Establishment of real-time quantitative PCR assay for the detection of Enterobacter sakazakii based on internal amplification control

Authors

WANG Jinfeng, The Technical Center of Hebei Entry-exit Inspection and Quarantine Bureau, Shijiazhuang, Hebei 050051, China
WANG Jianchang, The Technical Center of Hebei Entry-exit Inspection and Quarantine Bureau, Shijiazhuang, Hebei 050051, China
HU Lianxia, The Technical Center of Hebei Entry-exit Inspection and Quarantine Bureau, Shijiazhuang, Hebei 050051, China
SUN Xiaoxia, The Technical Center of Hebei Entry-exit Inspection and Quarantine Bureau, Shijiazhuang, Hebei 050051, China
CHEN Zhimin, The Technical Center of Hebei Entry-exit Inspection and Quarantine Bureau, Shijiazhuang, Hebei 050051, China
JIANG Yanfen, The Technical Center of Hebei Entry-exit Inspection and Quarantine Bureau, Shijiazhuang, Hebei 050051, China

Abstract

Based on ompA gene sequence of Enterobacter sakazakii, the specific primer and probe were designed. An internal amplification control (IAC) was added and used to monitor the performance of reaction system. The assay could be used reliably to detect E. sakazakii genome DNA with the sensitivity of 1 pg. The detection limit for E. sakazakii was 1×10⁴ CFU when used the DNA extracted by water boiling as the template. With the use of ompA, the detection limit can reach 10³ copies. Through the standard curves of ompA, the quantification was linear between Ct and template copy number (R²=0.999). For the artificially contaminated powered milk with the concentration of 10 CFU/25 g, the E. sakazakii could be detected after 10 hours culture when used the DNA extracted by commercial kit and water boiling. The E. sakazakii fluorescence quantitative PCR assay provides reference data for optimization and modification of the standardized molecular biochemical approach of E. sakazakii.

Publication Date

6-28-2016

First Page

68

Last Page

72

DOI

10.13652/j.issn.1003-5788.2016.06.016

Recommended Citation

Jinfeng, WANG; Jianchang, WANG; Lianxia, HU; Xiaoxia, SUN; Zhimin, CHEN; and Yanfen, JIANG (2016) "Establishment of real-time quantitative PCR assay for the detection of Enterobacter sakazakii based on internal amplification control," Food and Machinery: Vol. 32: Iss. 6, Article 16.
DOI: 10.13652/j.issn.1003-5788.2016.06.016
Available at: https://www.ifoodmm.cn/journal/vol32/iss6/16

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