Single tube multiplex PCR assay for the identification of banned meat species

Iqbal, Muhammad Kashif; Saleem, Muhammad Sulyman; Imran, Muhammad; Khan, Waseem; Ashraf, Kamran; Zahoor, Muhammad; Rashid, Imran; Rehman, Habib-Ur; Nadeem, Asif; Ali, Saadat; Naz, Sarwat; Shehzad, Wasim

doi:10.1080/19393210.2020.1778098

Cited by 15 publications

(11 citation statements)

References 49 publications

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“…The multiplex PCR technique is a highly effective method for detecting multiple targets in a single platform, which dramatically cuts the cost and time of analysis through a simple agarose gel analysis ( 18 – 20 ). Notably, with the increase of primers and multiplicity of PCR reaction, mutual interference of PCR components causes lower efficiency and even the failure of amplification ( 7 ).…”

Section: Discussionmentioning

confidence: 99%

Molecular Authentication of Twelve Meat Species Through a Promising Two-Tube Hexaplex Polymerase Chain Reaction Technique

et al. 2022

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Frequent meat frauds have aroused significant social attention. The aim of this study is to construct a two-tube hexaplex polymerase chain reaction (PCR) method offering accurate molecular authentication of twelve meat species in actual adulteration event. Deoxyribonucleic acid (DNA) sequencing demonstrates that designed primers can specifically amplify target species from genomic DNA mixture of six species in each tube reaction, which showed 100% accuracy of horse (148 bp), pigeon (218 bp), camel (283 bp), rabbit (370 bp), ostrich (536 bp), and beef (610 bp) as well as turkey (124 bp), dog (149 bp), chicken (196 bp), duck (277 bp), cat (380 bp), and goose (468 bp). A species-specific primer pair produced the target band in the presence of target genomic DNA but not non-target species. Through multiplex PCR assays with serial concentration of the DNA mixture of six species in each PCR reaction, the detection limit (LOD) of the two-tube hexaplex PCR assay reached up to 0.05–0.1 ng. Using genomic DNA isolated from both boiled and microwave-cooked meat as templates, PCR amplification generated expected PCR products. These findings demonstrate that the proposed method is specific, sensitive and reproducible, and is adequate for food inspection. Most importantly, this method was successfully applied to detect meat frauds in commercial meat products. Therefore, this method is of great importance with a good application foreground.

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Section: Discussionmentioning

confidence: 99%

Molecular Authentication of Twelve Meat Species Through a Promising Two-Tube Hexaplex Polymerase Chain Reaction Technique

et al. 2022

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“…Nowadays, adulteration practice has been ingeniously done with the morphological and physical characteristics similar to pure meat (6). Multiplex PCR assays have some advantages in discriminating animal origins, because they are easily accomplished through simple agarose gel analysis and dramatically minimizes the cost (32)(33)(34). As shown in Supplementary Table S1, much information is available on recently published multiplex PCR methods.…”

Section: Discussionmentioning

confidence: 99%

A Heptaplex PCR Assay for Molecular Traceability of Species Origin With High Efficiency and Practicality in Both Raw and Heat Processing Meat Materials

Zhou

Zhong²,

Zhou³

et al. 2022

Front. Nutr.

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Frequent meat frauds have become a global issue because adulteration risks the food safety, breaches market rules, and even threatens public health. Multiplex PCR is considered to be a simple, fast, and inexpensive technique that can be applied for the identification of meat products in food industries. However, relatively less is known about a multiplex PCR method authenticating seven animal species simultaneously in one reaction due to technological challenge. Through screening new species-specific primers and optimizing PCR system, a heptaplex PCR method was established, which could simultaneously detect seven meat ingredients of camel (128 bp), pigeon (157 bp), chicken (220 bp), duck (272 bp), horse (314 bp), beef (434 bp), and pork (502 bp) in a single-tube reaction. DNA sequencing solidly validated that each set of primers specifically amplified target species from total DNA mixtures of seven meat species. The developed multiplex assay was stable and sensitive enough to detect 0.01–0.025 ng DNA from various meat treatments including raw, boiled, and autoclaved meat samples or target meat content of 0.1% total meat weight, suggesting the suitability of the heptaplex PCR technique for tracing target meats with high accuracy and precision. Most importantly, a market survey validated the availability of this multiplex PCR technique in real-world meat products with a good application foreground.

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“…Nowadays, PCR-based techniques are the effective methods for species authentication. Real-time PCR techniques and microchip electrophoresis-dependent multiplex PCR methods require special infrastructures [ 11 , 29 , 30 ]; multiplex PCR assays through simple agarose gel analysis minimizes the cost drastically on a large scale and can be easily carried out to verify the identity of ingredients in foodstuffs [ 6 , 31 , 32 ]. As seen in Table 3 , much is known about multiplex PCR assays that simultaneously verify two to six meat ingredients in one reaction.…”

Section: Discussionmentioning

confidence: 99%

A Simple and Reliable Single Tube Septuple PCR Assay for Simultaneous Identification of Seven Meat Species

Cai

Zhou

Liu

et al. 2021

Foods

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Multiplex PCR methods have been frequently used for authentication of meat product adulteration. Through screening of new species-specific primers designed based on the mitochondrial DNA sequences, a septuple PCR method is ultimately developed and optimized to simultaneously detect seven species including turkey (110 bp), goose (194 bp), pig (254 bp), sheep (329 bp), beef (473 bp), chicken (612 bp) and duck (718 bp) in one reaction. The proposed method has been validated to be specific, sensitive, robust and inexpensive. Taken together, the developed septuple PCR assay is reliable and efficient, not only to authenticate animal species in commercial meat products, but also easily feasible in a general laboratory without special infrastructures.

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Single tube multiplex PCR assay for the identification of banned meat species

Cited by 15 publications

References 49 publications

Molecular Authentication of Twelve Meat Species Through a Promising Two-Tube Hexaplex Polymerase Chain Reaction Technique

Molecular Authentication of Twelve Meat Species Through a Promising Two-Tube Hexaplex Polymerase Chain Reaction Technique

A Heptaplex PCR Assay for Molecular Traceability of Species Origin With High Efficiency and Practicality in Both Raw and Heat Processing Meat Materials

A Simple and Reliable Single Tube Septuple PCR Assay for Simultaneous Identification of Seven Meat Species

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