APPLICATION MULTIPLEX PCR FOR EARLY DETECTION ESCHERICHIA COLI CONTAMINATION IN SOME DRINKING WATER RESOURCES IN ABEPURA, PAPUA INDONESIA

Arsyam Mawardi; I Made Budi; Daniel Lantang

doi:10.26418/jpmipa.v14i2.59129

APPLICATION MULTIPLEX PCR FOR EARLY DETECTION ESCHERICHIA COLI CONTAMINATION IN SOME DRINKING WATER RESOURCES IN ABEPURA, PAPUA INDONESIA

Arsyam Mawardi, I Made Budi, Daniel Lantang

Abstract

Abstract

Microbial detection takes a long time to produce positive results, so a quicker detection method was chosen. Multiplex polymerase chain reaction (m-PCR) identified bacterial strains in less than 24 hours, detects E.coli specifically, and is faster than traditional methods. The goal of this study was to use m-PCR to detect early pathogenic E.coli bacteria in several drinking water sources in the Abepura district of Papua Indonesia as a parameter of pollution and water quality. The Chelex100 and microwave combination method was used to extract DNA. The first round of testing was done at four different concentrations: 0.125, 0.250, 0.375, and 0.500 M. The purity of the extracted DNA was good, ranging from 1.80 to 1.94. The optimum primer concentration for multiplexing applications is 0.25 uM for lt primer; 0.125 uM for stx2 and eae primer, with an annealing temperature of 55oC. m-PCR has been shown to quickly detect pathogenic E. coli in water samples. In the PCR process, E.coli DNA template was obtained with high purity (1.80-1.94) and concentration (576.9-4301.6 ng/uL) . Each multiplex set included three primer pairs for the target gene lt-eae-stx2 on ETEC-EPEC and EHEC respectively. The m-PCR process showed excellent results, and this findings can be considered as a reference for water analysis in several drinking sources in Papua Province.

Keywords

Drinking water resources, early detection, m-PCR, pathogenic E.coli

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References

Anggreini, R. (2015). Analisis cemaran bakteri Escherichia coli (E.coli) O157:H7 pada daging sapi di kota Makassar. Makassar: Universitas Hasanuddin.

Arlita, Y. (2014). Identifikasi Bakteri Escherichia coli Dan Salmonella Sp. Pada Makanan Jajanan Bakso Tusuk Di Kota Manado. Jurnal E-Biomedik, 2(1), 9–14.

Bintari, S. H., Fibriana, F., Mustikaningtyas, D., & Iswari, R. S. (2014). PCR Approach for Rapid Detection of Escherichia coli in Tempe Using a Specific Primer. Berkala Penelitian Hayati, 19(2), 54–58.

Bio-Rad Laboratories. (2017). Chelex 100 Chelating Ion Exchange Resin Instruction Manual. Bio-Rad Laboratories, 1–24. https://www.bio-rad.com/webroot/web/pdf/lsr/literature/LIT200.pdf

Biswas, S., Parvez, M. A. K., Shafiquzzaman, M., Nahar, S., & Rahman, M. N. (2010). Isolation and characterization of Escherichia coli in ready-to-eat foods vended in Islamic University, Kushtia. Journal of Bio-Science, 18(1), 99–103.

BPPS. (2020). Kota Jayapura Dalam Angka 2019. Jayapura: Badan Pusat Statistik.

Chandra, B., & Widyastuti, P. (2007). Pengantar kesehatan lingkungan (1st ed.). Jakarta: EGC.

Fedio, W. M., Jinneman, K. C., Yoshitomi, K. J., Zapata, R., Wendakoon, C. N., Browning, P., & Weagant, S. D. (2011). Detection of E. coli O157:H7 in Raw Ground Beef by PathatrixTM Immunomagnetic-Separation, Real-Time PCR and Cultural Methods. International Journal of Food Microbiology, 148(2), 87–92.

Food and Drug Administration. (2012). Bad Bug Book, Foodborne Pathogenic Microorganisms and Natural Toxins (K. A. Lampel, S. Al-Khaldi, & S. M. Cahill (eds.); 2nd ed.). Silver Spring.

Fratamico, P. M., Bagi, L. K., Cray, W. C., Narang, N., Yan, X., Medina, M., & Liu, Y. (2011). Detection by Multiplex Real-Time Polymerase Chain Reaction Assays and Isolation of Shiga Toxin-Producing Escherichia coli Serogroups O26, O45, O103, O111, O121, and O145 in Ground Beef. Foodborne Pathogens and Disease, 8(5), 601–607.

Fratamico, P. M., & DebRoy, C. (2010). Detection of Escherichia coli O157:H7 in Food Using Real-Time Multiplex PCR Assays Targeting the stx 1, stx 2, wzy O157, and the fliC h7 or eae Genes. Food Analytical Methods, 3(4), 330–337.

