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A Novel Real-Time Loop-Mediated Isothermal Amplification Combined with Immunomagnetic Beads Separation and Ethidium Bromide Monoazide Treatment for Rapid and Ultrasensitive Detection of Viable Escherichia coli O157:H7 in Milk

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Abstract

In this study, a real-time loop-mediated isothermal amplification (Rti-LAMP) combined with immunomagnetic beads separation (IMS) and ethidium bromide monoazide (EMA) treatment was developed for the rapid and ultrasensitive detection of viable Escherichia coli (E. coli) O157:H7 in milk without enrichment. Polyclonal antibodies against E. coli O157:H7 flagellum and specific immunomagnetic beads (IMBs) were prepared. The capture efficiency of target bacteria was more than 95% by adding 50 μL of prepared IMBs into 1 mL of E. coli sample for incubation at 37 °C for 20 min. Then, Gly-HCl buffer (0.2 M, pH 2.2) was a key to the effective eluting and separating of bacteria from IMB-bacterial complexes. The eluted bacteria were resuspended with 20 μL TZ lysing solution and boiled at 99.5 °C for 10 min, as a DNA template of Rti-LAMP. The development of IMS-Rti-LAMP assay could specifically detect viable E. coli O157:H7 as low as 2.7 CFU/mL in pure culture. Furthermore, the optimum volume for detection of E. coli O157:H7 in milk was 1 mL of the sample by IMS-Rti-LAMP assay. Before Gly-HCl elution, 4 μg/mL of EMA was used to treat bacterial cells which significantly inhibited DNA amplification derived from dead cells in Rti-LAMP. The limit of detection (LOD) of viable E. coli O157:H7 in milk was 6 CFU/mL by the IMS-EMA-Rti-LAMP, and the whole assay could be completed in 4 h. The development of IMS-EMA-Rti-LAMP assay was a rapid, ultrasensitive, and specific detection of viable E. coli O157:H7 in milk without enrichment.

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Funding

This study was funded by the National Natural Science Foundation of China (No. 31760483); the Special Funds for Collaborative Innovation of Modern Agricultural Science and Research in Jiangxi Province, China (No. JXXTCX201703); and the Natural Science Foundation of Jiangxi Province, China (No. 20171ACB20013).

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Authors and Affiliations

  1. College of Food Science and Engineering, Jiangxi Agricultural University, Nanchang, 330045, China

    Hu Chen, Ya-Ke Li, Tian-Tian Zhang, Yan Bi, Mei Shu, Chan Zhong, Kai-Jie Tang & Guo-Ping Wu

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  1. Hu Chen
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  2. Ya-Ke Li
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  3. Tian-Tian Zhang
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  4. Yan Bi
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  5. Mei Shu
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  6. Chan Zhong
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  7. Kai-Jie Tang
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  8. Guo-Ping Wu
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Corresponding author

Correspondence to Guo-Ping Wu.

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Conflict of Interest

Hu Chen declares that he has no conflict of interest. Ya-Ke Li declares that he has no conflict of interest. Tian-Tian Zhang declares that he has no conflict of interest. Yan Bi declares that he has no conflict of interest. Mei Shu declares that he has no conflict of interest. Chan Zhong declares that he has no conflict of interest. Kai-Jie Tang declares that he has no conflict of interest. Guo-Ping Wu declares that he has no conflict of interest.

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Chen, H., Li, YK., Zhang, TT. et al. A Novel Real-Time Loop-Mediated Isothermal Amplification Combined with Immunomagnetic Beads Separation and Ethidium Bromide Monoazide Treatment for Rapid and Ultrasensitive Detection of Viable Escherichia coli O157:H7 in Milk. Food Anal. Methods 14, 944–956 (2021). https://doi.org/10.1007/s12161-020-01932-y

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