RSUSSH 2020
IN20-072 Multiplex PCR Assay Using Specific Primers for Species Identification in Meat Products
Presenter: Syed Muhammad Kamal Uddin
University of Malaya, Malaysia
Abstract
Recently, Polymerase Chain Reaction (PCR) has been widely used for species authentication, given their simplicity, specificity, and sensitivity. Multiplex PCR (mPCR) assays allow the simultaneous identification and differentiation of several species by using species-specific primers (forward and reverse primers). The present study aims to develop multiplex (heptaplex) PCR using newly designed specific primers for identification of seven commonly consumed meat, namely, beef, goat, sheep, buffalo, chicken, duck, and pork. Multiple-target detection in a single reaction saves both time and analytical costs. However, the design of primer sets used in mPCR assays is more critical and complicated since all biomarkers must be annealed to their respective targets under a single set of PCR conditions. In this study, seven short length biomarkers (73 - 263 bp) were designed for each of cow (106 bp), goat (236 bp), buffalo (138 bp), sheep (263 bp), chicken ( 161 bp), duck ( 203 bp), and pig (73 bp) to develop mPCR assay targeting mitochondrial ND5 and cytb genes. After downloading the sequences from the National Center for Biotechnology Information (NCBI) website, the primer3 v.0.4.0 software was used. The designed primers were verified for specificity in-silico against 7 targets and 19 non-target species. The complete sequence matched only with the target species whereas considerable mismatching was observed with other non-targets. The pairwise distances analyzed through the neighbor-joining method reflected enough genetic distances among the studied species, eliminating the possibility of any cross-target amplification and thus confirming the target detection by the developed mPCR assay system.
Citation format:
Uddin, S., Chowdhury, Z., Kazi, S., Hossain, M., & Johan, R.. (2020). Multiplex PCR Assay Using Specific Primers for Species Identification in Meat Products. Proceeding in RSU International Research Conference, May 1, 2020. Pathum Thani, Thailand.QUESTIONS & ANSWERS
- Your work is very interesting and useful to confirm Halal status. But your presentation is not interesting. You are lack of the slides presentation. Many audiences do not catch up your detail or you work. So in my opinion,the slide presentation is very important.
- This work is very interesting but there are many works that use PCR for species identification in meat products. So, what are the different points of this work? What are the advantages point of your work?
Answer to question:
Thanks to honourable chairperson for evaluating my presentation and providing valuable suggestions to make improvement.
Although there are many papers using PCR for species identification, our work is different and also advantageous in the context of the following points:
# First to report heptaplex PCR for detecting seven most commonly consumed meat items like beef, buffalo, goat, sheep, chicken, duck and pork.
# Use of seven newly designed species-specific primers for target amplification.
# Use of short-length biomarkers (< 263 bp) that could be amplified in case of DNA degradation even under severe heat treatment in processed food products instead of long-length ones that are relatively less stable.