Specific primer design of mitochondrial 12S rRNA for species identification in raw meats

Cahyadi, Muhammad; Puruhita,; Barido, Farouq Heidar; Hertanto, Bayu Setya

doi:10.1088/1755-1315/102/1/012038

Cited by 8 publications

(21 citation statements)

References 12 publications

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“…The characteristics of selected primer sets are presented in Table 1. A total of 15 sets of primer were generated in this study (Supplementary Material 1). Of these, primer pair for bovine was designed in previous studies (Cahyadi et al, 2018) while two sets of primer for dog and rat were novel finding and firstly reported in this work. Universal forward primer was a hundred percent similar for cattle, dog, and rat.…”

Section: Primer Characteristicsmentioning

confidence: 97%

“…Selected primer candidates for dog and rat are presented in Table 1. Primer pair for bovine used in this study was previously reported by Cahyadi et al (2018) while the new dog and rat reverse primers were synthesized by Integrated DNA Technologies, Singapore.…”

Section: Primer Designmentioning

confidence: 99%

“…A total of 10 reactions containing different DNA sample and primer pair were tested to evaluate specificity of the primers in this study ( Table 2). The PCR was carried out in total volume of 25 µL containing 12.5 µL of My Taq HS Red Mix solution (Bioline, United Kingdom), 1 µL (10 µM) of universal forward primer, 1 µL (10 µM) of reverse primers, 1 µL of DNA template, and 8.5 µL ddH 2 O (Cahyadi et al, 2018). The PCR was performed by following these steps: initial denaturation at 95 o C for 3 minutes, 30 cycles of denaturation at 95 o C for 15 seconds, annealing temperature at 64 o C for 30 seconds, and extension at 72 o C for 30 seconds.…”

Section: Evaluation Of Primer Using Polymerase Chain Reaction (Pcr) Amentioning

confidence: 99%

“…Sequence analysis of mitochondrial 12S rRNA has previously been used to identify species on the samples originated from animal tissue (Girish et al, 2004;Yang et al, 2014). Moreover, a current report revealed that 12S rRNA gene could be used for identification of chicken and pig in beef using multiplex PCR (Cahyadi et al, 2018). In the present work, specific primers from mitochondrial 12S rRNA were designed and tested for identification of dog and rat meat in beef using multiplex PCR assay.…”

Section: Introductionmentioning

confidence: 95%

“…Primer design should be carefully performed to get specific primer which is able to amplify DNA target in PCR (Dieffenbach et al, 1993;Pradnyaniti et al, 2013). For example, primers which are designed for mitochondrial genome should not anneal in the region of nuclear genome and vice versa (Cahyadi et al, 2018).…”

Section: Introductionmentioning

confidence: 99%

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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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Section: Primer Characteristicsmentioning

confidence: 97%

Section: Primer Designmentioning

confidence: 99%

Section: Evaluation Of Primer Using Polymerase Chain Reaction (Pcr) Amentioning

confidence: 99%

Section: Introductionmentioning

confidence: 95%

Section: Introductionmentioning

confidence: 99%

See 3 more Smart Citations

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

Barido,

Desti,

Pramono

et al. 2023

Food Chemistry: Molecular Sciences

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Mitochondrial COX2 primer for bovine identification using qPCR: Design and validation

Sintia,

Barido,

Volkandari

et al. 2024

IOP Conf. Ser.: Earth Environ. Sci.

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Beef is a prime livestock product that is prone to adulteration with other relatively lower price species. The occurrence causes losses to consumers and also violates consumer protection law. Quantitative PCR (qPCR) is a molecular approach technology that is suitable for detecting the presence of a broad spectrum of DNA in raw and processed animal-based foods. Mitochondrial DNA (mtDNA) is well known having unique nucleotide sequence which is able to be used as genetic markers in living things. The cytochrome C oxidase subunit II (COX2), a part of mtDNA, can be explored to be biomarker candidate for detection of contamination in various animal products. Therefore, this study was conducted to design and validate specific primer targeting mtDNA COX2 for bovine identification using qPCR. This study designed forward primer (5’-CATGAGCTGTGCCCTCTCTAG-3’) and reverse primer (5’-TAATATAAGCCTGGACGGGA-3’) using the National Center for Biotechnology Information (NCBI) website with accession number GU947020. Moreover, those primers were tested in vitro to optimize reaction condition, and component, and also to evaluate repeatability and sensitivity of the qPCR. Three species DNA, bovine, pig, and chicken were utilized in this study. This study found that the designed primer pair successfully amplified the DNA target in 32 cycles of qPCR, Cq value at 22.48±0.38, Tm at 82.55°C, and 20 ng/μL of bovine DNA. In addition, the limit of detection (LOD) was 2.08 ng/μL and the primer pair specifically attached to bovine DNA. This study concluded that the primer pair targeting mtDNA COX2 can be a strong biomarker candidate for the identification of bovine DNA in various food products.

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Specific primer design of mitochondrial 12S rRNA for species identification in raw meats

Cited by 8 publications

References 12 publications

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

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

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

Mitochondrial COX2 primer for bovine identification using qPCR: Design and validation

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