Quantification and Discrimination of Viable and Dead Escherichia coli O157:H7 Cells from Chicken Without Enrichment by E
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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 Yuan Yang Zhao 1 & Kai Jie Tang 1 & Tian Tian Zhang 1 & Yan Yan Gao 1 & Li Ping Lin 1 & Guo Ping Wu 1
Received: 18 July 2017 / Accepted: 3 November 2017 # Springer Science+Business Media, LLC, part of Springer Nature 2017
Abstract In this study, a rapid and sensitive method of realtime 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 × 10 3 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 × 10 1 to 1.0 × 10 4 CFU/g without enrichment. * Guo Ping Wu [email protected] 1
College of Food Science and Engineering, Jiangxi Agricultural University, Nanchang 330045, China
Keywords Escherichia coli O157:H7 . Ethidium bromide monoazide (EMA) . Rti-LAMP . Viable cells . Heat-killed cells . Chicken . Without enrichment
Introduction Escherichia coli O157:H7 is the common serotype in the enterophemorrhagic E. coli (EHEC), which also is the third most common bacterial food-borne pathogen after Salmonella and Campylobacter spp. They can be transmitted by contaminated food or water causing food-borne diseases (Wong et al. 2000). Infection with E. coli O157:H7 may lead to dysentery, hemorrhagic colitis (HC), hemolytic uremic syndrome (HUS), and thrombotic thrombocytopenic purpura (TTP) (Caprioli et al. 2005). Livestock, poultry, and their meat products are the main transmission route of E. coli O157:H7 around the world (Zhang et al. 2015). There is a need for the development of more sensitive, rapid, and inexpensive methods for detection of this pathogen associated with foods (Kokkinos et al. 2014; Techathuvanan and D’Souza 2012). To date, refere
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