A Novel Universal Primer Multiplex Real-Time PCR (UP-M-rtPCR) Approach for Specific Identification and Quantitation of Cat, Dog, Fox, and Mink Fractions Using Nuclear DNA Sequences

Overview

Journal Foods

Specialty Biotechnology

Date 2023 Feb 11

PMID 36766123

Authors

Wenjun Wang

Tiean Wei

Manna Shi

Yu Han

Yang Shen

Xiang Zhou

Bang Liu

Affiliations

Soon will be listed here.

Abstract

Adulteration of meat with carnivorous animals (such as cats, dogs, foxes, and minks) can cause ethical problems and lead to disease transmission; however, DNA quantitative methods for four carnivorous species in one tube reaction are still rare. In this study, a carnivore-specific nuclear DNA sequence that is conserved in carnivorous animals but has base differences within the sequence was used to design universal primers for its conserved region and corresponding species-specific probes for the hypervariable region. A novel universal primer multiplex real-time PCR (UP-M-rtPCR) approach was developed for the specific identification and quantitation of cat, dog, fox, and mink fractions in a single reaction, with a 0.05 ng absolute limit of detection (LOD) and 0.05% relative LOD. This approach simplifies the PCR system and improves the efficiency of simultaneous identification of multiple animal-derived ingredients in meat. UP-M-rtPCR showed good accuracy (0.48-7.04% relative deviation) and precision (1.42-13.78% relative standard deviation) for quantitative analysis of cat, dog, fox, and mink DNA as well as excellent applicability for the evaluation of meat samples.

Citing Articles

Double-Tube Multiplex TaqMan Real-Time PCR for the Detection of Eight Animal-Derived Dairy Ingredients.

Ma X, Xia H, Pan Y, Huang Y, Xu T, Guan F J Agric Food Chem. 2024; 72(20):11640-11651.

PMID: 38725129 PMC: 11117397. DOI: 10.1021/acs.jafc.4c01294.

References

Wang W, Fu M, Zhang Q, Zhen Y, Liu J, Xiang S . A novel quantitative real-time PCR method for the detection of mammalian and poultry species based on a shared single-copy nuclear DNA sequence. Food Chem. 2020; 341(Pt 2):128170. DOI: 10.1016/j.foodchem.2020.128170. View

Zhou S, Zhong G, Zhou H, Zhang X, Zeng X, Wu Z . A Heptaplex PCR Assay for Molecular Traceability of Species Origin With High Efficiency and Practicality in Both Raw and Heat Processing Meat Materials. Front Nutr. 2022; 9:890537. PMC: 9260169. DOI: 10.3389/fnut.2022.890537. View

Wu Q, Xiang S, Wang W, Zhao J, Xia J, Zhen Y . Species Identification of Fox-, Mink-, Dog-, and Rabbit-Derived Ingredients by Multiplex PCR and Real-Time PCR Assay. Appl Biochem Biotechnol. 2017; 185(1):1-12. DOI: 10.1007/s12010-017-2621-2. View

Cai Z, Zhou S, Liu Q, Ma H, Yuan X, Gao J . A Simple and Reliable Single Tube Septuple PCR Assay for Simultaneous Identification of Seven Meat Species. Foods. 2021; 10(5). PMC: 8153340. DOI: 10.3390/foods10051083. View

Rahman M, Ali M, Hamid S, Mustafa S, Hashim U, Hanapi U . Polymerase chain reaction assay targeting cytochrome b gene for the detection of dog meat adulteration in meatball formulation. Meat Sci. 2014; 97(4):404-9. DOI: 10.1016/j.meatsci.2014.03.011. View

Iqbal M, Saleem M, Imran M, Khan W, Ashraf K, Yasir Zahoor M . Single tube multiplex PCR assay for the identification of banned meat species. Food Addit Contam Part B Surveill. 2020; 13(4):284-291. DOI: 10.1080/19393210.2020.1778098. View

Yang C, Zhong G, Zhou S, Guo Y, Pan D, Wang S . Detection and characterization of meat adulteration in various types of meat products by using a high-efficiency multiplex polymerase chain reaction technique. Front Nutr. 2022; 9:979977. PMC: 9525214. DOI: 10.3389/fnut.2022.979977. View

Day M . One Health: the small animal dimension. Vet Rec. 2011; 167(22):847-9. DOI: 10.1136/vr.c6492. View

Li X, Guan Y . Specific Identification of the Adulterated Components in Beef or Mutton Meats Using Multiplex PCR. J AOAC Int. 2019; 102(4):1181-1185. DOI: 10.5740/jaoacint.18-0338. View

10.

Kirsten H, Teupser D, Weissfuss J, Wolfram G, Emmrich F, Ahnert P . Robustness of single-base extension against mismatches at the site of primer attachment in a clinical assay. J Mol Med (Berl). 2006; 85(4):361-9. DOI: 10.1007/s00109-006-0129-2. View

11.

Ballin N, Vogensen F, Karlsson A . Species determination - Can we detect and quantify meat adulteration?. Meat Sci. 2010; 83(2):165-74. DOI: 10.1016/j.meatsci.2009.06.003. View

12.

Guo L, Yu Y, Xu W, Li C, Liu G, Qi L . Simultaneous detection of ovine and caprine DNA in meat and dairy products using triplex Man real-time PCR. Food Sci Nutr. 2020; 8(12):6467-6476. PMC: 7723203. DOI: 10.1002/fsn3.1936. View

13.

Enserink M . Coronavirus rips through Dutch mink farms, triggering culls. Science. 2020; 368(6496):1169. DOI: 10.1126/science.368.6496.1169. View

14.

Li K, Luo Y, Huang K, Yang Z, Wan Y, Xu W . Single universal primer recombinase polymerase amplification-based lateral flow biosensor (SUP-RPA-LFB) for multiplex detection of genetically modified maize. Anal Chim Acta. 2020; 1127:217-224. DOI: 10.1016/j.aca.2020.06.001. View

15.

Iwobi A, Sebah D, Kraemer I, Losher C, Fischer G, Busch U . A multiplex real-time PCR method for the quantification of beef and pork fractions in minced meat. Food Chem. 2014; 169:305-13. DOI: 10.1016/j.foodchem.2014.07.139. View

16.

Xue C, Wang P, Zhao J, Xu A, Guan F . Development and validation of a universal primer pair for the simultaneous detection of eight animal species. Food Chem. 2016; 221:790-796. DOI: 10.1016/j.foodchem.2016.11.102. View

17.

Shi J, Wen Z, Zhong G, Yang H, Wang C, Huang B . Susceptibility of ferrets, cats, dogs, and other domesticated animals to SARS-coronavirus 2. Science. 2020; 368(6494):1016-1020. PMC: 7164390. DOI: 10.1126/science.abb7015. View

18.

Hanapi U, Desa M, Ismail A, Mustafa S . A higher sensitivity and efficiency of common primer multiplex PCR assay in identification of meat origin using NADH dehydrogenase subunit 4 gene. J Food Sci Technol. 2015; 52(7):4166-75. PMC: 4486532. DOI: 10.1007/s13197-014-1459-7. View

19.

Liu G, Luo J, Xu W, Li C, Guo Y, Guo L . Improved triplex real-time PCR with endogenous control for synchronous identification of DNA from chicken, duck, and goose meat. Food Sci Nutr. 2021; 9(6):3130-3141. PMC: 8194750. DOI: 10.1002/fsn3.2272. View

20.

Liu M, Boireau P . Trichinellosis in China: epidemiology and control. Trends Parasitol. 2002; 18(12):553-6. DOI: 10.1016/s1471-4922(02)02401-7. View