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

Abstract

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.

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

“…Oligonucleotide primers specific to the MBT cytochrome b gene sequence were designed following a standardized procedure published in our earlier report [ 26 ]. The designed primers were Forward-5′-AGCCCTTCTAACATCTCTGCTC-3′, and Reverse-5′—CTCACCAGACATCTCACTAGCA-3′, which targeted a 120-bp site of the cyt b gene, and their specificity was ensured by three different testing systems [ 50 ].…”

“…To simulate the complexity of the matrices, two types of binary mixtures, i.e., MBT-beef and MBT-goat, were made in a total volume of 100 g of specimen by mixing MBT meat in a proportion of 10%, 1%, 0.1% and 0.01% with an adjusted amount of beef and goat meat[ 26 , 39 , 52 ]. A ternary admixture composed of MBT meat, chicken and wheat flour was made by mixing 10%, 1%, 0.1% and 0.01% of turtle meat into chicken and wheat flour, in which the MBT:chicken:wheat flower contribution was 20:80:100, 2:98:100, 0.2:99.8:100 and 0.02:99.98:100, respectively[ 52 ].…”

“…Total DNA was extracted from 30 mg of muscle tissue of each species (21 species) and the admixed and commercial meat products using a Yeastern Genomic DNA Mini Kit (Yeastern Biotech Co., Ltd. Taipei, Taiwan), and plant DNA was extracted following Ma et al (2000). The purity and concentration of all of the extracted DNA was determined using a UV-VIS spectrophotometer (Biochrom Libra S70, Biochrom Ltd, Cambridge, UK) based on absorbance at A260/280 and calculating the ratios [ 26 ]. All of the extracted DNA was kept at -20°C until further use.…”

“…), parasites (such as Spirometra, Trichinella, Gnathostoma, and Pentastomids), and various type of biotoxins such as lyngbyatoxins, cyanotoxins, cytotoxins, haemotoxins, mycotoxins, and neurotoxins [ 25 ]. Furthermore, it is a sensitive social and religious issue because the consumption of turtle-derived materials is prohibited by certain religions such as Islam [ 26 ]. Therefore, there is a definite need for a reliable authentication method to detect turtle species in the food chain and traditional multicure medicinal products; we believe this would either prevent or reduce illegal trade and the chance of adulteration by or substitution of turtle ingredients in common food, thus mitigating the health hazards caused by turtle-borne zoonoses and allergens[ 11 ].…”

“…Recently, species-specific PCR and PCR–DNA sequencing[ 20 ], PCR-RFLP[ 49 ], and quantitative PCR assays[ 35 ], have been used in the detection of MBT ( C . amboinensis ) and other turtle species, but most of these assays are based on longer-targets that break down during food processing[ 26 , 39 ]. Here, for the first time, we described a very short-amplicon-length (120 bp) PCR-RFLP assay targeting the cytochrome b (cytb) gene for the confirmed identification of MBT in the food chain; the digestion of the PCR product (120 bp) by Bf1 restriction endonuclease produced distinctive fingerprints (72 bp, 43 bp and 5 bp fragments) for MBT ( Fig 1 ).…”