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Simultaneous detection of Salmonella spp., Pseudomonas aeruginosa, Bacillus cereus, and Escherichia coli O157:H7 in environmental water using PMA combined with mPCR

  • Microbial Ecology and Environmental Microbiology
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Abstract

A multiplex polymerase chain reaction (mPCR) with propidium monoazide (PMA) and internal amplification control (IAC) for the simultaneous detection of waterborne pathogens Salmonella spp., Pseudomonas aeruginosa, Bacillus cereus, and Escherichia coli O157:H7, was developed. This PMA-IAC-mPCR assay used four new specific primers based on the genes for invA, ecfX, cesB, and fliC, respectively. A 16S rRNA primer was chosen for IAC to eliminate false negative results. The photosensitive dye, propidium monoazide (PMA) was used to exclude signals from dead bacteria that could lead to false positive results. In pure culture, the limits of detection (LOD) were 101 CFU/ml for P. aeruginosa, 102 CFU/ml for both Salmonella spp. and E. coli O157:H7, and 103 CFU/ml for B. cereus, respectively. In addition, with a 6–8 h enrichment of all four bacteria that were combined in a mixture that was spiked in water sample matrix, the LOD was 3 CFU/ml for Salmonella spp., 7 CFU/ml for E. coli O157:H7, 10 CFU/ml for B. cereus and 2 CFU/ml for P. aeruginosa. This PMA-IAC-mPCR assay holds potential for application in the multiplex assay of waterborne pathogens.

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References

  • Aw, T.G. and Rose, J.B. 2012. Detection of pathogens in water: from phylochips to qPCR to pyrosequencing. Curr. Opin. Biotechnol.23, 422–430.

    Article  CAS  Google Scholar 

  • Bonetta, S., Borelli, E., Bonetta, S., Conio, O., Palumbo, F., and Carraro, E. 2011. Development of a PCR protocol for the detection of Escherichia coli O157: H7 and Salmonella spp. in surface water. Environ. Monit. Assess.177, 493–503.

    Article  CAS  Google Scholar 

  • Chen, S., Wang, F., Beaulieu, J.C., Stein, R.E., and Ge, B. 2011. Rapid detection of viable salmonellae in produce by coupling propidium monoazide with loop-mediated isothermal amplification. Appl. Environ. Microbiol.77, 4008–4016.

    Article  CAS  Google Scholar 

  • Chiang, Y.C., Yang, C.Y., Li, C., Ho, Y.C., Lin, C.K., and Tsen, H.Y. 2006. Identification of Bacillus spp., Escherichia coli, Salmonella spp., Staphylococcus spp. and Vibrio spp. With 16S ribosomal DNA-based oligonucleotide array hybridization. Int. J. Food Microbiol.107, 131–137.

    Article  CAS  Google Scholar 

  • de Freitas, C.G., Santana, Â.P., da Silva, P.H., Gonçalves, V.S., Barros, M., Torres, F.A., Murata, L.S., and Perecmanis, S. 2010. PCR multiplex for detection of Salmonella Enteritidis, Typhi and Typhimurium and occurrence in poultry meat. Int. J. Food Microbiol.139, 15–22.

    Article  Google Scholar 

  • Deer, D.M. and Lampel, K.A. 2010. Development of a multiplex real-time PCR assay with internal amplification control for the detection of Shigella species and enteroinvasive Escherichia coli. J. Food Prot.73, 1618–1625.

    Article  CAS  Google Scholar 

  • Feng, K., Hu, W., Jiang, A., Sarengaowa, Xu, Y., Zou, Y., Yang, L., and Wang, X. 2016. A dual filtration-based multiplex PCR method for simultaneous detection of viable Escherichia coli O157:H7, Listeria monocytogenes, and Staphylococcus aureus on fresh-cut cantaloupe. PLoS One11, e0166874.

    Article  Google Scholar 

  • Forghani, F., Langaee, T., Eskandari, M., Seo, K.H., Chung, M.J., and Oh, D.H. 2015. Rapid detection of viable Bacillus cereus emetic and enterotoxic strains in food by coupling propidium monoazide and multiplex PCR (PMA-mPCR). Food Control55, 151–157.

