The Establishment Of A Triplex Real-time PCR Assay For The Detection Of Shiga Toxin-Producing Escherichia Coli O157 And Clinical Detection Of STEC O157 In Fecal Samples Originated From Several Cattle Farms In Jiangsu Province

Shiga Toxin-Producing Escherichia coli(STEC)is a general term of several intestinal pathogens,it causes scattered infections in local areas or outbreaks in large areas.E.coli O157 strain a type of STEC induces gastrointestinal diseases in human,such as hemorrhagic colitis(bloody diarrhoea)and heamolytic uremic syndrome(HUS),and ultimately leads to high morbidity and mortality.STEC O157 is transmitted through fecal-oral route.When growing in intestine toxins were secreted which exerts the virulent effect and leads to diseases occurred.Conventional detection means,such as biochemical identification,serotype identification are complicated and time-consume.Recently,the real-time PCR assays have been developed.However,most of these methods can only identify one specific target gene and could not prevent the possible of come to a false-negative conclusion.Considering that some virulence genes are not STEC O157 strain specific,they also expressed in the top six non-O157 STEC serotypes(O26,O45,O103,O111,O121,O145),therefore further bacterial serotype identification would be required to confirm the results,which can not reach the purpose of rapid detection.Since,hylAy eaeA,fliCh7,and stx2 are commonly expressed in virulent STEC O157,a triplex qRT-PCR was developed to identify STEC O157 by the detection of rfbE,stx2,and eae.The specificity,sensitivity and efficiency for the developed triplex qRT-PCR were also analyzed.Clinical feces samples from cattle in some farms in Jiangsu province were tested by this developed method and confirmed the applicability of this method for the detection of STEC O157.1 The establishment of a triplex real-time PCR assay for the detection of Shiga Toxin-Producing Escherichia coli O157In this study,0 antigen gene rfbE,and stx2,eae were employed to establish a triplex qRT-PCR to detect STEC O157.The specific experiment showed that only for the O157:H7 strain can we get three amplification curves,suggesting that the method is specific for the detection of STEC O157.In the sensitive experiment,the minimum detection limit for STEC O157 detection is 101CFU/mL,indicating that this method is highly sensitive relative to the other method.During the repetitive experiment,with the same detected samples,Ct values of rfbE,stx2,eae are keeping stable,the variations among parallel groups are less than 1,suggesting the method is repeatable.In summary,in this study,we established a triplex qRT-PCR for rapid detection of STEC O157.2 Clinical detection of STEC O157 in fecal samples originated from several cattle farms in Jiangsu ProvinceTo understand the infection status of STEC O157 in Jiangsu Province and validate the established method,250 samples from two different cattle farms in Jiangsu Provine were collected and subjected to investigation of STEC O157 by the established method.As a result,the percentage of STEC O157 positive among these samples are 2.4%and 0%in the cattle farm of Yangda and Helong respectively.In addition,we noticed that there are still many samples showed amplification curves on stx2 and eae gene,suggesting the possibility of the other types of E.coli or non-O157 STEC strains.So the established method can also be used for the analysis of other types of E.coli or non-O157 STEC strains.Furthermore,among the six STEC O157 positive samples,we only got one positive result with conventional PCR(cPCR)targeting the same three genes,which further confirmed that our established triplex qRT-PCR is suit for the investigation of clinical samples from cattle farms.