Scientia Agricultura Sinica ›› 2016, Vol. 49 ›› Issue (23): 4619-4627.doi: 10.3864/j.issn.0578-1752.2016.23.014

• STORAGE·FRESH-KEEPING·PROCESSING • Previous Articles     Next Articles

Multiplex Enrichment Quantitative PCR Assays for the Detection of Vibrio alginolyticus, Vibrio parahaemolyticus, Vibrio vulnificus and Vibrio cholerae

WEI Shuang1,2, WANG Tian-jie3, LONG Yang4, ZHOU Guang-biao1, LIN Chun-gui1, HUANG Shuai1, WU Xi-yang2   

  1. 1Shantou Entry-Exit Inspection and Quarantine Bureau, Shantou 515041, Guangdong
    2Department of Food Science and Engineering, College of Science and Engineering, Jinan University, Guangzhou 510632
    3Guangzhou Entry-Exit Inspection and Quarantine Bureau, Guangzhou 510632
    4Zhanjiang Entry-Exit Inspection and Quarantine Bureau, Zhanjiang 524022, Guangdong
  • Received:2016-05-03 Online:2016-12-01 Published:2016-12-01

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Abstract: 【Objective】Vibrio parahaemolyticus, V. alginolyticus, V. cholerae and V. vulnificus have been recognized as the important foodborne pathogens causing human disease. It is important to establish a detection method to identify these 4 foodborne pathogenic Vibrio species in order to ensure food safety. To develop a multiplex enrichment quantitative PCR (ME-qPCR) assay that can simultaneously detect Vibrio parahaemolyticus, V. alginolyticus, V. cholerae and V. vulnificus in the presence of an internal amplification control (IAC), to make it possible for researchers and technical staff achieve simple detection of these 4 foodborne pathogens. 【Method】 Inner and outer species-specific PCR primers were designed based on gyrB gene for V. alginolyticus, collagenase gene for V. parahaemolyticus, vvhA gene for V. vulnificus and ompW gene for V. cholerae, 16S rRNA gene of bacteria as IAC primers was used to indicate false-negative results. All inner and outer primers were used in first round multiplex enrichment PCR with a small number of cycles so as to avoid competition between amplicons. Each gene has 4 types of probable products which can be the templates for the next nested real-time PCR. This can enrich the target genes from the genomic DNA template successfully. The reaction product was then diluted and analyzed individual real-time PCRs using inner primers. The results were analysed by amplification curve and melting curve. ME-qPCR method was developed after optimization of the first round multiplex enrichment PCR cycle numbers (10, 15 and 20 cycles). The specificity of ME-qPCR was validated by 14 bacteria standard strains. The sensitivity and quantitative capability of ME-qPCR were tested by using 10-fold serially diluted genomic DNA of 4 Vibrio strains. Strains as templates, and then, the ME-qPCR was used to detect 69 suspicious Vibrio strains and the results were compared with physiological and biochemical experiments. 【Result】 Fifteen cycles were determined to use in first round multiplex enrichment PCR in this study finally. The results showed that the ME-qPCR assay was rapid, high-throughput, sensitive and specificand the existence of IAC could successfully eliminate false-negative results. The sensitivity of ME-qPCR was 0.001ng per reaction, about 10 times higher than real-time PCR. The ME-qPCR was validated with 69 suspicious Vibrio strains. The result showed that 27 green bacteria colonies were V. parahaemolyticus, 22 yellow bacteria colonies were V. alginolyticus, 1 yellow bacteria colony was V. vulnificus, and V. cholerae was not detected. The results are consistent with physiological and biochemical experiments. 【Conclusion】 The ME-qPCR assay is specific, stable and reliable for the detection of V. parahaemolyticus, V. alginolyticus, V. cholerae and V. vulnificus. It’s sensitivity is high, and can effectively indicate the false negative of PCR reaction, the results without gel electrophoresis, and is suitable for the rapid screening of 4 common pathogenic vibrio. in food

Key words: Vibrio cholerae, Vibrio vulnificus, Vibrio parahaemolyticus, Vibrio alginolyticus, internal amplification control, ME-qPCR

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