cuora amboinensis) has been a wildlife-protected vulnerable turtle species in Malaysia since 2005.

However, because of its purported usage in traditional medicine, tonic foods and feeds, clandestine black market trade is rampant. Several polymerase chain reaction (PCR) assays for the taxonomic detection and classification of turtle species have been proposed. These assays are based on long-length target amplicons which are assumed to break down under compromised states and, hence, might not be suitable for the forensic tracing and tracking of turtle trafficking. For the first time this paper develops a very short-amplicon-length PCR assay (120 bp) for the detection of Malayan box turtle meat in raw, processed and mixed matrices, and experimental evidence is produced that such an assay is not only more stable and reliable but also more sensitive than those previously published. We checked the assay specificity against 20 different species and no cross-species detection was observed. The possibility of any false-negative detection was eliminated by a universal endogenous control for eukaryotes. The assay detection limit was 0.0001 ng of box turtle DNA from pure meat and 0.01% turtle meat in binary and ternary admixtures and commercial meatballs. Superior target stability and sensitivity under extreme treatments of boiling, autoclaving and microwave cooking suggested that this newly developed assay would be suitable for any forensic and/or archaeological identification of Malayan box turtle species, even in severely degraded specimens. Further, in silico studies indicated that the assay has the potential to be used as a universal probe for the detection of nine Cuora species, all of which are critically endangered.

Keywords: Malayan box turtle; wildlife protection; short-amplicon-length PCR assay; endogenous control; forensic and archaeological identification

Introduction

Authentication of ingredients in foods, medicines and consumer products is an undeniable need in the perspectives of health, religion, fair trade and wildlife conservation (Fajardo et al. 2010; Triantafyllidis et al. 2010;

Amaral et al. 2014; Cheng et al. 2014). Some recent events, such as horse meat adulteration in beef burgers and school meals in Europe (Premanandh 2013), and rat and pork meat selling as lamb and mutton in China (Ali et al. 2014a), reflect a shivering diversion on human ethics and have raised consumers? doubts about labelled foods, medicine and their ingredients. Despite an international embargo, illegal trades of certain endangered species have been widespread at both national and international levels (Schoppe 2008a). According to the International