Huang, D. B., Mohanty, A., DuPont, H. L., Okhuysen, P. C., & Chiang, T. (2006). A Review of an Emerging Enteric Pathogen: Enteroaggregative Escherichia coli. Journal of Medical Microbiology, 55(10), 1303–1311.

Kagkli, D. M., Folloni, S., Barbau-Piednoir, E., van den Eede, G., & van den Bulcke, M. (2012). Towards a Pathogenic Escherichia coli Detection Platform Using Multiplex SYBR®green Real-Time PCR Methods and High Resolution Melting Analysis. PLoS ONE, 7(6).

Luyt, C. E., Hékimian, G., Bonnet, I., Bréchot, N., Schmidt, M., Robert, J., Combes, A., & Aubry, A. (2020). Usefulness of Point-Of-Care Multiplex PCR to Rapidly Identify Pathogens Responsible for Ventilator-Associated Pneumonia and Their Resistance to Antibiotics: an Observational Study. Critical Care, 24(1), 378.

Madic, J., Vingadassalon, N., de Garam, C. P., Marault, M., Scheutz, F., Brugère, H., Jamet, E., & Auvray, F. (2011). Detection of Shiga toxin-Producing Escherichia coli Serotypes O26:H11, O103:H2, O111:H8, O145:H28, and O157:H7 in Raw-Milk Cheeses by Using Multiplex Real-Time PCR. Applied and Environmental Microbiology, 77(6), 2035–2041.

Mohammed, M. A. M. (2012). Molecular Characterization of Diarrheagenic Escherichia coli Isolated from Meat Products Sold at Mansoura City, Egypt. Food Control, 25(1), 159–164.

Radji, M., Puspaningrum, A., & Sumiati, A. (2010). Deteksi Cepat Bakteri Escherichia coli dalam Sampel Air dengan Metode Polymerase Chain Reaction Menggunakan Primer 16E1 dan 16E2. Makara of Science, 14(1), 39–43.

Rathnayaka, R. M. U. S. K. (2011). Evaluation of Five Dna Extraction Metods in the Detection of. The Journal of Agricultural Sciences, 6(1), 24–31.

Reyes-Escogido, L., Balam-Chi, M., Rodríguez-Buenfil, I., Valdés, J., Kameyama, L., & Martínez-Pérez, F. (2010). Purification of Bacterial Genomic DNA in less than 20 min Using Chelex-100 Microwave: Examples from Strains of Lactic Acid Bacteria Isolated from Soil Samples. Antonie van Leeuwenhoek, International Journal of General and Molecular Microbiology, 98(4), 465–474.

Russo, P., Botticella, G., Capozzi, V., Massa, S., Spano, G., & Beneduce, L. (2014). A Fast, Reliable, and Sensitive Method for Detection and Quantification of Listeria Monocytogenes and Escherichia coli O157:H7 in Ready-to-Eat Fresh-Cut Products by MPN-qPCR. BioMed Research International, 2014.

Sen, K., Sinclair, J. L., Boczek, L., & Rice, E. W. (2011). Development of a Sensitive Detection Method for Stressed E. coli O157:H7 in Source and Finished Drinking Water by Culture-qPCR. Environmental Science and Technology, 45(6), 2250–2256.

Shen, Z., Hou, N., Jin, M., Qiu, Z., Wang, J., Zhang, B., Wang, X., Wang, J., Zhou, D., & Li, J. (2014). A Novel Enzyme-Linked Immunosorbent Assay for Detection of Escherichia coli O157:H7 Using Immunomagnetic and Beacon Gold Nanoparticles. Gut Pathogens, 6(1), 1–8.

Sjöling, Å., Sadeghipoorjahromi, L., Novak, D., & Tobias, J. (2015). Detection of Major Diarrheagenic Bacterial Pathogens by Multiplex PCR Panels. Microbiological Research, 172, 34–40.

Soemirat, J. (2018). Kesehatan Lingkungan. Yogyakarta: UGM Press.

Teertstra, W. R., Tegelaar, M., Dijksterhuis, J., Golovina, E. A., Ohm, R. A., & Wösten, H. A. B. (2017). Maturation of Conidia on Conidiophores of Aspergillus niger. Fungal Genetics and Biology, 98, 61–70.

Torres, A. G., Arenas-Hernandez, M. M., & Martinez-Laguna, Y. (2010). Overview of Escherichia coli: Pathogenic Escherichia coli Latin in America: Bentham Sciences Publisher.

Yunita, N. A. (2015). Identifikasi dan Karakterisasi Bahaya Bakteri Patogen pada Pangan Jajanan Anak Sekolah di Bogoritle. Tesis: Institut Pertanian Bogor

Yuwono, T. (2006). Teori dan Aplikasi Polymerase Chain Reaction: Panduan Eksperimen PCR untuk Memecahkan Masalah Biologi Terkini. Yogyakarta: ANDI.

DOI: http://dx.doi.org/10.26418/jpmipa.v14i2.59129