title: 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.
authors.affiliation: Nanotechnology and Catalysis Research Center (NANOCAT), University of Malaya, Kuala Lumpur, Malaysia.
authors.lastname: Asing
authors.affiliation: Nanotechnology and Catalysis Research Center (NANOCAT), University of Malaya, Kuala Lumpur, Malaysia.|Centre for Research in Biotechnology for Agriculture (CEBAR), University of Malaya, Kuala Lumpur, Malaysia.
authors.forename: Md Eaqub
authors.initials: ME
authors.lastname: Ali
authors.affiliation: Nanotechnology and Catalysis Research Center (NANOCAT), University of Malaya, Kuala Lumpur, Malaysia.
authors.forename: Sharifah Bee
authors.initials: SB
authors.lastname: Abd Hamid
authors.affiliation: Nanotechnology and Catalysis Research Center (NANOCAT), University of Malaya, Kuala Lumpur, Malaysia.
authors.forename: M A Motalib
authors.initials: MA
authors.lastname: Hossain
authors.affiliation: Institute of Halal Products Research, University of Putra Malaysia, UPM Serdang, Selangor, Malaysia.
authors.forename: Shuhaimi
authors.initials: S
authors.lastname: Mustafa
authors.affiliation: School of Aquaculture and Fisheries, University of Malaysia Terrenganu, Kuala Terrenganu, Terrenganu, Malaysia.
authors.forename: Md Abdul
authors.initials: MA
authors.lastname: Kader
authors.affiliation: Department of Pharmaceutical Technology, Faculty of Pharmacy, International Islamic University, Kuantan, Pahang, Malaysia.
authors.forename: I S M
authors.initials: IS
authors.lastname: Zaidul
mesh_terms: D000818:Animals; D001681:Biological Assay / Q000379:methods; D004247:DNA / Q000235:genetics; D020387:Food Chain; D005506:Food Contamination / Q000032:analysis; D005511:Food Handling / Q000379:methods; D056656:Lab-On-A-Chip Devices; D008461:Meat Products / Q000032:analysis*; D008516:Medicine, Chinese Traditional / Q000379:methods; D016133:Polymerase Chain Reaction / Q000379:methods*; D012150:Polymorphism, Restriction Fragment Length / Q000235:genetics*; D015203:Reproducibility of Results; D013045:Species Specificity; D014426:Turtles / Q000235:genetics*
chemical_list: D004247:DNA