Study on the rapid detection of five strains of diarrheagenic Escherichia coli by real-time fluorescence quantitative PCR

Authors

WANG Fangmei, Hunan Province Produced Commodity Quality Supervision and Inspection Institute, Changsha, Hunan 410007, China; College of Life Science, Hunan Normal University, Changsha, Hunan 410081, China; Key Laboratory of Protein Chemistry and Development Biology of
ZHONG Wentao, Hunan Province Produced Commodity Quality Supervision and Inspection Institute, Changsha, Hunan 410007, China
WANG Shuhao, Hunan Province Produced Commodity Quality Supervision and Inspection Institute, Changsha, Hunan 410007, China
LU Lin, Hunan Province Produced Commodity Quality Supervision and Inspection Institute, Changsha, Hunan 410007, China
HAN Fenling, Hunan Province Produced Commodity Quality Supervision and Inspection Institute, Changsha, Hunan 410007, China

Abstract

Primers, and probes were designed for the conserved sequences of five virulence genes involved in Escherichia coli diarrhea mentioned in the new standard of GB 4789.6—2016 national food safety standard. Four kits of multiple real-time fluorescence PCR were established to detect five diarrhea-causing E. coli simultaneously. The results showed that 12 pairs of virulence genes of the four kits were verified by the standard reserve strains of the five diarrhea-causing E. coli strains. In addition, these 12 pairs of virulence genes only play a role in the specific role of corresponding diarrhea-causing E. coli, and have no amplification curve for common foodborne pathogens. The detection limit of escV, bfpB, stx₁, ipaH and invE genes is 10 CFU/mL, and that of aggR gene is 10² CFU/mL. The detection limit of astA and Pic genes was 10³ CFU /mL. When the concentration of stx₂A, ipaH and stIb genes was less than 10 CFU/mL, a significant “S” type amplification curve could be observed, and the line shape was better. In addition, 40 samples including meat, milk, animal diarrhea, and some artificial contamination samples were tested, and a total of 11 positive samples were found. Consistent with the test results of the new standard 4789.6—2016, The results showed that the four kit methods were simple, specific, sensitive and practical.

Publication Date

5-28-2019

First Page

88

Last Page

95

DOI

10.13652/j.issn.1003-5788.2019.05.016

Recommended Citation

Fangmei, WANG; Wentao, ZHONG; Shuhao, WANG; Lin, LU; and Fenling, HAN (2019) "Study on the rapid detection of five strains of diarrheagenic Escherichia coli by real-time fluorescence quantitative PCR," Food and Machinery: Vol. 35: Iss. 5, Article 16.
DOI: 10.13652/j.issn.1003-5788.2019.05.016
Available at: https://www.ifoodmm.cn/journal/vol35/iss5/16

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