Development of rapid and simultaneous detection of four major foodborne pathogens using a multiplex PCR method

Document Type : Original Article

Authors

1 Food and Drug Administration, Shiraz University of Medical Sciences, Shiraz, Iran

2 Department of Biology, Marvdasht Branch, Islamic Azad University, Marvdasht, Iran

3 Center for Nanotechnology in Drug Delivery, Shiraz University of Medical Sciences, Shiraz, Iran

4 Department of Pharmacology and Toxicology, Faculty of Pharmacy, Shiraz University of Medical Sciences, Shiraz, Iran

Abstract

Food borne diseases are an important public health problem has major impacts on human health, also affect trade and economic issues. Developing microbial cultures to detect foodborne pathogens is time-consuming and expensive. The aim of this study is to develop a multiplex (mPCR) method for the simultaneous detection of Staphylococcus aureus, Escherichia coli, Listeria monocytogenes and Salmonella enteritidis. Buffered peptone water (BPW) was used as pre-enrichment. Simplex and multiplex PCR settings were optimized and applied to both pure co-cultures and artificially inoculated ready-to-eat food samples (falafel and chicken nugget). The four microorganisms could be detected individually and in enrichment media artificially inoculated at 101 CFU/mL by mPCR. In conclusion, the individual and combined growth of E. coli, S. enterica, S. aureus and, L. monocytogenes with low levels of contamination in the presence of food matrices such as falafel and chicken nuggets is effectively supported by BPW broth as co-culture medium before mPCR detection. The proposed protocol for pre-enrichment of E. coli, S. enterica, S. aureus and, L. monocytogenes in takes approximately 34 hours , compared to culture methods that require at least 7 days. This notably reduces analysis time, effort, and cost.

Highlights

Marzieh Rashedinia (Google Scholar)

 

