Establishment And Clinical Application Of Loop-mediated Isothermal Amplification Detection Method For E. Coli Virulence Factor Of Weaned Piglet Edema Disease

Edema disease of swine(ED)is an important disease of weaned pigs caused by Shiga toxin-producing E.coli,the incidence of acute and rapid death,poor clinical treatment,high mortality.At present,there are many methods for detecting E.coli,but these methods can not meet the requirements of rapid field test.Ring-mediated isothermal amplification is a new type of nucleic acid amplification technology developed in recent years.Because of its simple,rapid and convenient operation,strong specificity,high sensitivity and low cost,it has been widely used in the detection of pathogenic microorganisms widely usedIn this study,two set of LAMP primers were designed based on the conserved sequence of the Stx2e gene of E.coli and the fimbriae F18ab gene published in the GenBank and online primer design software(http://primerexplorer.jp/e/).LAMP methods for detection of Stx2e gene and F18ab gene of shiga toxigenic escherichia coli were established based on a series of optimization of the concentration of Mg2+,the concentration of dNTP,the concentration of betaine,the concentration of primer,the amount of Bst DNA polymerase,the reaction temperature and the reaction time were selected in the reaction system.The positive control was Stx2e genotype and F18ab bacterial hair genotype of shiga toxigenic escherichia coli,with Pasteurella,staphylococcus aureus,salmonella,duck plague creamer’s bacillus,streptococcus and proteus,gas capsule clostridium,swine erysipelas coli,F18ac pili type as a negative control to ultrapure water as a blank control,respectively,the established LAMP method for specific experiments,the results showed that only the shiga toxigenic escherichia coli was positive,the other strains were negative and no cross-reaction occurred.The LAMP methods established in this study were used to detect 83 samples of suspected edema disease of weaned piglets,and conventional PCR method was used as the control method.The results showed that the detection results and detection rate of the two methods were consistent,but the established LAMP sensitivity was significantly higher than the conventional PCR method.