Detection of meat from horse, donkey and their hybrids (mule/hinny) by duplex real-time fluorescent PCR

Abstract

Meat adulteration is currently a common practice worldwide. In China, adulteration of donkey meat products with the similar species (horse and mule/hinny) meat and mislabeling are becoming widespread concerns. In this study, a sensitive and species-specific duplex real-time PCR assay based on the simultaneous amplification of fragments of the creatine kinase muscle gene family, was developed and optimized for the identification of horse, donkey and mule /hinny species in raw and heat-processed meat products. Duplex real-time PCR results showed different fluorescence amplification curves for horse and donkey. Both kinds of fluorescence amplification curves appeared simultaneously for mule/hinny. The limit of detection (LOD) of the method was up to 0.01 ng /μL. The method and strategy developed in this study could be applied to detect the presence of adulterants from horse and mule /hinny meat in raw donkey meat and meat products.

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

“…Thus, Druml et al (2016) tested their reference RT-PCR assay for relative quantification of game meat on raw and cooked deer sausage, whereas the PCR-RFLP assay developed by Asing et al (2016b) successfully amplified Malaysian box turtle products of all adulteration levels, including 1% (w/w) and autoclaved samples. Several other authors validated their assays to test their robustness and performance in the field (Kim et al, 2020;Wang et al, 2020;Wang, Hang, et al, 2019), to verify the authenticity of wildlife products offered for sale (quality control and traceability) (Szemethy et al, 2021), to ensure the submission of a credible analytical test report for forensic or law enforcement purposes (Vaithiyanathan et al, 2021), to ensure the efficiency of the method and its suitability for daily use or routine laboratory work (Iwobi et al, 2017;Szemethy et al, 2021), and to detect wildlife trafficking (Ali et al, 2017).…”

Meta-analysis data of the accuracy of tests for meat adulteration by real-time PCR

“… General study details LOD Specificity # Authors Target species Method Gene Number of samples Сt ± SD Concentration [ng/μL] Number of samples Сt of target species False-positive True-positive False-negative True-negative 1a Wang et al. [4] horse duplex qPCR creatine kinase muscle (MCK) 90 36 0.01 21 22 0 3 0 18 1b Wang et al. [4] donkey duplex RT PCR creatine kinase muscle (MCK) 90 38 0.01 21 24 0 3 0 18 3 Li et al.…”

“… [4] horse duplex qPCR creatine kinase muscle (MCK) 90 36 0.01 21 22 0 3 0 18 1b Wang et al. [4] donkey duplex RT PCR creatine kinase muscle (MCK) 90 38 0.01 21 24 0 3 0 18 3 Li et al. [5] mutton qPCR housekeeping gene replication protein A1 (RPA1) 18 29.91±0.00 0.5 6 26 0 1 0 5 74 Al-Kahtani et al.…”