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A pilot study on PCR-based detection of four foodborne pathogenic microorganisms

  • Qiang GongEmail author
  • Zhanli Li
  • Mingfu Niu
Original Paper
  • 219 Downloads

Abstract

To establish PCR-based detection methods for Staphylococcus aureus, Shigella, Pasteurella multocida and Pseudomonas aeruginosa, the nuc, ipah, ptfa and oprl genes were amplified by singleplex PCRs and multiplex PCR using specific primers that were designed according to the DNA sequences retrieved from GenBank. Then the annealing temperature was optimized, accompanied by a study of the specificity and sensitivity of the singleplex PCRs and multiplex PCR. The results showed that DNA fragments of 280, 474, 150 and 331 bp were specifically amplified from the four pathogenic bacteria mentioned above. No target DNA fragments were obtained from other pathogenic bacteria, including Salmonella typhimurium, Campylobacter jejuni, Clostridium perfringens and pathogenic Escherichia coli. The sensitivity of the singleplex PCRs were 100, 1, 1 and 10 pg/μL respectively. The detection limits of the four pathogenic bacteria in the multiplex PCR were 100, 1, 10 and 10 pg/µL respectively. These results showed that singleplex PCRs and multiplex PCR have good specificity and sensitivity. In conclusion, this experiment has laid a foundation for further research on rapid detection methods against these four pathogenic bacteria in food.

Keywords

Foodborne pathogenic microorganism Polymerase chain reaction Singleplex Multiplex 

Notes

Acknowledgements

The authors gratefully acknowledge the grant from the production-study-research cooperation projects of Henan Province, China (No. 2014HNCXY008).