    Article  CAS  Google Scholar 

  • Fricker, M., Messelhäußer, U., Busch, U., Scherer, S., and Ehling-Schulz, M. 2007. Diagnostic real-time PCR assays for the detection of emetic Bacillus cereus strains in foods and recent food-borne outbreaks. Appl. Environ. Microbiol.73, 1892–1898.

    Article  CAS  Google Scholar 

  • Golpayegani, A., Douraghi, M., Rezaei, F., Alimohammadi, M., and Nodehi, R.N. 2019. Propidium monoazide-quantitative polymerase chain reaction (PMA-qPCR) assay for rapid detection of viable and viable but non-culturable (VBNC) Pseudomonas aeruginosa in swimming pools. J. Environ. Health Sci. Eng.17, 407–416.

    Article  CAS  Google Scholar 

  • Hixon, S.C., White, W.E.Jr., and Yielding, K.L. 1975. Selective covalent binding of an ethidium analog to mitochondrial DNA with production of petite mutants in yeast by photoaffinity labeling. J. Mol. Biol.92, 319–329.

    Article  CAS  Google Scholar 

  • Kong, R.Y.C., Lee, S.K.Y., Law, T.W.F., Law, S.H.W., and Wu, R.S.S. 2002. Rapid detection of six types of bacterial pathogens in marine waters by multiplex PCR. Water Res.36, 2802–2812.

    Article  CAS  Google Scholar 

  • Li, F., Xie, G., Zhou, B., Yu, P., Yu, S., Aguilar, Z.P., Wei, H., and Xu, H. 2016. Rapid and simultaneous detection of viable Cronobacter sakazakii, Staphylococcus aureus, and Bacillus cereus in infant food products by PMA-mPCR assay with internal amplification control. LWT-Food Sci. Technol.74, 176–182.

    Article  CAS  Google Scholar 

  • Liang, T., Zhou, P., Zhou, B., Xu, Q., Zhou, Z., Wu, X., Aguilar, Z.P., and Xu, H. 2019. Simultaneous quantitative detection of viable Escherichia coli O157:H7, Cronobacter spp., and Salmonella spp. Using sodium deoxycholate-propidium monoazide with multiplex real-time PCR. J. Dairy Sci.102, 2954–2965.

    Article  CAS  Google Scholar 

  • Maynard, C., Berthiaume, F., Lemarchand, K., Harel, J., Payment, P., Bayardelle, P., Masson, L., and Brousseau, R. 2005. Waterborne pathogen detection by use of oligonucleotide-based microarrays. Appl. Environ. Microbiol.71, 8548–8557.

    Article  CAS  Google Scholar 

  • Nocker, A., Cheung, C.Y., and Camper, A.K. 2006. Comparison of propidium monoazide with ethidium monoazide for differentiation of live vs. dead bacteria by selective removal of DNA from dead cells. J. Microbiol. Methods67, 310–320.

    Article  CAS  Google Scholar 

  • Pan, R., Jiang, Y., Sun, L., Wang, R., Zhuang, K., Zhao, Y., Wang, H., Ali, M.A., Xu, H., and Man, C. 2018. Gold nanoparticle-based enhanced lateral flow immunoassay for detection of Cronobacter sakazakii in powdered infant formula. J. Dairy Sci.101, 3835–3843.

    Article  CAS  Google Scholar 

  • Sánchez-Parra, B., Núñez, A., and Moreno, D.A. 2019. Preventing legionellosis outbreaks by a quick detection of airborne Legionella pneumophila. Environ. Res.171, 546–549.

    Article  Google Scholar 

  • Tang, Y., Ali, Z., Zou, J., Yang, K., Mou, X., Li, Z., Deng, Y., Lu, Z., Ma, C., Shah, M.A.A., et al. 2014. Detection of Pseudomonas aeruginosa based on magnetic enrichment and nested PCR. J. Nanosci. Nanotechnol.14, 4886–4890.

