Short targeting multiplex PCR assay to detect and discriminate beef, buffalo, chicken, duck, goat, sheep and pork DNA in food products

Uddin, Syed Muhammad Kamal; Hossain, M. A. Motalib; Chowdhury, Zaira Zaman; Johan, Mohd Rafie

doi:10.1080/19440049.2021.1925748

Cited by 27 publications

(16 citation statements)

References 50 publications

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“…Meat products contribute essential nutrients to human such as proteins, fatty acids, trace elements, and vitamins, especially for the richest protein source ( 1 ). Based on the growing demand for animal protein-based foods, meat frauds such as counterfeiting and mislabeling have become a severe global issue ( 2 – 4 ).…”

Section: Introductionmentioning

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: Introductionmentioning

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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“…In addition, using a species-specific primer, molecular methods such as polymerase chain reaction (PCR) have been widely used in porcine DNA detection in meatballs, including the PCR-RFLP [ 20 , 21 ], species-specific PCR [ 18 ] and real-time PCR [ 11 , 22 ]. However, there are some limitations to using PCR techniques.…”

Section: Introductionmentioning

confidence: 99%

Rapid Detection of Porcine DNA in Meatball Using Recombinase Polymerase Amplification Couple with Lateral Flow Immunoassay for Halal Authentication

et al. 2022

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Point-of-care diagnostic methods for animal species determination are critical for rapid, simple, and accurate enforcement of food labelling. PCR is the most common method for species identification. However, the requirement of using a thermal cycler created drawbacks for the PCR application, particularly in low-resource settings. Hence, in this study, a method for porcine DNA detection using recombinase polymerase amplification (RPA), coupled with nucleic acid lateral flow immunoassay (NALFIA), was developed. Porcine-specific primers targeting pig (Sus scrofa) cytochrome b gene fragments specifically amplify a 197 bp fragment of the mitochondrial gene as being visualized by 2% agarose gel and PCRD NALFIA. The reaction temperature and time were 39°C and 20 min, respectively. Herein, the specificity of the primers to porcine was confirmed after being assayed against six animal species, namely cow, goat, chicken, duck, dog, and rabbit. The porcine-specific RPA assay shows a high limit of detection of 0.01 ng/µL pork DNA. Based on the preliminary performance data obtained from this study, the potential of this method as a rapid and sensitive tool for porcine DNA detection in meat-based products is foreseen.

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“…Furthermore, the amplification capacity of nucleic acids can improve the detection sensitivity of corresponding DNA analytical methods. Commonly used nucleic acid-based detection methods include polymerase chain reaction (PCR) [ 13 ], real-time fluorescent PCR [ 14 ], digital PCR [ 15 ], multiplex PCR [ 16 ], loop-mediated isothermal amplification (LAMP) [ 17 ], cross-priming amplification (CPA) [ 18 ], and recombinase polymerase amplification (RPA) [ 19 , 20 ]. PCR is considered the “gold standard” for identifying adulterated meat products and has been successfully employed to identify beef, lamb, pork, and chicken [ 21 ].…”

Section: Introductionmentioning

confidence: 99%

Visual Detection of Chicken Adulteration Based on a Lateral Flow Strip-PCR Strategy

Lan

Pan

et al. 2022

Foods

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The aim of this study was to develop an accurate, easy-to-use, and cost-effective method for the detection of chicken adulteration based on polymerase chain reaction (PCR) and lateral flow strip (LFS). We compared six DNA extraction methods, namely the cetyltrimethylammonium bromide (CTAB) method, salt method, urea method, SDS method, guanidine isothiocyanate method, and commercial kit method. The chicken cytb gene was used as a target to design specific primers. The specificity and sensitivity of the PCR-LFS system were tested using a self-assembled lateral flow measurement sensor. The results showed that the DNA concentration obtained by salt methods is up to 533 ± 84 ng µL−1, is a suitable replacement for commercial kits. The PCR-LFS method exhibits high specificity at an annealing temperature of 62 °C and does not cross-react with other animal sources. This strategy is also highly sensitive, being able to detect 0.1% of chicken in artificial adulterated meat. The results of the test strips can be observed with the naked eye within 5 min, and this result is consistent with the electrophoresis result, demonstrating its high accuracy. Moreover, the detection system has already been successfully used to detect chicken in commercial samples. Hence, this PCR-LFS strategy provides a potential tool to verify the authenticity of chicken.

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Short targeting multiplex PCR assay to detect and discriminate beef, buffalo, chicken, duck, goat, sheep and pork DNA in food products

Cited by 27 publications

References 50 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

Rapid Detection of Porcine DNA in Meatball Using Recombinase Polymerase Amplification Couple with Lateral Flow Immunoassay for Halal Authentication

Visual Detection of Chicken Adulteration Based on a Lateral Flow Strip-PCR Strategy

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