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

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.

Heptaplex Polymerase Chain Reaction Assay for the Simultaneous Detection of Beef, Buffalo, Chicken, Cat, Dog, Pork, and Fish in Raw and Heat-Treated Food Products

“…Total DNA extraction was done from meat and fish samples by utilizing the Yeastern Genomic DNA Mini Kit (Yeastern Biotech Co., Ltd., Taipei, Taiwan) and adhering to the manufacturer’s instructions . Approximately 20 mg of muscle tissue from each sample was pounded and homogenized in an Eppendorf tube by a micropestle.…”

A systematic review of DNA‐based methods in authentication of game and less common meat species

“…Publicly available web domains provide online tools for researchers to perform in silico analyses to determine species-specific endonuclease and endonuclease sites for their targets (Ali et al, 2017). To detect Malaysian box turtle in food and traditional Chinese medicines, Asing et al (2016b) used the PCR-RFLP technique to restrict the 120 base pair (bp) long product of the cyt b gene with the Bfa1 endonuclease. According to them, the result of their RFLP assay is highly reliable and useful for legal and forensic applications.…”

“…In primer design, one way to ensure product specificity is the presence of base mismatches at the 3′ end of primers (Zhao, Xu, et al, 2019a). The following are some of the methods the authors used to ensure specificity in their experiments: (1) close observation of primer and product sequences using the multiple sequence alignment software-CLC genomic workbench (Druml et al, 2016); (2) use of the online tool NCBI-BLAST to access regions of similarity and dissimilarity, sequence alignment using ClustalW and MEGA software, and construction of a phylogenetic tree; and (3) in vitro laboratory testing of primers with target and nontarget species (Asing et al, 2016a;Asing et al, 2016b). Meira et al (2017) used the NCBI primer-BLAST tool to check their primer specificity in silico; Zhao, Xu, et al (2019a) intentionally added mismatches at the penultimate base at the 3′ end of their primer to improve its specificity.…”

Double gene targeting multiplex PCR-RFLP detects Crocodylus porosus in chicken meatball and traditional medicine

“…Then, these model chicken meatball products were tested under raw and heat treated conditions to evaluate the reliability and accuracy of the method to demonstrate the common forms of adulteration in the commercial food industry. [35] Additionally, the 10% crocodile-adulterated model chicken meatball products were subjected to boiling at 98°C for 90 min, autoclaving for 120°C at 2.5 h under 45 psi and microwave oven cooking at 700 W for 30 min because these treatments were known to break down DNA (Asing et al, 2016;Hossain et al, 2016). To the best of our knowledge, no other studies have tested the double gene mPCR assay for crocodile under food processing conditions such as boiling, extreme autoclaving (2.5 h) and microwave cooking (700 W for 30 min).…”