In Silico Analysis of Actin Gene as a Candidate for DNA Non-Halal Detection Base on Real-Time PCR

Seagames Waluyo; Jekmal Malau; Muhareva Raekiansyah; Edwin Yulian; Imam Hardiman

doi:10.15575/ijhar.v3i2.12123

Authors

Seagames Waluyo PT. Sciencewerke Indonesia, Indonesia
Jekmal Malau PT. Sciencewerke Indonesia, Indonesia
Muhareva Raekiansyah PT. Sciencewerke Indonesia, Indonesia
Edwin Yulian PT. Sciencewerke Indonesia, Indonesia
Imam Hardiman PT. Sciencewerke Indonesia & Universitas Prof. Dr. HAMKA, Indonesia

DOI:

https://doi.org/10.15575/ijhar.v3i2.12123

Keywords:

Actin gene, halal product, real-time PCR, uMelt

Abstract

Actin genes are genes that are common in organisms, and their expression is constitutive. These genes are used for gene normalization and internal control of DNA extraction, but the actin gene is not widely used for halal certification tests. Bioinformatic studies help to analyze the experiment through in silico more deeply before the experiment is carried out in laboratory, making it more efficient and time effective. uMelt is an analysis to predict the melting curve of target amplification in real-time PCR. Real-time PCR has been widely used for screening and detection of pork content in a product. This research aimed to explore actin gene as a candidate for testing pork using qPCR. The study was carried out in two main stages, namely alignment of the DNA sequence and analysis of the melting curve using the uMelt approach. The results showed a set of actin genes containing conserved regions that can be used as degenerate primers with different family-type coverages. Melting curve prediction with uMelt shows differences in tm peaks so as the types of samples can be easily identified. The use of bioinformatic applications such as uMelt helps in the simulation of predicting the melting curve to increase the precision of the analysis.

References

Aminuddin, M. Z. (2016). Sertifikasi Produk Halal: Studi Perbandingan Indonesia dan Thailand. SHAHIH: Journal of Islamicate Multidisciplinary, 1(1), 2527-8126.

Ballin, N. Z., Vogensen, F. K., & Karlsson, A. H. (2009). Species determination - Can We Detect and Quantify Meat Adulteration?. Meat Science 83(2), 165-174.

Bustin, S. A. (2004). A-Z of quantitative PCR. La Jolla, CA: International University Line.

Bustin, S., & Huggett, J. (2017). qPCR Primer Design Revisited. In Biomolecular Detection and Quantification 14, 19-28.

Cai, Y., Li, X., Lv, R., Yang, J., Li, J., He, Y., & Pan, L. (2014). Quantitative Analysis of Pork and Chicken Products by Droplet Digital PCR. BioMed Research International, 14, 1-6.

Dwight, Z. L., Palais, R., Kent, J., & Wittwer, C. T. (2014). Heterozygote PCR Product Melting Curve Prediction. Human Mutation, 35(3), 278-282.

Faridah, H. D. (2019). Sertifikasi Halal Di Indonesia: Sejarah, Perkembangan dan Implementasi. Journal of Halal Product and Research, 2(2), 68-78.

Hall, T. A. (2007). BioEdit: a User-Friendly Biological Sequence Alignment Editor and Analysis Program for Windows 95/98/NT. Nucl Acids Symp Ser 41: 95-98 (1999). Nucleic Acids Symposium Series, 41(41), 95-98.

Kim, M. J., & Kim, H. Y. (2019). A fast multiplex real-time PCR assay for simultaneous detection of pork, chicken, and beef in commercial processed meat products. LWT, 114, 1-6.

Kim, Y. S., Yu, H. K., Lee, B. Z., & Hong, K. W. (2018). Effect of DNA extraction methods on the detection of porcine ingredients in halal cosmetics using real-time PCR. Applied Biological Chemistry, 61(5), 549-555.

