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Novel Development of a qPCR Assay Based on the rpoB Gene for Rapid Detection of Cronobacter spp.

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Abstract

A novel real-time PCR (qPCR) assay with internal amplification control based on the rpoB gene was developed for the detection and quantification of Cronobacter spp. Inclusivity and exclusivity of the qPCR assay were tested on a strain collection containing 19 Cronobacter and 26 non-Cronobacter strains. All Cronobacter strains were successfully identified, whereas no cross-reactivity was observed with non-Cronobacter strains. The sensitivity of the qPCR assay for pure culture and powdered infant formula (PIF) without enrichment was 3.44 log CFU/ml(g) (2.74 × 103 CFU/ml(g)). When the qPCR assay was applied to artificially contaminated PIF after a 12-h enrichment step, as few as 0.03 log CFU/ml (1.06 × 100 CFU/ml) of C. sakazakii could be detected. The limit of detection of the qPCR assay was not reduced by the presence of 8 log CFU/ml of Salmonella Enteritidis in PIF. A total of 70 food samples were analyzed for the presence of Cronobacter spp., out of which 3 dry cereal products, 5 maternal milk, and 1 infant food formula were found as positive by qPCR. The results obtained by qPCR were consistent with those obtained by culture-based method. Results from this study demonstrate that the qPCR assay is a rapid, specific, and accurate method suitable for Cronobacter detection in foods.

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Acknowledgments

This work was supported by the National Twelfth Five-Year Research Program of China (2012BAK08807), the National Natural Science Foundation of China (31401595), the Jiangsu Technology of Agricultural Innovation Fund CX(12)3087, and a project funded by the Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD).

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    Authors and Affiliations

    1. College of Food Science and Technology, Nanjing Agricultural University, 1 Weigang, Jiangsu, 210095, Nanjing, People’s Republic of China

      Yuanhong Li, Qiming Chen, Hua Jiang, Fengxia Lu, Xiaomei Bie & Zhaoxin Lu

    2. School of Public Health, Xuzhou Medical College, 209 Tongshan Road, Xuzhou, 221004, Jiangsu, People’s Republic of China

      Yuanhong Li, Hua Jiang & Yang Jiao

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    1. Yuanhong Li
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    2. Qiming Chen
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    3. Hua Jiang
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    4. Yang Jiao
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    5. Fengxia Lu
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    6. Xiaomei Bie
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    7. Zhaoxin Lu
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    Correspondence to Zhaoxin Lu.

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    Yuanhong Li and Qiming Chen have contributed equally to this work.

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    Li, Y., Chen, Q., Jiang, H. et al. Novel Development of a qPCR Assay Based on the rpoB Gene for Rapid Detection of Cronobacter spp.. Curr Microbiol 72, 436–443 (2016). https://doi.org/10.1007/s00284-015-0971-y

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