Validating duplex-PCR targeting ND2 for bovine and porcine detection in meat products

Barido, Farouq Heidar; Desti, Desti; Pramono, Ahmad; Abdurrahman, Zakaria Husein; Volkandari, Slamet Diah; Cahyadi, Muhammad

doi:10.1016/j.fochms.2023.100181

Cited by 3 publications

(5 citation statements)

References 36 publications

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“…This analysis was done using Basic Local Alignment Search Tool (BLAST) (https://blast.ncbi.nlm.nih.gov/Blast.cgi) and oligonucleotide analyzer for dimer and hairpin analysis (http://sg.idtdna.com/site) [22]. Furthermore, the primer specificity was assessed using Clustal Omega to find out the total mismatch of the primers and provide a phylogenetic tree toward other species (https://www.geneious.com/prime/) [22,[24][25]. The primers macaque targeting 97-bp D-loop gene were chosen in this work, namely forward: 5'-TGACTCCCACCACATCCC-3' and reverse: 5'-GTGTGGAGCTAGAATATTGAACCG-3'.…”

Section: Primer Designmentioning

confidence: 99%

“…The primer BLAST result had 100% query coverage and an expectation value (E-value) of 1 × 10 −5 . E-value ≤ 0.01 denotes homologous sequences with macaque D-loop gene [24], and this E-value is widely accepted for establishing the primer [33]. Secondly, the establishment of pair-wise distance and the phylogenetic tree was conducted with multiple aligned between the developed primer, 6 other meat species and 2 non-meat species.…”

Section: Primer Designmentioning

confidence: 99%

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Specific Real-Time PCR Assay Targeting D-loop Gene and Short Amplicon Sequencing for Identification of Monkey Meat in Beef Meatballs

Kurniawati,

Sismindari,

Rumiyati

et al. 2024

Indones. J. Chem.

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Macaque monkey (Macaca fascicularis) meat (MM) has been reported to be consumed as meatball and soup products in Indonesia. MM is not allowed to be traded in Indonesia and is considered not halal meat; therefore, MM is not allowed to be consumed by Muslim communities. In this study, species-specific primer (SSP) targeting on mitochondrial displacement (D-loop) region coupled with real-time PCR assay was used to identify monkey meat in beef meatballs. The PCR product was also subjected to sequencing in order to ensure the adulteration practice of MM in beef meatballs. The primers were designed and subjected to in silico specificity test using BLAST. The used primers were: forward: 5’-TGACTCCCACCACATCCC-3’ and reverse: 5’-GTGTGGAGCTAGAATATTGAACCG-3’. Real-time PCR assay using SSP targeting on mt-D-loop primers was capable of identifying MM in meatball products, specifically with a detection limit of 0.0078 ng corresponding to 1% MM in beef meatballs. The developed method can be proposed as the standard method for detecting MM in food products intended for halal authentication analysis, provided that DNAs are available in food products.

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Section: Primer Designmentioning

confidence: 99%

Section: Primer Designmentioning

confidence: 99%

Specific Real-Time PCR Assay Targeting D-loop Gene and Short Amplicon Sequencing for Identification of Monkey Meat in Beef Meatballs

Kurniawati,

Sismindari,

Rumiyati

et al. 2024

Indones. J. Chem.

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“…Studies exemplify diverse methods addressing these challenges. Techniques like duplex‐polymerase chain reaction (PCR) in Indonesia for meatballs (Barido et al., 2023) and targeted metabolomics in Japan and Indonesia for coffee authentication (Aurum et al., 2023) highlight concerns over potential food fraud. Advanced tools such as nuclear magnetic resonance (NMR) spectroscopy for edible bird's nests in Malaysia (Lee et al., 2022) and ultrahigh‐performance liquid chromatography (UHPLC)–mass spectrometry (MS) for Withania somnifera authentication in Pakistan (Tousif et al., 2022) emphasize the need for sophisticated techniques.…”

Section: Food Authentication and Contamination In Asian Continentsmentioning

confidence: 99%

“…Authenticating food products becomes crucial for ensuring safety in the global market amid challenges like contamination, mislabeling, and adulteration. Various studies (Aurum et al., 2023; Barido et al., 2023; Lee et al., 2022; Tousif et al., 2022) highlight the use of advanced techniques such as DNA barcoding, NMR spectroscopy, and MS in different countries to address authenticity issues. Mycotoxin detection methods, like LC–MS–MS and HPLC, have been applied in Malaysia and Indonesia (Soleimany et al., 2012; Jalili & Jinap, 2012; Sharmili et al., 2016), whereas innovative techniques like molecularly imprinted polymer nanoparticles were used in Indonesia to tackle specific contaminations (Munawar et al., 2020).…”