    Article  CAS  Google Scholar 

  • Velusamy, V., Arshak, K., Korostynska, O., Oliwa, K., and Adley, C. 2010. An overview of foodborne pathogen detection: In the perspective of biosensors. Biotechnol. Adv.28, 232–254.

    Article  CAS  Google Scholar 

  • Wang, L., Ye, C., Xu, H., Aguilar, Z.P., Xiong, Y., Lai, W., and Wei, H. 2015. Development of an SD-PMA-mPCR assay with internal amplification control for rapid and sensitive detection of viable Salmonella spp., Shigella spp. and Staphylococcus aureus in food products. Food Control57, 314–320.

    Article  CAS  Google Scholar 

  • Wei, C., Zhong, J., Hu, T., and Zhao, X. 2018. Simultaneous detection of Escherichia coli O157:H7, Staphylococcus aureus and Salmonella by multiplex PCR in milk. 3 Biotech.8, 76.

    Google Scholar 

  • Xie, X., Wang, S., Jiang, S.C., Bahnemann, J., and Hoffmann, M.R. 2016. Sunlight-activated propidium monoazide pretreatment for differentiation of viable and dead bacteria by quantitative realtime polymerase chain reaction. Environ. Sci. Technol. Lett.3, 57–61.

    Article  CAS  Google Scholar 

  • Yang, I.C., Shih, D.Y.C., Wang, J.Y., and Pan, T.M. 2007. Development of rapid real-time PCR and most-probable-number realtime PCR assays to quantify enterotoxigenic strains of the species in the Bacillus cereus group. J. Food Prot.70, 2774–2781.

    Article  CAS  Google Scholar 

  • Yu, S., Yan, L., Wu, X., Li, F., Wang, D., and Xu, H. 2017. Multiplex PCR coupled with propidium monoazide for the detection of viable Cronobacter sakazakii, Bacillus cereus, and Salmonella spp. in milk and milk products. J. Dairy Sci.100, 7874–7882.

    Article  CAS  Google Scholar 

  • Yu, Q., Zhai, L., Bie, X., Lu, Z., Zhang, C., Tao, T., Li, J., Lv, F., and Zhao, H. 2016. Survey of five food-borne pathogens in commercial cold food dishes and their detection by multiplex PCR. Food Control59, 862–869.2

    Article  CAS  Google Scholar 

  • Zhang, Z., Wang, L., Xu, H., Aguilar, Z.P., Liu, C., Gan, B., Xiong, Y., Lai, W., Xu, F., and Wei, H. 2014. Detection of non-emetic and emetic Bacillus cereus by propidium monoazide multiplex PCR (PMA-mPCR) with internal amplification control. Food Control35, 401–406.

    Article  CAS  Google Scholar 

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Acknowledgments

The work was supported by the National Key R&D Program of China (2018YFC1602500), Research Foundation from Academic and Technical Leaders of Major Disciplines in Jiangxi Province, China (20194BCJ22004) and Research Foundation from State Key Laboratory of Food Science and Technology, Nanchang University, China (SKLF-ZZA-201912).

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Authors and Affiliations

  1. State Key Laboratory of Food Science and Technology, Nanchang University, Nanchang, 330047, P. R. China

    Guoyang Xie, Shuang Yu, Wen Li, Dan Mu & Hengyi Xu

  2. Zystein, LLC., Fayetteville, Arkansas, 72703, USA

    Zoraida P. Aguilar

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  1. Guoyang Xie
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  2. Shuang Yu
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  3. Wen Li
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  4. Dan Mu
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  5. Zoraida P. Aguilar
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  6. Hengyi Xu
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Correspondence to Hengyi Xu.

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Xie, G., Yu, S., Li, W. et al. Simultaneous detection of Salmonella spp., Pseudomonas aeruginosa, Bacillus cereus, and Escherichia coli O157:H7 in environmental water using PMA combined with mPCR. J Microbiol. 58, 668–674 (2020). https://doi.org/10.1007/s12275-020-0084-6

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  • DOI: https://doi.org/10.1007/s12275-020-0084-6

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