A multiplex PCR method mediated by universal primers for the identification of eight meat ingredients in food products

Liu, Wanwan; Tao, Jing; Xue, Mingzhu; Ji, Jiangang; Zhang, Yahan; Zhang, Lijun; Sun, Wanping

doi:10.1007/s00217-019-03350-9

Cited by 21 publications

(10 citation statements)

References 16 publications

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“…However, to our knowledge, little information is available on a universal eukaryotic primer set amplifying the fragment with more than 700 bp length. As alternatives, we chose three pairs of universal eukaryotic primers, which target different mitochondrial DNA sequences, including 12S rRNA, 16S rRNA, 18S rRNA genes, and amplifies distinguishable 456, 240 and 99 bp PCR fragments in all meat species, respectively [ 26 , 27 , 28 ]. In addition, the control primer set should be inserted in the multiplex assay; these PCR fragments were too close to that of turkey (110 bp), pig (254 bp) and beef (473 bp) to discriminate each other.…”

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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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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“…Adulteration of meat with products from multiple species is increasingly being reported (Ali et al, 2015; Di Pinto et al, 2015; Izadpanah et al, 2018; Kitpipit et al, 2014; O’Mahony, 2013), raising the demand for affordable and faster techniques for their detection. Many studies describing multi-species analysis of vertebrates in meat have utilized multiplex PCR (Ali et al, 2015; Balakrishna et al, 2019; Izadpanah et al, 2018; Kitpipit et al, 2014; Li et al, 2019; Liu et al, 2019; Qin et al, 2019; Wang et al, 2020). While useful, multiplex PCR requires use of expensive probes and post-PCR procedures such as agarose gel electrophoresis for size separation of amplicons and/or DNA sequencing, thereby increasing analysis time, cost, and risk of cross-contamination.…”

Section: Discussionmentioning

confidence: 99%

Species substitution in the meat value chain by high-resolution melt analysis of mitochondrial PCR products

Njaramba

Wambua

Mukiama

et al. 2021

Preprint

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Food fraud in several value chains including meat, fish, and vegetables has gained global interest in recent years. In the meat value chain, substitution of high commercial-value meats with similar cheaper or undesirable species is a common form of food fraud that raises ethical, religious, and dietary concerns. The presence of undeclared species could also pose public health risks caused by allergic reactions and the transmission of food-borne or zoonotic pathogens. Measures to monitor meat substitution are being put in place in many developed countries. However, information about similar efforts in sub-Saharan Africa is sparse. In this study, we used PCR coupled with high-resolution melting (PCR-HRM) analysis targeting the three mitochondrial genes, cytochrome oxidase 1 (CO1), cytochrome b (cyt b), and 16S rRNA, to detect species substitution in meat sold to consumers in Nairobi, Kenya’s capital city. Out of 107 meat samples from seven common livestock animals (cattle, goat, sheep, pig, chicken, rabbit, and camel), 11 (10.3%) had been substituted. Of 61 samples sold as beef, two were goat and one was camel. Of 30 samples sold as goat meat, four were mutton (sheep) and three were beef. One of nine samples purchased as pork was beef. Our results indicate that PCR-HRM analysis is a cost and time effective technique that can be employed to detect species substitution. The combined use of the three markers produced PCR-HRM profiles that successfully allowed the distinction of species. We demonstrate its utility not only in analysis of raw meat samples, but also of cooked, dried, and rotten samples, meat mixtures, and with the use of different DNA extraction protocols. We propose that this approach has broad applications in authentication of meat products and protection of consumers from food fraud in the meat industry in low- and middle-income countries such as Kenya, as well as in the developed world.