Keywords

References

1.    Li P, Feng X, Chen B, Wang X, Liang Z, Wang L. The Detection of Foodborne Pathogenic Bacteria in Seafood Using a Multiplex Polymerase Chain Reaction System. Foods. 2022; 11(23):3909.
2.    Ruppitsch W, Pietzka A, Cabal A, Chakeri A, Schmid D, Lakicevic B, et al. Advances in foodborne outbreak investigation and source tracking using whole genome sequencing.  IOP Conf. Ser.: Earth Environ Sci. 2019: 333
3.    Wei C, Zhong J, Hu T, Zhao X. Simultaneous detection of Escherichia coli O157:H7, Staphylococcus aureus and Salmonella by multiplex PCR in milk. 3 Biotech. 2018 Jan;8(1):76. doi: 10.1007/s13205-018-1086-5. Epub 2018 Jan 13. PMID: 29354387; PMCID: PMC5767162.
4.    Lei I-F, Roffey P, Blanchard C, Gu K. Development of a multiplex PCR method for the detection of six common foodborne pathogens. J Food Drug Anal. 2008;16(4):6.
5.    Ding T, Suo Y, Zhang Z, Liu D, Ye X, Chen S, Zhao Y. A Multiplex RT-PCR Assay for S. aureus, L. monocytogenes, and Salmonella spp. Detection in Raw Milk with Pre-enrichment. Front Microbiol. 2017 May 31;8:989. doi: 10.3389/fmicb.2017.00989. PMID: 28620364; PMCID: PMC5449760.
6.    Okafor AC, Ogbo FC. Occurrence and enumeration of multiple bacterial pathogens in edible snails from South East Nigeria. Food Sci Technol. 2019;7:23-30.
7.    Zeng D, Chen Z, Jiang Y, Xue F, Li B. Advances and Challenges in Viability Detection of Foodborne Pathogens. Front Microbiol. 2016 Nov 22;7:1833. doi: 10.3389/fmicb.2016.01833. PMID: 27920757; PMCID: PMC5118415.
8.    Latha C, Anu CJ, Ajaykumar VJ, Sunil B. Prevalence of Listeria monocytogenes, Yersinia enterocolitica, Staphylococcusaureus, and Salmonella enterica Typhimurium in meat and meat products using multiplex polymerase chain reaction. Vet World. 2017 Aug;10(8):927-931. doi: 10.14202/vetworld.2017.927-931. Epub 2017 Aug 16. PMID: 28919685; PMCID: PMC5591481.
9.    Dwivedi HP, Jaykus LA. Detection of pathogens in foods: the current state-of-the-art and future directions. Crit Rev Microbiol. 2011 Feb;37(1):40-63. doi: 10.3109/1040841X.2010.506430. Epub 2010 Oct 7. PMID: 20925593.
10.    Morshedi G, Ghanbarinejad V, Zareei M, Poornajaf A, Hassanian-Moghaddam H, Hadi V, Isvand A, Saadati MR, Maleki H, Keshavarzi M. PCR based identification of exotoxin A-producing Pseudomonas aeroginosa isolated from burn wound infection. Trends in Pharmaceutical Sciences. 2022 Mar 1;8(1):51-6.
11.    Odooli S, Safari A, Kargar M, Ghasemi Y. Selective detection, isolation and enumeration of Bifidobacterium animalis subsp. lactis BB-12 from Iranian commercial probiotic yoghurts. Trends in Pharmaceutical Sciences. 2016 Dec 1;2(4):265-76.
12.    Chauhan N, Singh C, Chauhan D, Chaudhary J, Rawat P. Modern techniques and developments in the detection of foodborne pathogens. InBiosensors for Foodborne Pathogens Detection 2024 Jan 1 (pp. 17-55). Academic Press.
13.    Chen Y, Wang Z, Shi Q, Huang S, Yu T, Zhang L, Yang H. Multiplex PCR method for simultaneous detection of five pathogenic bacteria closely related to foodborne diseases. 3 Biotech. 2021 May;11(5):219. doi: 10.1007/s13205-021-02759-y. Epub 2021 Apr 16. PMID: 33968564; PMCID: PMC8052391.
14.    Boukharouba A, González A, García-Ferrús M, Ferrús MA, Botella S. Simultaneous Detection of Four Main Foodborne Pathogens in Ready-to-Eat Food by Using a Simple and Rapid Multiplex PCR (mPCR) Assay. Int J Environ Res Public Health. 2022 Jan 18;19(3):1031. doi: 10.3390/ijerph19031031. PMID: 35162055; PMCID: PMC8834630.
15.    Martzy R, Bica-Schröder K, Pálvölgyi ÁM, Kolm C, Jakwerth S, Kirschner AKT, et al. Simple lysis of bacterial cells for DNA-based diagnostics using hydrophilic ionic liquids. Sci Rep. 2019 Sep 30;9(1):13994. doi: 10.1038/s41598-019-50246-5. PMID: 31570727; PMCID: PMC6768989.
16.    Park YS, Lee SR, Kim YG. Detection of Escherichia coli O157:H7, Salmonella spp., Staphylococcus aureus and Listeria monocytogenes in kimchi by multiplex polymerase chain reaction (mPCR). J Microbiol. 2006 Feb;44(1):92-7. PMID: 16554723.
17.    Wu SJ, Chan A, Kado CI. Detection of PCR amplicons from bacterial pathogens using microsphere agglutination. J Microbiol Methods. 2004 Mar;56(3):395-400. doi: 10.1016/j.mimet.2003.11.005. PMID: 14967231.
18.    Molinaro AM. Diagnostic tests: how to estimate the positive predictive value. Neurooncol Pract. 2015 Dec;2(4):162-166. doi: 10.1093/nop/npv030. Epub 2015 Sep 7. PMID: 31386059; PMCID: PMC6664615.
19.    Hernández Hernández O, Gutiérrez-Escolano AL, Cancio-Lonches C, Iturriaga MH, Pacheco-Aguilar JR, Morales-Rayas R, et al. Multiplex PCR method for the detection of human norovirus, Salmonella spp., Shigella spp., and shiga toxin producing Escherichia coli in blackberry, coriander, lettuce and strawberry. Food Microbiol. 2022 Apr;102:103926. doi: 10.1016/j.fm.2021.103926. Epub 2021 Oct 19. PMID: 34809952.
20.    Wang Y, Suo B. A new 7-plex PCR assay for simultaneous detection of shiga toxin-producing Escherichia coli O157 and Salmonella Enteritidis in meat products.J Verbrauch Lebensm. 2011;6:441-7.
21.    Alarcón B, García-Cañas V, Cifuentes A, González R, Aznar R. Simultaneous and sensitive detection of three foodborne pathogens by multiplex PCR, capillary gel electrophoresis, and laser-induced fluorescence. J Agric Food Chem. 2004 Nov 17;52(23):7180-6. doi: 10.1021/jf049038b. PMID: 15537335.
22.    Chen J, Tang J, Bhunia AK, Tang C, Wang C, Shi H. Development of a multi-pathogen enrichment broth for simultaneous growth of five common foodborne pathogens. J Gen Appl Microbiol. 2015;61(6):224-31. doi: 10.2323/jgam.61.224. PMID: 26782652.
23.    Zilelidou E, Manthou E, Skandamis P. Growth differences and competition between Listeria monocytogenes strains determine their predominance on ham slices and lead to bias during selective enrichment with the ISO protocol. Int J Food Microbiol. 2016 Oct 17;235:60-70. doi: 10.1016/j.ijfoodmicro.2016.07.016. Epub 2016 Jul 12. PMID: 27434679.
24.    Garrido A, Chapela MJ, Román B, Fajardo P, Vieites JM, Cabado AG. In-house validation of a multiplex real-time PCR method for simultaneous detection of Salmonella spp., Escherichia coli O157 and Listeria monocytogenes. Int J Food Microbiol. 2013 Jun 3;164(1):92-8. doi: 10.1016/j.ijfoodmicro.2013.03.024. Epub 2013 Apr 3. PMID: 23624537.
Trends in Pharmaceutical Sciences
Volume 10, Issue 2
June 2024
Pages 83-90

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