A pilot study on PCR-based detection of four foodborne pathogenic microorganisms
- 219 Downloads
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.
KeywordsFoodborne pathogenic microorganism Polymerase chain reaction Singleplex Multiplex
The authors gratefully acknowledge the grant from the production-study-research cooperation projects of Henan Province, China (No. 2014HNCXY008).
- 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.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.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
- 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
- 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
- 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.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
- 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
- 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.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
- 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