References

  1. 1.
    F.J. He, S.Q. Liu, Detection of P. aeruginosa using nano-structured electrode-separated piezoelectric DNA biosensor. Talanta 62, 271–277 (2004)CrossRefGoogle Scholar
  2. 2.
    S. Furukawa, T. Watanabe, T. Koyama, M. Yamasaki, Inactivation of food poisoning bacteria and Geobacillus stearothermophilus spores by high pressure carbon dioxide treatment. Food Control 20(1), 53–58 (2009)CrossRefGoogle Scholar
  3. 3.
    M.A. Fernández-Rojas, S. Vaca, M. Reyes-López, M. de la Garza, F. Aguilar-Romero, E. Zenteno, E. Soriano-Vargas, E. Negrete-Abascal, Outer membrane vesicles of Pasteurella multocida contain virulence factors. Microbiologyopen 3(5), 711–717 (2014)CrossRefGoogle Scholar
  4. 4.
    C.M. Shih, C.L. Chang, M.Y. Hsu, J.Y. Lin, C.M. Kuan, H.K. Wang, C.T. Huang, M.C. Chung, K.C. Huang, C.E. Hsu, C.Y. Wang, Y.C. Shen, C.M. Cheng, Paper-based ELISA to rapidly detect Escherichia coli. Talanta 145, 2–5 (2015)CrossRefGoogle Scholar
  5. 5.
    B. Balakrishnan, S. Barizuddin, T. Wuliji, M. El-Dweik, A rapid and highly specific immunofluorescence method to detect Escherichia coli O157:H7 in infected meat samples. Int. J. Food Microbiol. 231, 54–62 (2016)CrossRefGoogle Scholar
  6. 6.
    M. Barkallah, Y. Gharbi, M. Hmani, L.I. Fendri, Locked nucleic acid probe-based real-time PCR for the diagnosis of Listeria monocytogenes in ruminants. Mol. Cell. Probes 30(3), 138–145 (2016)CrossRefGoogle Scholar
  7. 7.
    M. Uyttendaele, A. Rajkovic, S. Ceuppens, L. Baert, E.V. Coillie, L. Herman, V. Jasson, H. Imberechts, PCR applications in food microbiology. Encycl. Food Microbiol. 2, 1033–1041 (2014)CrossRefGoogle Scholar
  8. 8.
    X.X. Chen, M. Gan, H. Xu, F. Chen, X. Ming, H.Y. Xu, H. Wei, F. Xu, C.W. Liu, Development of a rapid and sensitive quantum dot-based immunochromatographic strip by double labeling PCR products for detection of Staphylococcus aureus in food. Food Control 46, 225–232 (2014)CrossRefGoogle Scholar
  9. 9.
    K. Gokduman, M.D. Avsaroglu, A. Cakiris, L.C. Gurakan, Recombinant plasmid-based quantitative Real-Time PCR analysis of Salmonella enterica serotypes and its application to milk samples. J. Microbiol. Methods 122, 50–58 (2016)CrossRefGoogle Scholar
  10. 10.
    A. Garrido-Maestu, M.J. Chapela, E. PeñaraÑda, J.M. vieites, A.G. Cabado, In-house validation of novel multiplex real-time PCR gene combination for the simultaneous detection of the main human pathogenic vibrios (Vibrio cholerae, Vibrio parahaemolyticus, and Vibrio vulnificus). Food Control 37, 371–379 (2014)CrossRefGoogle Scholar
  11. 11.
    Y.G. Xu, L.M. Sun, Y.S. Wang, P.P. Chen, Z.M. Liu, Y.J. Li, L.J. Tang, Simultaneous detection of Vibrio cholerae, Vibrio alginolyticus, Vibrio parahaemolyticus and Vibrio vulnificus in seafood using dual priming oligonucleotide (DPO) system-based multiplex PCR assay. Food Control 71, 64–70 (2017)CrossRefGoogle Scholar
  12. 12.
    R. Ushio, M. Yamamoto, K. Nakashima, H. Watanabe, K. Nagai, Y. Shibata, K. Tashiro, T. Tsukahara, H. Nagakura, N. Horita, T. Sato, M. Shinkai, M. Kudo, A. Ueda, T. Kaneko, Digital PCR assay detection of circulating Mycobacterium tuberculosis DNA in pulmonary tuberculosis patient plasma. Tuberculosis 99, 47–53 (2016)CrossRefGoogle Scholar
  13. 13.
    J. Rhein, N.C. Bahr, A.C. Hemmert, D.R. Boulware, Diagnostic performance of a multiplex PCR assay for meningitis in an HIV-infected population in Uganda. Diagn. Microbiol. Infect. Dis. 84(3), 268–273 (2016)CrossRefGoogle Scholar
  14. 14.
    T. Ojima, K. Hirano, K. Honda, M. Kusumoto, Development of a multiplex PCR assay for rapid virulence factor profiling of extraintestinal pathogenic Escherichia coli isolated from cattle. J. Microbiol. Methods 128, 31–33 (2016)CrossRefGoogle Scholar
