Simultaneous identification of animal-derived components in meats using high-throughput sequencing in combination with a custom-built mitochondrial genome database

Yinan Zhang,Mingzhen Rao,Yu Zhao,Qinfeng Qu,Fei Tao,Nana Zhang

doi:10.1038/s41598-020-65724-4

Abstract

Currently, the inspection and supervision of animal ingredients relies primarily upon specific amplification-dependent methods, whose efficiency and accuracy are being seriously challenged by the increasing diversity and complexity of meat products. High-throughput sequencing (HTS) technology was employed to develop an alternative method to detect animal-derived ingredients in meat products. A custom-built database containing 2,354 complete mitochondrial genomic sequences from animals, an identification analysis pipeline based on short-sequence alignment, and a web-based server were built to facilitate this detection. The entire process, including DNA extraction, gene amplification, and sequencing, was established and optimized for both marker gene (part of the CYTB gene)-based detection and total DNA-based detection. Using simulated samples containing various levels of pig, cattle, sheep, chicken, rabbit, and mice ingredients, the detection capability and accuracy of this method were investigated. The results of this study indicated that the method is capable of detecting animal components in meats that are present at levels as low as 1%. Our method was then tested using 28 batches of real meat products such as raw meat slices, raw meat mince, cooked dried meat, cooked meat sausage, and other supermarket samples, with a traditional qPCR method as the control. The results demonstrated an accuracy of 97.65% for the qualitative detection method, which indicate that the developed method is reliable for the detection of animal components. The method is also effective for the identification of unknown food samples containing mixed animal components, which suggests a good future in application.

Highlights

The inspection and supervision of animal ingredients relies primarily upon specific amplification-dependent methods, whose efficiency and accuracy are being seriously challenged by the increasing diversity and complexity of meat products
The CYTB gene is a typical mitochondria gene that contains phylogenetic information extending from the intraspecific level to the intergeneric level[8], and it has been used as a common target gene for meat component identification in a number of studies
The primer pairs of CB1-5 and CB3A were more universal than the other two primer pairs, as a single band of approximately 385 bp was amplified from chicken, sheep, pig, rat, cattle, and rabbit genomes using the CYTB gene derived primer pairs (Fig. 1)

Summary

Introduction

The inspection and supervision of animal ingredients relies primarily upon specific amplification-dependent methods, whose efficiency and accuracy are being seriously challenged by the increasing diversity and complexity of meat products. With the development of high-throughput sequencing, the DNA meta-barcoding approach has emerged as a promising method for the identification of unknown and multiple species[3]. This method has been investigated extensively for the identification of organisms in the environment[4], characterization of traditional Chinese medicines[5], diet assessment[6], and fisheries applications[7]. The D-loop region, COI gene, and rRNA genes are good targets for species identification, and they have been reported to be effective for food identification[14,15,16] These studies presented the advantages and potential of the identification methods that are based on mitochondrial genetic information

Methods

Results

Conclusion