Section: Food Authentication and Contamination In Asian Continentsmentioning

confidence: 99%

Food authentication, current issues, analytical techniques, and future challenges: A comprehensive review

Haider,

Iqbal,

Bhatti

et al. 2024

Comp Rev Food Sci Food Safe

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Food authentication and contamination are significant concerns, especially for consumers with unique nutritional, cultural, lifestyle, and religious needs. Food authenticity involves identifying food contamination for many purposes, such as adherence to religious beliefs, safeguarding health, and consuming sanitary and organic food products. This review article examines the issues related to food authentication and food fraud in recent periods. Furthermore, the development and innovations in analytical techniques employed to authenticate various food products are comprehensively focused. Food products derived from animals are susceptible to deceptive practices, which can undermine customer confidence and pose potential health hazards due to the transmission of diseases from animals to humans. Therefore, it is necessary to employ suitable and robust analytical techniques for complex and high‐risk animal‐derived goods, in which molecular biomarker‐based (genomics, proteomics, and metabolomics) techniques are covered. Various analytical methods have been employed to ascertain the geographical provenance of food items that exhibit rapid response times, low cost, nondestructiveness, and condensability.

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“…In previous studies, mitochondrial DNA (mtDNA) has been explored to gure out a primer for porcine detection in food and feed. Various genes have been utilized for primer design such as cytochrome b (Ali et (Barido et al 2023), and COI (Boyrusbianto et al 2023). Researchers continue exploring and studying other genes in mitochondrial DNA or nuclear DNA to obtain biomarkers (primers) that are species-speci c, sensitive, stable, robust, speci c, and reliable (Soares et al 2013).…”

Section: Introductionmentioning

confidence: 99%

Validation of NADH Dehydrogenase 4L (ND4L) Primers Gene for Porcine Detection Using A SYBR Green Real-Time PCR Analysis on Commercial Processed Meat Products

Volkandari,

Jenie,

Rohman

et al. 2024

Preprint

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Porcine contamination in halal products is known to be found in commercial food markets in Indonesia. Detection methods are needed, one of which is Real-Time PCR with robust biomarkers in various types of processed food. In this study, we focused on designing a novel primer from NADH Dehydrogenase 4L (ND4L) gene that has never been explored as a porcine detection. Primers were assembled using GenBank NCBI (MK251046 Sus scrofa), and their efficacy (repeatability, sensitivity, and specificity) was evaluated. The capability primer performance was also compared with a commercial kit. Results showed that primer effectively amplified porcine DNA with a melting curve (Tm) of 78.17 ± 0.05oC and a Cq value of 11.95 ± 0.48 at a 20 ng/µL DNA concentration. CV values of Cq and Tm repeatability were 4,01 and 0.09%, respectively. The limit of detection primer reached 5 pg/µL DNA and 0.001% w/w binary meat mixture of pork-beef. This primer is highly specific to pig and wild boar species (against 30 species (non-pig). A comparative study on the ND4L primer with a commercial porcine detection kit (FAM label) revealed similar results in detecting porcine DNA in food products. ND4L primer successfully detected porcine DNA in 20 of 52 commercial meat products with various types of processing (according to their claims). Primer performed satisfactorily in all validation parameters with high sensitivity and specificity. Hence, this finding of the specific primers on the ND4L gene could be promising for detecting porcine DNA in food products.

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Validating duplex-PCR targeting ND2 for bovine and porcine detection in meat products

Cited by 3 publications

References 36 publications

Specific Real-Time PCR Assay Targeting D-loop Gene and Short Amplicon Sequencing for Identification of Monkey Meat in Beef Meatballs

Specific Real-Time PCR Assay Targeting D-loop Gene and Short Amplicon Sequencing for Identification of Monkey Meat in Beef Meatballs

Food authentication, current issues, analytical techniques, and future challenges: A comprehensive review

Validation of NADH Dehydrogenase 4L (ND4L) Primers Gene for Porcine Detection Using A SYBR Green Real-Time PCR Analysis on Commercial Processed Meat Products

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