Establishment And Application Of A Loop-mediated Isothermal Amplification Assay For Different Virulence Factors In E. Coli From Newborn Piglets

Newborn pig diarrhea has always been an important disease against the pig industry,although the causes of piglets diarrhea is more complex,pathogenic Escherichia coli is an important cause of piglets diarrhea pathogens.At present,the detection methods of Escherichia coli are mainly isolated and identified,immune serological tests and traditional PCR methods,but these methods can not meet the requirements of the rapid detection of grassroots on-site.Loop-mediated isothermal amplification technology is a new type of nucleic acid amplification technology developed by Notomi et al.Because of its simple operation,fast and convenient,high specificity,high sensitivity and low cost,it has been widely used in pathogenic microorganisms test.Therefore,to meet the clinical rapid detection,the study establishedthe LAMP technology for the piglet pathogenic Escherichia coli detection and virulence analysis.Loop-mediated isothermal amplification technology is developed by Notomi et al,it is a new type of nucleic acid amplification technology.Because of its simple operation,fast and convenient,high specificity,high sensitivity and low cost,in recent years,it has been widely used virus detection,parasite detection,fungal and mycoplasma detection,animal embryo sex identification,genetically modified food testing and tumor gene detection.Therefore,the establishment of LAMP technology to detect E.coli for the prevention and treatment of piglets diarrhea is of great significance.This study was based on the conserved genes of Escherichia coli enterotoxin(LT1 ST1,ST2)gene,fimbriae(K88,K99,987P)gene,virulence island(HPI,ETT2,LEE)gene published by GenBank Sequence,a set of LAMP primer were designed by using the online primer design software(http://primerexplorer.jp/e/).The concentrations of Mg2+,the concentration of betaine,the concentration of dNTP,the concentration of primer,the amount of Bst DNA polymerase and the reaction temperature and reaction time were optimized.The LAMP technology for Escherichia coli enterotoxin gene,fimbriae gene and irulence island gene is established.In this study,taking enterotoxin(LT1,ST1,ST2)genotype,fimbriae(K88,K99,987P)genotype,virulence island(HPI,ETT2,LEE)genotype E.coli as positive,eight common bacteria incloding Salmonella,Staphylococcus,Riemerella anatipestosis,Pasteurella,Streptococcus,Proteus,Clostridium perfringens and Pseudomonas aeruginosa and ultrapure were used as negative control,water were used as blank control,verificating specificity of the LAMP method.The results showed that only positive bacteria were positive and the other strains did not cross-react.In addition,the sensitivity of LAMP method was evaluated by PCR method.The results showed that the positive detection rate of traditional PCR and the LAMP is consistent,but the sensitivity of LAMP method is significantly higher than PCR method.The study detects 117 samples of newborn pig diarrhea by PCR and LAMP,The results showed that was detected 42 samples of(35.90%)ETEC,and 66 samples of(56.41%)HPI+E.coli,18 samples of(15.38%)LEE+E coli,117 samples of(100%)ETT2+E coli,19 samples(16.24%)were infected with HPI+E coli and ETEC,18samples(15.38%)were infected with LEE+E coli and ETEC,42 samples(35.89%)were infected with ETT2+ E coli and ETEC,10 samples(8.55%)were infected with HPI+,ETT2+ E coli and ETEC,9 samples(7.69%)were infected with LEE+,ETT2+ E coli and ETEC,9 samples(7.69%)were infected with HPI+,LEE+,ETT2+E coli and ETEC.