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“…Uji lain pada spesies sapi dan babi menggunakan primer gen 12S-, 16S-rRNA, dan tRNA-Val memiliki batas deteksi antara 0,0025-0,025 ng (Dalmasso et al, 2004), primer gen ND5 spesies sapi dan cyt b spesies babi (Hossain et al, 2017) serta primer gen cyclic-GMP-phosphodiesterase pada spesies sapi dan beta-actin pada spesies babi dengan limit deteksi 0,003 ng (Iwobi et al, 2017). Pengujian sensitivitas untuk spesies anjing dan babi telah dilakukan oleh Liu et al (2019) menggunakan primer gen ATPase 6 untuk spesies anjing dan cyt b untuk spesies babi memiliki limit deteksi 0.05 ng/µL. Penggunaan primer lain dalam pengujian sensitivitas pada gen 16S rRNA untuk spesies anjing dan COI untuk spesies babi memiliki sensitivitas hingga 0.002 ng (Li et al, 2019), primer gen mitochondrial ATPase subunit 8 untuk spesies anjing dan babi dengan limit deteksi 0.03 ng (Prusakova et al, 2018).…”

Section: Simplex-dan Multiplex-pcrunclassified

“…Penggunaan primer gen 12S rRNA sebagai deteksi sensitivitas telah dilakukan pada spesies sapi dan babi, namun belum ditemukan deteksi sensitivitas untuk spesies anjing dan tikus. Penelitian ini apabila dibandingkan dengan penelitian terdahulu, primer gen 12S rRNA lebih sensitif (Matsunaga et al, 1999;Dalmasso et al, 2004;Yin et al, 2009;Ali et al, 2015;Hossain et al, 2017;Iwobi et al, 2017;Prusakova et al, 2018;Liu et al, 2019;Li et al, 2019). Primer gen 12S rRNA untuk spesies babi sama sensitif dengan primer gen COI yaitu 0,001 ng (Wang et al, 2019), kurang sensitif untuk spesies babi apabila dibandingkan dengan primer cyt b oleh Lubis et al (2018), dan kurang sensitif untuk spesies tikus oleh Chen et al (2020).…”

Section: Simplex-dan Multiplex-pcrunclassified

Sensitivitas Multiplex-Polymerase Chain Reaction Gen 12S rRNA dalam Mendeteksi Pemalsuan Daging Sapi dengan Daging Babi, Anjing dan Tikus

et al. 2021

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Daging sapi dan olahannya merupakan sasaran pemalsuan. Pelaku pemalsuan daging sapi mencampur daging sapi dengan daging spesies hewan lain, sehingga berakibat pada berubahnya status kehalalan daging. Selain itu, kondisi tersebut dapat menjadi penyebab serius pada risiko kesehatan, seperti potensi keracunan, sumber penyakit maupun alergi. Pengawasan yang efektif diperlukan guna menjamin keaslian dan keamanan daging. Metode deteksi secara molekuler yang umum digunakan adalah multiplex-PCR karena efisien dan sensitif. Tujuan penelitian ini untuk mendeteksi sensitivitas multiplex-PCR primer gen 12S rRNA yang spesifik untuk spesies sapi, anjing, babi, dan tikus. Template DNA dibuat masing-masing enam konsentrasi, yaitu 25; 10; 1; 0,1; 0,01; 0,001 ng/ìL untuk proses simplex dan multiplex-PCR. Hasil uji sensitivitas menggunakan metode simplex-PCR menunjukkan bahwa primer gen 12S rRNA dapat mengamplifikasi spesies sapi, anjing, babi, dan tikus dengan akurat hingga konsentrasi 0,001 ng/ìL. Pengujian sensitivitas multiplex-PCR gen 12S rRNA dapat mengamplifikasi spesies sapi, anjing, babi hingga konsentrasi 0,001 ng/ìL dan pada spesies tikus hingga konsentrasi 1 ng/ìL. Metode PCR ini dapat digunakan sebagai solusi alternatif dalam studi halal dan verifikasi label pangan

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A multiplex PCR method mediated by universal primers for the identification of eight meat ingredients in food products

Cited by 21 publications

References 16 publications

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

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

Species substitution in the meat value chain by high-resolution melt analysis of mitochondrial PCR products

Sensitivitas Multiplex-Polymerase Chain Reaction Gen 12S rRNA dalam Mendeteksi Pemalsuan Daging Sapi dengan Daging Babi, Anjing dan Tikus

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