Primer design, in silico PCR and optimum annealing temperature for Escherichia coli detection in refillable drinking water samples

Afifatul Achyar; Annisa Irna   Putri; Dwi Hilda Putri; Yuni Ahda

Afifatul Achyar Universitas Negeri Padang
Annisa Irna Putri Universitas Negeri Padang
Dwi Hilda Putri Universitas Negeri Padang
Yuni Ahda Universitas Negeri Padang

Keywords: E.coli, primer design, PCR in silico, annealing

Abstract

Refill Drinking Water Depots (DAMIU) in the community are easy to find at affordable prices, which is a concern in the feasibility of refill drinking water quality. E.coli is one of the pathogenic bacteria present in drinking water that has poor quality. When it enters the body it can cause symptoms of diarrhea, fever, vomiting and others. The purpose of this study was to create a specific primer for E.coli that can be used to detect E.coli and determine the optimum temperature of primer annealing. Researchers do a new primer design because the existing design does not necessarily produce the same results due to various factors of different experimental conditions. The primer design was carried out on an in silico-based and had to meet the criteria for a good primer because it would be used in vitro. E.coli gene sequences was aligned with Shigella sp. using Pairwise Alignment. Primer candidates were analyzed using NCBI's Primer3 and Geneious Prime tools. The result is that the first primer pair is forward 5'–ATGCAGTGGTTCCTTATCTCACA-3' reverse 5'- ATCCTTAATGGCACTGCGCT-3', amplifying the amplicons along 417 bp in the yraJ gene. The second primer pair, forward 5'-CAGAACGTTTTTCATTCAGCAGG-3' reverse 5'-GCCACTACCAGATCGAGTCA-3' amplifies the 573 bp amplicon in the rne gene. The optimum annealing temperature for both pairs of primers was 59.5^oC.

References

Abd-El-Haleem, D., Kheiralla, Z. H., Zaki, S., Rushdy, A. A., & Abd-El-Rahiem, W. (2003). Multiplex-PCR and PCR-RFLP assays to monitor water quality against pathogenic bacteria. J. Environ. Monit, 5, 865–870.

Achyar, A., Atifah, Y., & Putri, D. H. (2021). In silico study of developing a method for detecting pathogenic bacteria in refillable drinking water samples. Journal of Physics: Conference Series (Vol. 1940, No. 1, p.012061). IOP Publishing.

Agrippina, Fidela Devina. (2019). Identifikasi coliform dan Escherichia coli pada air minum dalam kemasan (AMDK) di Bandar Lampung. Majalah Teknologi Agro Industri (Tegi), 11(2), 54-57.

Agustia, T., Zahra, V. D., Hardin, W. M., Irfanolla, Y., Saputra, A., Edgar, N. R., et al. (2019). Kualitas Air Minum Yang Diproduksi Depot Air Minum Isi Ulang di Universitas Negeri Padang Berdasarkan Persyaratan Mikrobiologi. Jurnal Kapita Selekta Geografi, 2(5), 1-6

Athena, Sukar, & Haryono. (2004). Kandungan bakteri total coli dan Eschereclria coli/ fecal coli air minum dari depot air minum isi ulang di jakarta, tangerang, dan bekasi. Bul. Penel. Kesehatan, 32(3), 135-143.

Borah, P., (2011). Primer designing for PCR. Science Vision., vol. 11(3): 134-136.

BPOM. RI. (2008). Pengujian mikrobiologi pangan. Pusat Pengujian Obat dan Makanan. Jakarta: Badan Pengawasan Obat dan Makanan Republik Indonesia.

Bustin, S., & Huggett, J. (2017). qPCR primer design revisited. Biomolecular Detection and Quantification, 14, 19-28.

Entjang, I. (2003). Mikrobiologi dan Parasitologi untuk Akademi Keperawatan dan Sekolah Tenaga Kesehatan yang Sederajat. Bandung: PT. Citra Aditya Bakti.

Handoyo, D., & Rudiretna, A. (2000). Prinsip umum dan pelaksanaan polymerase chain reaction (PCR) [General principles and implementation of polymerase chain reaction]. Unitas, 9(1), 17-29.

Kearse Matthew, Richard M, Amy W, S Stones-Havas, M Cheung, S Sturrock, S Buxton, A Cooper, S Markowitz, C Duran, Tobias T, B Ashton, Peter M, and A Drummond. (2012). Geneious Basic: An integrated and extendable desktop software platform for the organization and analysis of sequence data. Bioinformatics. 28(12): 1647–1649.

Kementrian Kesehatan Republik Indonesia. (2010). Peraturan Menteri Kesehatan Republik Indonesia Nomor 492/MENKES/PER/IV/2010 Tentang Persyaratan Kualitas Air Minum. Jakarta, Indonesia.

Melliawati, R. (2009). Escherichia coli dalam kehidupan manusia. BioTrends, 4(1), 10-14.

Muhsinin, S., Sulastri, M. M., & Dadih, S. (2018). Deteksi cepat gen InvA pada Salmonella spp. dengan metode PCRm. Jurnal Sains Farmasi & Klinis, 5(3), 191-200.

Pesurnay, Y. (2018). Deteksi keberadaan bakteri Escherichia coli O157:H7 pada air galon penderita diare dengan metode polymerase chain reaction (PCR). Bimafika, 9, 23-26.

Raharja, Z. T. (2015). Identifikasi Escherichia coli pada Air Minum Isi Ulang dari Depot di Kelurahan Pisangan dan Cirendeu Tahun 2015. Jakarta: Program Studi Pendidikan Dokter Fakultas Kedokteran dan Ilmu Kesehatan Universitas Islam Negeri Syarif Hidayatullah.

Stadhouders, R., Pas, S. D., Anber, J., Voermans, J., Mes, T. H., & Schutten, M. (2010). The effect of primer-template mismatches on the detection and quantification of nucleic acids using the 5′ nuclease assay. The Journal of Molecular Diagnostics, 12(1), 109-117.

Sutiknowati, L. I. (2016). Bioindikator pencemar, bakteri Escherichia coli. Jurnal Oseana, 41(4), 63-71.

Syamsurizal, S., Ardi, A., M, D., Fevria R., Atifah, Y., Badriyya, E., Achyar, A. 2021. Design of primer Ipomoea batatas chloroplast gene matK. Tropical Genetics, 1(1): 12-16.

Winata, E., & Hartantyo, E. (2013). Kualitas air tanah disepanjang kali gajah wong ditinjau dari pola sebaran Escherichia coli (studikasus kecamatan umbulharjo). Jurnal Fisika Indonesia, XVII (50), 9-11.