A multiplex PCR method for detection of five animal species in processed meat products using novel species-specific nuclear DNA sequences


The continuous development of fast and simple new methods to identify animal-derived ingredients is very important for the authentication of meat products. This study intended to develop a multiplex PCR method using new species-specific nuclear DNA (nDNA) sequences for the detection of ingredients derived from sheep/goat, bovine, chicken, duck and pig in meat products. Sequence alignment analysis in 53 species showed high specificity of species-specific nDNA. Species-specific primers were designed on the conservative region of each species-specific nDNA sequence. The specificity and conservation of the sequences and primers were verified by PCR reaction and sequencing with the limit of detection down to 0.5 ng. Then, a species-specific multiplex PCR method was developed and optimized to simultaneously detect sheep/goat (237 bp), bovine (223 bp), chicken (192 bp), duck (168 bp) and pig (154 bp) in one reaction. Various processed meat products containing one or more animal-derived ingredients were detected by the developed multiplex PCR method, and the results were consistent with their labeled meat species. Our study provides a fast and simple detection method for regulating labeling of animal-derived ingredients in meat products.

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This work was supported by the National Science and Technology Major Project of China (2018ZX08012-001-010).

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Correspondence to Xiang Zhou or Bang Liu.

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Wenjun Wang declares that he has no conflict of interest. Xiaokang Wang declares that he has no conflict of interest. Qingde Zhang declares that he has no conflict of interest. Zuhong Liu declares that he has no conflict of interest. Xiang Zhou declares that he has no conflict of interest. Bang Liu declares that he has no conflict of interest. We have filed patents for primers to detect bovine and pig specific nuclear DNA sequences.

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Supplementary file1 Supplementary Fig. 1. All SNPs in the species-specific sequences of target species collected from NCBI, Ensembl and UCSC databases, as well as from the PCR sequencing results of different breeds and individuals. The underlined sequences were primer binding regions; degenerate bases M=A/C, S=G/C, Y=C/T, R=A/G, K=G/T. (PDF 119 kb)

Supplementary file2 Supplementary Fig. 2. Conservation analysis of PCR for sheep/goat (A), bovine (B), chicken (C), duck (D), and pig (E). In the agarose gel, M, BM2000 DNA Marker, B, blank control, N, negative control, 1-44, samples of different breeds or individuals. (PDF 299 kb)

Supplementary file3 Supplementary Fig. 3. Detection results in lamb, beef, chicken, duck, pork and mixed meat products. M, BM2000 DNA Marker; B, blank control; N, negative control; P1, positive control of sheep/goat; P2, positive control of bovine; P3, positive control of chicken; P4, positive control of duck; P5, positive control of pig; RLK, roasted lamb kebab; BLR, boiled lamb roll; BH, beef ham; BJ, beef jerky; DCC, deep-fried chicken chop; GCK, grilled chicken kebab; SDN, stewed duck neck; RD, roasted duck; PS, pork sausage; CBS, cooked bacon slices; (a), mixed meat rolls; (b), beef dumplings; (c), beef ham slices; (d), western ham; (e), pork ham slices; (f), sausage; (g), homemade meatball. (PDF 250 kb)

Supplementary file4 (DOCX 15 kb)

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Wang, W., Wang, X., Zhang, Q. et al. A multiplex PCR method for detection of five animal species in processed meat products using novel species-specific nuclear DNA sequences. Eur Food Res Technol 246, 1351–1360 (2020). https://doi.org/10.1007/s00217-020-03494-z

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  • Species-specific nuclear DNA sequence
  • Multiplex PCR
  • Meat products
  • Species identification