  15. 15.
    Y. Yan, X.J. Jia, H.H. Wang, X.F. Fu, J.M. Ji, P.Y. He, L.X. Chen, J.Y. Luo, Z.W. Chen, Dynamic quantification of avian influenza H7N9(A) virus in a human infection during clinical treatment using droplet digital PCR. J. Virol. Methods 234, 22–27 (2016)CrossRefGoogle Scholar
  16. 16.
    Q. Yu, L.G. Zhai, X.M. Bie, Z.X. Lu, C. Zhang, T.T. Tao, J.J. Li, F.X. Lv, H.Z. Zhao, Survey of five food-borne pathogens in commercial cold food dishes and their detection by multiplex PCR. Food Control 59, 862–869 (2016)CrossRefGoogle Scholar
  17. 17.
    X.H. He, X.B. Xu, K. Li, B. Liu, T.L. Yue, Identification of Salmonella enterica Typhimurium and variants using a novel multiplex PCR assay. Food Control 65, 152–159 (2016)CrossRefGoogle Scholar
  18. 18.
    C.S.M.L. Estrada, L.D.C. VeLÁzquez, M.E. Escudero, G.I. Favier, V. Lazarte, A.M.S. de Guzmán, Pulsed field, PCR ribotyping and multiplex PCR analysis of Yersinia enterocolitica strains isolated from meat food in San Luis Argentina. Food Microbiol. 28(1), 21–28 (2011)CrossRefGoogle Scholar
  19. 19.
    K. Yamada, A. Ibata, M. Suzuki, R. Kurane, Designing multiplex PCR system of Campylobacter jejuni for efficient typing by improving monoplex PCR binary typing method. J. Infect. Chemother. 21(1), 50–54 (2015)CrossRefGoogle Scholar
  20. 20.
    S. Yonogi, M. Kanki, T. Ohnishi, M. Shiono, T. Iida, Y. Kumeda, Development and application of a multiplex PCR assay for detection of the Clostridium perfringens enterotoxin- encoding genes cpe and becAB. J. Microbiol. Methods 127, 172–175 (2016)CrossRefGoogle Scholar
  21. 21.
    A. Garrido-maestu, M.J. Chapela, J.M. Vieites, A.G. Cabado, Application of real-time PCR to detect Listeria monocytogenes in a mussel processing industry: impact on control. Food Control 46, 319–323 (2014)CrossRefGoogle Scholar
  22. 22.
    F. Cattani, V.C. Barth Jr., J.S.R. Nasário, S. Oliveira, Detection and quantification of viable Bacillus cereus group species in milk by propidium monoazide quantitative real-time PCR. J. Dairy Sci. 99(4), 2617–2624 (2016)CrossRefGoogle Scholar
  23. 23.
    X.C. Lv, Y. Li, W.W. Qiu, X.Q. Wu, B.X. Xu, Y.T. Liang, Development of propidium monoazide combined with real-time quantitative PCR (PMA-qPCR) assays to quantify viable dominant microorganisms responsible for the traditional brewing of Hong Qu glutinous rice wine. Food Control 66, 69–78 (2016)CrossRefGoogle Scholar
  24. 24.
    F. Postollec, H. Falentin, S. Pavan, D. Sohier, Recent advances in quantitative PCR (qPCR) applications in food microbiology. Food Microbiol. 28, 848–861 (2011)CrossRefGoogle Scholar
  25. 25.
    S.M.D. Silva, L.K. Vang, N.D. Olson, S.P. Lund, A.S. Downey, Z. Kelman, M.L. Salit, N.J. Lin, J.B. Morrow, Evaluation of microbial qPCR workflows using engineered Saccharomyces cerevisiae. Biomol. Detect. Quantif. 7, 27–33 (2016)CrossRefGoogle Scholar
  26. 26.
    A.K. Chiefari, M.J. Perry, C. Kelly-Cirino, C.T. Egan, Detection of Staphylococcus aureus enterotoxin production genes from patient samples using an automated extraction platform and multiplex real-time PCR. Mol. Cell. Probes 29(6), 461–467 (2015)CrossRefGoogle Scholar
  27. 27.
    Y.J. Zhang, S. Zhang, X.Z. Liu, H.A. Wen, M. Wang, A simple method of genomic DNA extraction suitable for analysis of bulk fungal strains. Lett. Appl. Microbiol. 51, 114–118 (2010)Google Scholar
  28. 28.
    P. Ghosh, S.L. Bodhankar, Determination of risk factors and transmission pathways of Helicobacter pylori in asymptomatic subjects in Western India using polymerase chain reaction. Asian Pac. J. Trop. Dis. 2, 12–17 (2012)CrossRefGoogle Scholar
  29. 29.
