Development and Validation of Multiplex PCR for the Simultaneous Detection of Total Coliform Bacteria, Escherichia coli and Clostridium perfringens in Water
Abstract
Background: Water is an important source of numerous infectious diseases in humans. The most important indicator bacteria include Escherichia coli and total coliform species. Clostridium perfringens is a general indicator of treatment efficiency.
Methods: Diagnosing microbial contamination of water by culture method, in addition to high cost, has low speed and many limitations. The PCR (Polymerase Chain Reaction) method is a promising alternative for simultaneous detection of these bacteria. In this research, we developed multiplex PCR for detection of lacZ, uidA and plc genes as an indicator of the presence of total coliforms, Escherichia coli and Clostridium perfringens. In the following, 33 samples of water and wastewater from Razavi Khorasan province were tested using standard culture methods and this multiplex PCR for comparison.
Results: Multiplex PCR was not only confirmed by cultural results but also has more accuracy and sharpness. The results of the validation tests showed that our multiplex PCR method had a significant advantage over the conventional culture method.
Conclusion: The developed multiplex PCR method demonstrated superior accuracy and efficiency compared to traditional culture techniques, offering a reliable alternative for rapid detection of waterborne pathogens.
2. Mishra RK. Fresh water availability and its global challenge. British J Multidis Adv Stud 2023; 4(3):1-78.
3. Babuji P, Thirumalaisamy S, Duraisamy K, et al. Human health risks due to exposure to water pollution: a review. Water 2023; 15(14):2532.
4. Some S, Mondal R, Mitra D, et al. Microbial pollution of water with special reference to coliform bacteria and their nexus with environment. Energy Nexus 2021; 1:100008.
5. Rice EW, Bridgewater L, Association APH. Standard methods for the examination of water and wastewater: American public health association Washington, DC; 2012.
6. Hijnen WA. Quantitative methods to assess capacity of water treatment to eliminate micro-organisms: IWA publishing; 2010.
7. Lamy MC, Sanseverino I, Niegowska M, et al. Microbiological parameters under the drinking water directive. Publications Office of the European Union: Luxembourg. 2020.
8. Wilkes G, Edge T, Gannon V, et al. Seasonal relationships among indicator bacteria, pathogenic bacteria, Cryptosporidium oocysts, Giardia cysts, and hydrological indices for surface waters within an agricultural landscape. Water Res 2009; 43(8):2209-23.
9. Hijnen WA, Dullemont YJ, Schijven JF, et al. Removal and fate of Cryptosporidium parvum, Clostridium perfringens and small-sized centric diatoms (Stephanodiscus hantzschii) in slow sand filters. Water Res 2007; 41(10):2151-62.
10. Kheiri R, Ranjbar R, Memariani M, et al. Multiplex PCR for detection of water-borne bacteria. Water Sci Tech 2017; 17(1):169-75.
11. Molina F, López-Acedo E, Tabla R, et al. Improved detection of Escherichia coli and coliform bacteria by multiplex PCR. BMC Biotechnol 2015; 15:1-9.
12. Tantawiwat S, Tansuphasiri U, Wongwit W, et al. Development of multiplex PCR for the detection of total coliform bacteria, Escherichia coli, and Clostridium perfringens in drinking water. Southeast Asian J Trop Med Public Health 2005; 36(1):162-9.
13. Bej AK, McCarty SC, Atlas R. Detection of coliform bacteria and Escherichia coli by multiplex polymerase chain reaction: comparison with defined substrate and plating methods for water quality monitoring. Appl Environ Microbiol 1991; 57(8):2429-32.
14. Bisson J, Cabelli V. Membrane filter enumeration method for Clostridium perfringens. Appl Environ Microbiol 1979; 37(1):55-66.
15. Franco-Duarte R, Černáková L, Kadam S, et al. Advances in chemical and biological methods to identify microorganisms—from past to present. Microorganisms 2019; 7(5):130.
16. Abo-Amer A, Soltan ES, Abu-Gharbia M. Molecular approach and bacterial quality of drinking water of urban and rural communities in Egypt. Acta Microbiol Immunol Hung 2008; 55(3):311-26.
17. Rompre A, Servais P, Baudart J, et al. Detection and enumeration of coliforms in drinking water: current methods and emerging approaches. J Microbiol Methods 2002; 49(1):31-54.
18. Tung HY, Chen WC, Ou BR, et al. Simultaneous detection of multiple pathogens by multiplex PCR coupled with DNA biochip hybridization. Lab Anim 2018; 52(2):186-95.
19. Elnifro EM, Ashshi AM, Cooper RJ, et al. Multiplex PCR: optimization and application in agnostic virology. Clin Microbiol Rev 2000; 13(4):559-70.
20. Zhao X, Zhong J, Wei C, et al. Current perspectives on viable but non-culturable state in foodborne pathogens. Front Microbiol 2017; 8:580.
21. Bej AK, Dicesare JL, Haff L, et al. Detection of Escherichia coli and Shigella spp. in water by using the polymerase chain reaction and gene probes for uid. Appl Environ Microbiol 1991; 57(4):1013-7.
22. Dehghan F, Zolfaghari MR, Arjomandzadegan M, et al. Comparison of PCR with standard method (MPN) for detection of bacterial contamination in drinking water. Iran South Med J 2014; 17(5):867-78.
23. Pandove G, Sahota P, Vikal Y, et al. Multiplex PCR water testing kit for rapid, economic and simultaneous detection of Escherichia coli, Yersinia enterocolitica and Aeromonas hydrophila from drinking water. Curr Sci 2013:352-8.
24. El-Leithy M, El-Shatoury EH, El-Senousy W, et al. Detection of six E. coli O157 virulence genes in water samples using multiplex PCR. Egypt J Microbiol 2012; 47:171-88.
25. Zimoń B, Psujek M, Matczak J, et al. Novel multiplex-PCR test for Escherichia coli detection. Microbiol Spec 2024:e03773-23.
| Files | ||
| Issue | Vol 13 No 3 (2025) | |
| Section | Original Articles | |
| DOI | https://doi.org/10.18502/jmb.v13i3.19508 | |
| Keywords | ||
| Clostridium perfringens E. coli Multiplex PCR Coliforms Total coliform Water | ||
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