Quantification and Discrimination of Viable and Dead Escherichia coli O157:H7 Cells from Chicken Without Enrichment by Ethidium Bromide Monoazide Real-time Loop-Mediated Isothermal Amplification
In this study, a rapid and sensitive method of real-time loop-mediated isothermal amplification (Rti-LAMP) assays was developed for quantification and discrimination of viable and heat-killed E. coli O157:H7 cells treated with low concentration of ethidium bromide monoazide (EMA). Four micrograms per milliliter of EMA was chosen as the optimal concentration which did not inhibit DNA amplification derived from viable cells, but significantly increased the Tt values of dead cells in Rti-LAMP assays. When the DNA from 2.0 × 103 viable CFU of E. coli O157:H7 was subjected to EMA-Rti-LAMP, the resulting Tt value was 17.73 min. In contrast, the DNA from 2.0 × 103 CFU completely heat destroyed CFU of E. coli O157:H7 did not yield a positive amplification which Tt value was regarded as 60 min. When the DNA from viable plus heat-killed CFU at a ratio of 5:2995 was subjected to EMA-Rti-LAMP, the resulting Tt value was 23.06 min, which was statistically identical (P < 0.05) to the Tt value of 24.07 min obtained with the DNA from only 5 viable CFU. The results indicate that even though 3.0 × 103 dead cells yielded a negative amplification setting the Tt value as 60 min, low numbers of viable cells in the presence of much higher numbers of dead cells still yielded a linear plot for enumerating viable CFU from Tt values. Detection of E. coli O157:H7 derived from contaminated chicken samples, the EMA-Rti-LAMP could notably distinguish viable and heat-killed cells from 5.0 × 101 to 1.0 × 104 CFU/g without enrichment.
KeywordsEscherichia coli O157:H7 Ethidium bromide monoazide (EMA) Rti-LAMP Viable cells Heat-killed cells Chicken Without enrichment
This study was funded by the National Natural Science Foundation of China (No. 31560480, 31760483), Jiangxi Natural Science Foundation of China (No. 20171ACB20013), and the special funds for collaborative innovation of modern agricultural science and research in Jiangxi, China (No. JXXTCX201703).
Compliance with Ethical Standards
Conflict of Interest
Yuan Yang Zhao declares that he has no conflict of interest. Kai Jie Tang declares that she has no conflict of interest. Tian Tian Zhang declares that she has no conflict of interest. Yan Yan Gao declares that she has no conflict of interest. Li Ping Lin declares that she has no conflict of interest. Guo Ping Wu declares that he has no conflict of interest.
This article does not contain any studies with human participants or animals performed by any of the authors.
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