    K.A. Lampel, P.A. Orlandi, Polymerase chain reaction detection of invasive Shigella and Salmonella enterica in food. Gene Probes 179, 235–244 (2002)CrossRefGoogle Scholar
  30. 30.
    D.M. Silva, L. Domingue, On the track for an efficient detection of Escherichia coli in water: a review on PCR-based methods. Ecotoxicol. Environ. Saf. 113, 400–411 (2015)CrossRefGoogle Scholar
  31. 31.
    O.G. Brakstad, J.A. Maeland, Generation and characterization of moloclonal antibodies against Staphylococcus aureus thermonuclease. Acta Pathol. Microbiol. Immunol. Scand. 97, 166–174 (1989)CrossRefGoogle Scholar
  32. 32.
    O.G. Brakstad, Detection of Staphylococcus aureus by polymerase chain reaction of the nuc gene. J. Clin. Microbiol. 22, 67 (1992)Google Scholar
  33. 33.
    S.M. Faruque, R. Khan, M. Kamruzzaman, S. Yamasaki, Q.S. Ahmad, T. Azim, G.B. Nair, Y. Takeda, D.A. Sack, Isolation of Shigella dysenteriae type 1 and S. flexneri strains from surface waters in Bangladesh: comparative molecular analysis of environmental Shigella isolates versus clinical strains. Appl. Environ. Microbiol. 68(8), 3908–3913 (2002)CrossRefGoogle Scholar
  34. 34.
    V.D. Thiem, O. Sethabutr, L.V. Seidlein, D.T. Dang, Detection of Shigella by a PCR assay targeting the ipaH gene suggests increased prevalence of shigellosis in Nha Trang, Vietnam. J. Clin. Microbiol. 42(5), 2031–2035 (2004)CrossRefGoogle Scholar
  35. 35.
    S.W. Doughty, C.G. Ruffolo, B. Adler, The type 4 fimbrial subunit gene of Pasteurella multocida. Vet. Microbiol. 72(1–2), 79–90 (2000)CrossRefGoogle Scholar
  36. 36.
    A. Gholami, A. Majidpour, M. Talebi-Taher, M. Boustanshenas, M. Adabi, PCR-based assay for the rapid and precise distinction of Pseudomonas aeruginosa from other Pseudomonas species recovered from burns patients. J. Prev. Med. Hyg. 57(2), E81–E85 (2016)Google Scholar
  37. 37.
    A.V. Maerle, D.Y. Ryazantsev, O.A. Dmitrenko, E.E. Petrova, R.L. Komaleva, I.V. Sergeev, D.Y. Trofimov, S.K. Zavriev, Detection of Staphylococcus aureus toxins using immuno-PCR. Russ. J. Bioorg. Chem. 40(5), 526–531 (2014)CrossRefGoogle Scholar
  38. 38.
    S.C. Ojha, C.Y. Yean, A. Ismail, K.K.B. Singh, A pentaplex PCR assay for the detection and differentiation of Shigella species. Biomed. Res. Int. 2013, 1–9 (2013)CrossRefGoogle Scholar
  39. 39.
    S. Nagai, S. Someno, T. Yagihashi, Differentiation of toxigenic from nontoxigenic isolates of Pasteurella multocida by PCR. J. Clin. Microbiol. 32(4), 1004–1010 (1994)Google Scholar
  40. 40.
    H. Wang, Q. Huang, D.C. Shi, Z.R. Chuai, W.L. Fu, Rapid detection of Pseudomonas aeruginosa by color loop-mediated isothermal amplification. Acta Acad. Med. Militaris Tertiae 34(22), 2264–2268 (2012)Google Scholar
  41. 41.
    F.A. Nassar, F.H. Abu-Elamreen, M.E. Shubair, F.A. Sharif, Detection of Chlamydia trachomatis and Mycoplasma hominis, genitalium and Ureaplasma urealyticum by polymerase chain reaction in patients with sterile pyuria. Adv. Med. Sci. 53(1), 80–86 (2008)CrossRefGoogle Scholar
  42. 42.
    D.W. Li, C.P. Huang, Y.M. Zhang, J.H. Xie, Z.J. Ran, Z.L. Xiong, W.X. Fan, Establishment and application of the triplex PCR for detecting M. Bovis, M. mycoides subsp. mycoides small colony type and M. agalactiae. Acta Vet. Zootech. Sin. 42(2), 306–310 (2011)Google Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2017

Authors and Affiliations

  1. 1.College of Food and BioengineeringHenan University of Science and TechnologyLuoyangPeople’s Republic of China
  2. 2.Henan Engineering Laboratory of Livestock Disease Diagnosing and Food Safety TestingLuoyangPeople’s Republic of China
  3. 3.Henan Engineering Research Center of Food MaterialLuoyangPeople’s Republic of China

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