Establishment And Application Of Multiple Fluorescence Quantitative PCR Detection Method For Seven Serotype Of STEC

Shigella toxin-producing Escherichia coli(STEC)is a group of enteric pathogens that can cause scattered infections worldwide.Cattle are the most important host of STEC.STEC lives in cattle intestines and is excreted in feces' which could contaminate food,water and soil.It is highly pathogenic to humans and can cause human gastrointestinal diseases such as hemorrhagic enteritis,hemolytic uremic,and even death in severe cases.O157 serotype is the main pathogenic serotype of STEC.However,in recent years,the prevalence of some non-O157 serotypes(such as O26,O45,O103,O111,O121,O145)has become increasingly serious,even exceeding the O157 serotype in some countries and regions.In order to quickly and accurately detect 7 serotypes of STEC,including O26,O45,O103,O111,O121,O145,O157 in cow feces,in this paper,a detection method using multiple fluorescence quantitative PCR after enrichment have been established,which can effectively detect 7 serotypes of STEC in cow feces.After verifying the feasibility of the method,the method was applied to the detection of fecal samples from dairy farms in some areas of Jiangsu Province.1 Establishment of detection methods for 7 serotypes of STECThe traditional isolation,biochemical identification and detection methods are complicated in process,the detection cycle is relatively long,and it is easy to cause missed detection.In this paper,the Taqman probe is used to establish a multiple fluorescence quantitative PCR detection method,which can be used with enrichment culture to quickly and accurately detect samples.In this paper,seven serotypes of STEC were divided into three groups:?O157,target genes are highly specific O antigen gene rfbE and stx1,stx2,eae;?O26,O103,and O111,wzxO26?wzxO103,wzxO111 were chosen for detection;?O45,O121,O145,wzxO45?wbqE+FO121,wzxO145 were target genes.The results of the specificity test showed that the three groups of multiplex fluorescence quantitative PCR had no amplification curve except for the amplification of the target gene in the positive control group.The sensitivity test results showed that the detection limit concentration of each strain reached 101 CFU/mL.The results of repeatability test showed that the Ct value difference was less than 1 with the same target gene concentration.The above results prove that the multiple fluorescence quantitative PCR detection method has good specificity,high sensitivity and good repeatability,and the detection method is feasible.After the preliminary establishment of the test method,in order to further verify the feasibility of the method,100 samples of cow feces were collected from the dairy farm and tested by classical PGR(cPGCR)and quantitative fluorescence PCR(qPCR)before and after enrichment step.The positive rates of O26,O45,O103,O111,O121,O145 and O157 in cPCR before enrichment were 2%,2%,3%,0%,1%,5%and 1%.The positive rates of mqPCR before enrichment were 4%,5%,7%,1%,4%,17%and 5%.After enrichment,the positive rates of cPCR were 5%,3%,6%,1%,4%,11%and 2%.After enrichment,the positive rates of mqPCR were 8%,11%,10%,3%,7%,25%and 7%.The data shows that enrichment samples before multiplex fluorescence quantitative PCR detection can effectively improve the detection rate,and is more sensitive and accurate than the multiple classical PCR methods widely used internationally.2 Clinical testing of stool samples in some dairy farms in Jiangsu Province100 cow feces samples from each dairy farms in parts of Jiangsu Province were collected,and a total of 400 samples were obtained.The sample was cultured by shaking to enrich the bacteria,and the culture after the enrichment was taken to prepare the DNA template,and the multiplex fluorescence quantitative PCR was used for detection.The results showed that the positive rates of O26,O45,O103,O111,O121,O145 and O157 from A dairy farm samples were 6%,11%,7%,0%,4%,14%and 5%.The positive rates from B dairy farm samples were 9%,4%,11%,2%,8%,18%and 6%.The positive rates from C dairy farm samples were 3%,5%,4%,0%,3%,0%and 0%.The positive rates from D dairy farm samples were 2%,3%,2%,0%,1%,16%,and 0%.The data showed that the dominant serotype of the four dairy farms was STEC O145,and the positive rates were 14%,18%,27%and 16%,respectively.In summary,the multiple fluorescence quantitative PCR detection method established in this article has high sensitivity and good specificity;the prevalence of STEC is different among different cattle farms in Jiangsu Province.