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

Abstract

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.

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

“…This result was in agreement with previous studies that successfully detected non-halal meats in raw meats (Cahyadi et al, 2018;Cahyadi et al, 2019).…”

“…The primer pairs of the 12S rRNA gene used in the present study can amplify the target area of the 12S rRNA gene using genomic DNA extracted from meatball samples. This result was in agreement with previous studies that successfully detected non-halal meats in raw meats ( Cahyadi et al, 2018 ; Cahyadi et al, 2019 ).…”

“…The 12S rRNA gene primer in the present study has advantages, namely using universal forward primers and specific reverse primers to produce specific results. The primary 12S rRNA gene also has the same annealing temperature, so the multiplex-PCR, which is using more than one primer pairs in one tube reaction, generates more accurate amplification targets (Cahyadi et al, 2019). The PCR product size for each species was consistent in the simplex-, duplex-, and also multiplex-PCR.…”

“…The genomic DNA extracted from the meatball sample was used as a DNA template for the purpose of obtaining specific segments of the 12S rRNA gene as target using primers previously reported by Cahyadi et al (2018) and Cahyadi et al (2019). Primer pairs are shown in Table 2.…”

“…Most of the previous studies develop mitochondrial genome such as Cytochrome b and 12S rRNA genes as target sites for phylogenetic, evolution, and species identification studies due to their unique nucleotides and maternally inherited to the offspring. The 12S rRNA region has been used for species identification in raw meats using multiplex-PCR assay ( Cahyadi et al, 2018 ; Cahyadi et al, 2019 ). Therefore, the objective of this study was to detect intentionally contaminated beef meatball with three non-halal meats (dog, pork, and rat) using multiplex-PCR with the 12S rRNA region designed as target sites.…”