Establishment and Validation of a Multiplex PCR Detection System for the Identification of Six Common Edible Meat Components

doi:10.12116/j.issn.1004-5619.2023.531002

Abstract

Abstract:

Objective To establish a rapid， accurate， and sensitive multiplex PCR detection method for the simultaneous identification of the six common edible meats （beef， lamp， chicken， pork， goose， duck）， and to evaluate its application value in meat adulteration identification. Methods Based on complete mitochondrial genomic sequences of six species in the GenBank database， DNA sequences （cattle： 16S rRNA; sheep： COX-1; chickens： Cytb; pig： COX-1; goose： NADH2; duck： 16S rRNA） with intra-species conservation and inter-species specificity were screened， and species-specific primers were designed to construct a multiplex PCR detection system that can simultaneously detect the meat of six common species. The species specificity， sensitivity and reproducibility of the system were studied, and the simulated mixture sample detection was performed. Results This study successfully constructed a multiplex PCR detection system that can detect the meats of six common species simultaneously. The system was not effective in DNA amplification of non-target species. When the DNA template sizes were 0.062 5-2 ng/μL， the amplified products of all six species could be detected. The duck component was still detected when the mixing ratio of duck and beef was as low as 0.5%. Conclusion This study constructs and establishes a multiplex PCR detection system with strong specificity， high sensitivity， and good reproducibility. It can accurately identify the components of animal origin in common edible meats and provide a simple and practical method for identifying adulteration of common edible meats and meat products in China.

Key words: forensic genetics, mitochondrial DNA, multiplex PCR, species identification, edible meat

CLC Number:

Zhi-wei JIANG, Ruo-cheng XIA, Rui-yang TAO, Cheng-tao LI. Establishment and Validation of a Multiplex PCR Detection System for the Identification of Six Common Edible Meat Components[J]. Journal of Forensic Medicine, 2024, 40(3): 254-260.

Figures/Tables 4

References 29

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物种	目标基因	引物序列（5´→3´）	扩增长度/bp	引物浓度/（μmol·L^-1）
牛	16S rRNA	正向：ACACAGGAGTGCATCTAAGG	104	0.40
牛	16S rRNA	反向：AATACTGGGAATGCTGGAGG	104	0.40
羊	COX-1^[28]	正向：CACGACGATACTCTGATTAC	157	0.40
	COX-1^[28]	反向：GTGGTTAGGTCTACAGTTAG	157	0.40
鸡	Cytb	正向：ACACTTGCCGGAACGTACAA	205	0.16
鸡	Cytb	反向：CCCCATGGGAGAACATAGCC	205	0.16
猪	COX-1^[28]	正向：CGGGTACACACTCAACCAAG	268	0.16
猪	COX-1^[28]	反向：TGTGCTTGTCAGTTCTACTGC	268	0.16
鹅	NADH2	正向：TGCTACTCTCGACCCTCAT	313	0.32
鹅	NADH2	反向：GTTGTGATCATGGATAGGTTAAGG	313	0.32
鸭	16S rRNA	正向：CTAGCTCAGCCGCTTAAACA	495	0.56
鸭	16S rRNA	反向：TCCACTAGTCGAGGTTGTGT	495	0.56

物种	目标基因	引物序列（5´→3´）	扩增长度/bp	引物浓度/（μmol·L^-1）
牛	16S rRNA	正向：ACACAGGAGTGCATCTAAGG	104	0.40
牛	16S rRNA	反向：AATACTGGGAATGCTGGAGG	104	0.40
羊	COX-1^[28]	正向：CACGACGATACTCTGATTAC	157	0.40
	COX-1^[28]	反向：GTGGTTAGGTCTACAGTTAG	157	0.40
鸡	Cytb	正向：ACACTTGCCGGAACGTACAA	205	0.16
鸡	Cytb	反向：CCCCATGGGAGAACATAGCC	205	0.16
猪	COX-1^[28]	正向：CGGGTACACACTCAACCAAG	268	0.16
猪	COX-1^[28]	反向：TGTGCTTGTCAGTTCTACTGC	268	0.16
鹅	NADH2	正向：TGCTACTCTCGACCCTCAT	313	0.32
鹅	NADH2	反向：GTTGTGATCATGGATAGGTTAAGG	313	0.32
鸭	16S rRNA	正向：CTAGCTCAGCCGCTTAAACA	495	0.56
鸭	16S rRNA	反向：TCCACTAGTCGAGGTTGTGT	495	0.56

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