| Escherichia coli(E.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,and the dose of infection is less than 10 CFU.Infection with E.coli O157:H7 may lead to dysentery,hemorrhagic colitis(HC),hemolytic uremic syndrome(HUS),and thrombotic thrombocytopenic purpura(TTP).Livestock,poultry,and their meat products are the main transmission route of E.coli O157:H7 around the world.Therefore,the research and development of rapid,sensitive and sensitive detection methods for E.coli O157:H7 and other pathogens is particularly important.Firstly,a real-time loop amplified DNA assay system(Rti-LAMP)was developed for the rapid detection of E.coli O157:H7 on the chicken.It showed that the lowest level of 3.5CFU/reaction for pure E.coli culture could be detected and finished in 45 min by the Rti-LAMP targeting the VT2 gene using Midori green as nucleic acid dye.Furthermore,seeding 25 g portions of chicken with various numbers of E.coli O157:H7 CFU,followed enrichment culture at 37 ~oC for 4 h,then filtration through Whirl-pak bag and pelleting the bacterial cells by centrifugation at 13,000 g.The resulting pellets were suspended in saline and processed for cell lysis and DNA purification.2μL purified DNA samples was incorporated into 25μL Rti-LAMP reactions at 65 ~oC for 60 min.The lowest level 140CFU/g of E.coli O157:H7 consistently detected by the Rti-LAMP assay.The entire assay could be completed in 7 h.Moreover,the samples were pretreated with bentonite coated activated carbon(BCAC)and without enrichment for Rti-LAMP reactions.The results showed that the sensitivity of the sample treated with BCAC was 12 CFU/g and finished in4.5 h.Fifty-one chicken samples purchased from market were detected by the Rti-LAMP assay with enrichment,the Rti-LAMP with BCAC pretreated but without enrichment,as the control of E.coli O157:H7 culture.The same one sample was E.coli O157:H7 positive by the Rti-LAMP assay with enrichment and E.coli O157:H7 culture,and other 7 samples(total 8 samples)were also E.coli O157:H7 positive detected by the Rti-LAMP pretreated with BCAC.These data suggested that the Rti-LAMP combined with BCAC pretreatment was more sensitive,rapid,simple and specific than that of culture for detection of E.coli O157:H7 retrieved from Chicken.However,this method is difficult to distinguish between dead and viable cells of E.coli O157:H7.Secondly,a rapid and sensitive method for quantification and discrimination of E.coli O157:H7 between viable and heat-killed cells was developed and applied to the detection of chicken using ethidium bromide monoazide(EMA)in combination with a real-time loop amplified(Rti-LAMP)assay.4μg/mL 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 2.0×10~3 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 of5:2995 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 3.0×10~3 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.Thirdly,E.coli O157:H7 will be converted to viable but non-culturable state(VBNC)in response to stressful environmental.VBNC state of pathogens cannot be detected by traditional culture methods,pathogens in this state pose a challenge to food safety.In order to detect the VBNC state of E.coli O157:H7,the EMA-Rti-LAMP established in the previous study was used to rapidly monitor the culturable and VBNC state of E.coli O157:H7 induced by low temperature with the fluorescence microscopy direct viable counting and plate counting method were used as controls.The results showed that the number of viable cells(include VBNC)obtained by EMA-Rti-LAMP and fluorescence microscopy direct viable counting was almost the same.All cells died after E.coli O157:H7was frozen-thawed 3 cycles at a concentration of 1.7×10~4 CFU/mL.In contrast,after1.7×10~6 and 1.7×10~8 CFU/mL of E.coli O157:H7 were freeze-thawed 6 cycles,both still retain 0.04%and 0.24%of the cells in the VBNC state,respectively.E.coli O157:H7(1.7×10~8 CFU/mL)was stored at 4 ~oC and-20 ~oC,the number of culturable cells decreased,but the number of VBNC state cells increased gradually with the storage time prolonged.After 258 d of storage,the number of culturable cells decreased to 0,and the concentration of VBNC cells was 1.1×10~6 CFU/mL at 4 ~oC.And at-20 ~oC,the concentrations of culturable cell decreased to 1.3×10~4 CFU/mL,but the concentrations of viable cell was 5.5×10~6 CFU/mL,and higher by the order of 2-3 compared to the concentration of culturable cell.The E.coli O157:H7 induced at-20 ~oC and 4 ~oC for 258 d was seeded on chicken and the sample was pretreated with BCAC,the sensitivity of viable E.coli O157:H7 from the contaminated chicken samples was 25 CFU/g by EMA-Rti-LAMP without enrichment.The entire assay could be completed in about 5 h.A total of 24 frozen chicken samples were detected by the GB method(GB4789.36—2016),BCAC-Rti-LAMP,and BCAC-EMA-Rti-LAMP.The results shown that one of indentical sample(4.17%)was detected as positive for E.coli O157:H7 by the GB method and BCAC-EMA-Rti-LAMP,while the other three of E.coli O157:H7 positive sample were detected by the BCAC-Rti-LAMP(a total of 4 positive samples,16.67%).VBNC state of E.coli O157:H7 cannot be detected by traditional culture methods,and the fluorescence microscopy direct vialbe counting cannot specifically detect target bacteria from foods containing other bacteria.Therefore,this study provides a rapid,simple and sensitive detection method for food contaminated viable(include VBNC)E.coli O157:H7 without enrichment. |
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