Mohamad, N. A., Mustafa, S., Khairil Mokhtar, N. F., & El Sheikha, A. F. (2018). Molecular beacon-based real-time PCR method for detection of porcine DNA in gelatin and gelatin capsules. Journal of the Science of Food and Agriculture, 98(12), 4570-4577.

Mustaqimah, D. N., Septiani, T., & Roswiem, A. P. (2021). Deteksi Dna Babi Pada Produk Sosis Menggunakan Real Time-Polymerase Chain Reaction (Rt-Pcr). Indonesia Journal of Halal, 3(2), 106-111.

Panina, Y., Germond, A., Masui, S., & Watanabe, T. M. (2018). Validation of Common Housekeeping Genes as Reference for qPCR Gene Expression Analysis During iPS Reprogramming Process. Scientific Reports, 8(1), 1-8.

Rachmawati, Y., Rokhim, S., Munir, M., & Agustina, E. (2018). Deteksi Kontaminan Fragmen Dna Pengkode Cyt B Babi Pada Sampel Softgell Candy Tak Berlabel Halal. Indonesia Journal of Halal, 1(1), 25-30.

Raharjo, T. J., Chudori, Y. N. C., & Agustina, F. W. (2019). TaqMan Probe Real-Time Polymerase Chain Reaction Targeting the ATPase 6 Gene for the Detection of Pork Adulteration in Meat and Meatballs. Journal of Food Safety, 39(6), 1-6.

RebouÃ§as, E. de L., Costa, J. J. do N., Passos, M. J., Passos, J. R. de S., Hurk, R. van den, & Silva, J. R. V. (2013). Real Time PCR and Importance of Housekeepings Genes for Normalization and Quantification of mRNA Expression in Different Tissues. Brazilian Archives of Biology and Technology, 56(1), 143-154.

Septiani, T. (2021). Identification of Rat Meatballs in Traditional Market in Area of Jakarta Using Real Time - Pcr. Indonesia Journal of Halal, 3(1), 94-99.

Sudjadi, Wardani, H. S., Sepminarti, T., & Rohman, A. (2016). Analysis of Porcine Gelatin DNA in a Commercial Capsule Shell Using Real-Time Polymerase Chain Reaction for Halal Authentication. International Journal of Food Properties, 19(9), 2127-2134.

Tan, L. L., Ahmed, S. A., Ng, S. K., Citartan, M., Raabe, C. A., Rozhdestvensky, T. S., & Tang, T. H. (2020). Rapid Detection of Porcine DNA in Processed Food Samples Using a Streamlined DNA Extraction Method Combined With the SYBR Green Real-Time PCR assay. Food Chemistry, 309, 1-27.

Tanabe, S., Hase, M., Yano, T., Sato, M., Fujimura, T., & Akiyama, H. (2007). A real-time quantitative PCR detection method for pork, chicken, beef, mutton, and horseflesh in foods. Bioscience, Biotechnology and Biochemistry, 71(12), 3131-3135.

Taylor, S., Wakem, M., Dijkman, G., Alsarraj, M., & Nguyen, M. (2010). A Practical Approach to RT-qPCR-Publishing Data that conform to the MIQE guidelines. In Methods 50(4),

Waluyo, S., Sustiprijatno, S., & Suharsono, S. (2016). Transformasi Genetik Tembakau Dengan Gen Cold Shock Protein Melalui Perantara Agrobacterium tumefaciens. Jurnal AgroBiogen, 9(2), 58-65.

Widayat, W., Winarni Agustini, T., Suzery, M., Ni'matullah Al-Baarri, A., & Rahmi Putri, S. (2019). Real Time-Polymerase Chain Reaction (RT-PCR) sebagai Alat Deteksi DNA Babi dalam Beberapa Produk Non-Pangan. Indonesia Journal of Halal, 2(1), 26-33.

Zhu, L., Zhang, Y., Hu, Y., Wen, T., & Wang, Q. (2013). Dynamic actin gene family evolution in primates. BioMed Research International, 2013, 1-11.