Identification of pork contamination in meatball using genetic marker mitochondrial DNA cytochrome b gene by duplex-PCR

Novianty, Elfrida; Kartikasari, Lilik Retna; Lee, Jun Heon; Cahyadi, Muhammad

doi:10.1088/1757-899x/193/1/012002

Cited by 12 publications

(10 citation statements)

References 8 publications

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“…Detection of a mixture of beef, dog, pig, and rat in raw meat and processed products such as meatballs has also been developed previously using the Cyt b gene (Novianty et al, 2016;Primasari, 2011;Rahman et al, 2014). The 12S rRNA gene primer in the present study has advantages, namely using universal forward primers and specific reverse primers to produce specific results.…”

Section: Discussionmentioning

confidence: 99%

“…Utilization of the 12S rRNA gene for species identification is previously reported that it can identify species of origin in the feedstuffs up to 0.01% of the DNA genome containing in the sample (Safdar and Junejo, 2015). In terms of using meatballs, earlier reports explained that duplex-PCR could detect up to only 1% chicken and pork contamination in the meatball made from beef (Novianty et al, 2016;Sari et al, 2017).…”

Section: Discussionmentioning

confidence: 99%

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A Novel Multiplex-PCR Assay to Detect Three Non-Halal Meats Contained in Meatball using Mitochondrial 12S rRNA Gene

Cahyadi¹,

Wibowo²,

Pramono³

et al. 2020

Food Sci Anim Resour

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The objective of this study was to detect three non-halal meat products consisted of dog, pork, and rat species in meatball using novel multiplex-PCR with 12S rRNA gene as target sites. A total of 33 self-made meatballs were used, and they were grouped into eleven types of meatball based on meat species origin contained in the meatballs. Each type consisted of three meatballs. Extraction of genomic DNA from the meatballs was used as a DNA template for simplex-, duplex-, and multiplex-PCR processes. The result of simplex-PCR, duplex-PCR, and multiplex-PCR showed that the 12S rRNA primer gene successfully amplified DNA for each species bovine, dog, pig, and rat, which are respectively indicated by 155, 244, 357, and 491 bp of DNA bands. In addition, multiplex-PCR with 12S rRNA gene primers can be uniquely and accurately used for detection bovine, dog, pig, and rat species on beef meatball in one reaction.

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Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

A Novel Multiplex-PCR Assay to Detect Three Non-Halal Meats Contained in Meatball using Mitochondrial 12S rRNA Gene

Cahyadi¹,

Wibowo²,

Pramono³

et al. 2020

Food Sci Anim Resour

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show abstract

“…This analysis performed to ensure that oligonucleotide primers designed in this study and other components of PCR were working together and they could be used as a marker for identification of dog and rat in food products containing beef and bovine derivatives. The studies in food authentication using PCR technology were reported using duplex PCR mitochondrial Cytochrome b gene to detect pork and chicken contamination in raw meats and meatballs using published primers (Ni'mah et al, 2016;Hertanto et al, 2017;Novianty et al, 2017). The use of mitochondrial 12S rRNA gene as a marker for species identification had been successfully conducted using sequence analysis of processed meats samples.…”

Section: Species Identification Using Multiplex Pcr 12s Rrna Genementioning

confidence: 99%

Development of mitochondrial 12S rRNA gene for identification of dog and rat in beef using multiplex PCR

Cahyadi

Taufik

Pramono

et al. 2019

J. Indonesian Trop. Anim. Agric.

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The 12S rRNA gene is one of unique regions in mitochondrial genome usually used for phylogenetic studies and species identification. The objective of present study was to develop species specific primers from mitochondrial 12S rRNA gene for identification of dog and rat in beef by using multiplex PCR assay. Three primer pairs of mitochondrial 12S rRNA gene specific for bovine, dog and rat were designed and selected to evaluate their specificity and fidelity. Moreover, a total of twelve DNA samples extracted from meat tissue were also prepared to test those primers using simplex and multiplex PCR. The PCR products were then visualized using 2% of agarose gel under the UV light and three of them were sequenced. In addition, sequence data were analyzed using Clustal Omega software and BLAST. The result showed that simplex PCR assay successfully amplified DNA targets which are respectively indicated by 155 bp (bovine), 244 bp (dog), and 491 bp (rat) of DNA bands. Furthermore, DNA sample sequences were identically similar to reference sequence used in this study. Multiplex and simplex PCR analyses also indicated that these primer pairs specifically amplified DNA target for each species in the samples containing various species. The results suggested that designed primers in this study could be used to identify dog and rat in raw beef containing these species meat. Further experiment should be conducted using meat-processed products and commercial meat products as samples.

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“…A study done by Ni'mah et al [17] using commercial extraction kit, Genomic DNA Mini Kit, was also able to detect pork contamination in both fresh and cooked beef up to 1% of contamination. A study by Novianty et al [18] has successfully identified pork contamination until the level of 1% in meatball. Samples were extracted using Quick-DNA Universal Kit.…”

Section: Resultsmentioning

confidence: 99%

Efficiency of Traditional Dna Extraction Method in PCR Detection of Porcine Dna in Meat Mixtures

et al. 2020

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Through the advancement of biotechnology, DNA-based methods are the most effective techniques in species identification, as they are rapid and have higher stability in harsh conditions compared to protein-based methods. This study was conducted to determine the efficiency of the traditional DNA extraction method, phenol/chloroform/isoamyl alcohol (PCIA), and comparing it with the commercially available kit by evaluating the purity, concentration, and suitability for amplification of porcine DNA in raw chicken and beef mixtures. The quantity and quality of the DNA extracts were assessed using a UV-Vis spectrophotometer. Polymerase chain reaction (PCR) was performed using species-specific primers targeting mitochondrial DNA cytochrome b (cyt b) gene of chicken (227-bp), beef (274-bp), and pork (398-bp), to confirm the template usability and quality of the DNA extracts. High DNA concentrations and purity were obtained from meat samples extracted using the PCIA method. The visualization of pork DNA on 2% agarose gel was able to detect pork contamination in raw meat mixtures up to minute proportion (1%). The existence of pork in chicken and beef was indicated with the presence of a specific 398-bp DNA band. Thus, the PCIA method can be recommended as a cost-effective and an excellent alternative to more expensive extraction kits in detecting pork DNA in raw meat mixtures.

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Identification of pork contamination in meatball using genetic marker mitochondrial DNA cytochrome b gene by duplex-PCR

Cited by 12 publications

References 8 publications

A Novel Multiplex-PCR Assay to Detect Three Non-Halal Meats Contained in Meatball using Mitochondrial 12S rRNA Gene

A Novel Multiplex-PCR Assay to Detect Three Non-Halal Meats Contained in Meatball using Mitochondrial 12S rRNA Gene

Development of mitochondrial 12S rRNA gene for identification of dog and rat in beef using multiplex PCR

Efficiency of Traditional Dna Extraction Method in PCR Detection of Porcine Dna in Meat Mixtures

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