Lab-on-a-Chip-Based PCR-RFLP Assay for the Detection of Malayan Box Turtle (Cuora amboinensis) in the Food Chain and Traditional Chinese Medicines.

Asing Asing,Md Abdul Kader,Shuhaimi Mustafa,M A Motalib Hossain,I S M Zaidul,Sharifah Bee Abd Hamid,Md Eaqub Ali,Panagiotis Kalaitzis

doi:10.1371/journal.pone.0163436

Asing Asing, Md Abdul Kader + Show 6 more

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https://doi.org/10.1371/journal.pone.0163436

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Journal: PloS one	Publication Date: Oct 7, 2016
Citations: 18	License type: CC BY 4.0

Affiliation: Universiti Putra Malaysia, University of Malaya

Abstract

The Malayan box turtle (Cuora amboinensis) (MBT) is a vulnerable and protected turtle species, but it is a lucrative item in the illegal wildlife trade because of its great appeal as an exotic food item and in traditional medicine. Although several polymerase chain reaction (PCR) assays to identify MBT by various routes have been documented, their applicability for forensic authentication remains inconclusive due to the long length of the amplicon targets, which are easily broken down by natural decomposition, environmental stresses or physiochemical treatments during food processing. To address this research gap, we developed, for the first time, a species-specific PCR-restriction fragment length polymorphism (RFLP) assay with a very short target length (120 bp) to detect MBT in the food chain; this authentication ensured better security and reliability through molecular fingerprints. The PCR-amplified product was digested with Bfa1 endonuclease, and distinctive restriction fingerprints (72, 43 and 5 bp) for MBT were found upon separation in a microfluidic chip-based automated electrophoresis system, which enhances the resolution of short oligos. The chances of any false negative identifications were eliminated through the use of a universal endogenous control for eukaryotes, and the limit of detection was 0.0001 ng DNA or 0.01% of the meat under admixed states. Finally, the optimized PCR-RFLP assay was validated for the screening of raw and processed commercial meatballs, burgers and frankfurters, which are very popular in most countries. The optimized PCR-RFLP assay was further used to screen MBT materials in 153 traditional Chinese medicines of 17 different brands and 62 of them were found MBT positive; wherein the ingredients were not declared in product labels. Overall, the novel assay demonstrated sufficient merit for use in any forensic and/or archaeological authentication of MBT, even under a state of decomposition.

Highlights

Farm-to-fork food safety and quality has long been a goal, but to ensure it, both regulatory and market monitoring measures must be transparent across the globe [1]
Stray dog (Canis lupus familiaris), cat (Felis catus) and rat (Rattus rattus) muscle meats were donated by Kuala Lumpur City Hall (KLCH) or (Dewan Bandaraya Kuala Lumpur (DBKL)), Air Panas, Kuala Lumpur and monkey (Macaca fascicularis sp) meat was a gift from the Department of Wildlife and National Park Malaysia (PERHILITAN/DWNPM), Cheras, Kuala Lumpur
The Malayan box turtle (Cuora amboinensis) is one of 18 native freshwater turtle and tortoise species in Malaysia, and despite being protected, it has been overexploited for its various organs, such shells and bones, which are believed to have healing properties for use in analgesic, antipyretic and invigorating traditional medicines

Summary

Introduction

Farm-to-fork food safety and quality has long been a goal, but to ensure it, both regulatory and market monitoring measures must be transparent across the globe [1]. The recent entry of some alien species, such as rat meat, into the food chain [3] is highly alarming for public health, religious faith and the fair-trade economy, and the illegal trade of certain wild and endangered species especially threaten biodiversity, ecology and food safety [4]. The belief in certain purported health benefits such as the distinctive flavor, high protein content, low fat and cholesterol contents and the absence of health-threatening anabolic steroids in bush meat have continued to encourage the overhunting of wild species [5, 6]. According to the Convention on International Trade in Endangered Species (CITES) of Wild Fauna and Flora, the value of the illegal trade in wildlife was US $5–20 billion per year in 2007[9], and expert reports on international and internal security and illicit economies have revealed that approximately US $8–10 billion of the annual trade in protected species happens in Southeast Asia alone [10, 11]

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