Establishment And Application Of Visual Loop-mediated Isothermal Amplification Detecting Porcine Transmissible Gastroenteritis Virus

Transmissible gastroenteritis（TGE）caused by transmissible gastroenteritis virus（TGEV）,that is an acute and contact intestinal infectious disease.TGEV belong to Coronavirus family coronavirus genus.The research found that pigs of different ages and breeds are susceptible to TGEV infection,and usually shows vomiting,watery diarrhea,and dehydration.Pig infection is often manifested as recessive infection or mixed infection with other viral diarrhea diseases.There is no effective treatment measures for the time being.It weigh heavily against the healthy and sustainable of China’s pig industry.Therefore,it is of great significance for detect and confirm the pathogen as soon as possible.The operating time of conventional etiological diagnosis technique is too long to meet the requirements of rapid detection.The molecular biology and serological diagnostic techniques are costly and difficult to operate,which are not applicable for grass-roots units.Thus,it is necessary to explore and establish more convenient and sensitive method for TGEV detection.In this study,M gene sequences of major epidemic TGEV strain was compared and then both inside and outside specific primers for Loop-Mediated Isothermal Amplification（LAMP）were designed according to the highly conserved region of TGEV M gene.A visual Loop-mediated isothermal amplification method for TGEV detection was established using hydroxyl naphthol blue（HNB）as indicator.The detection can be completed by incubating the reaction system for 45 min at 63℃to amplify,then 10 min under 85℃to inactivate Bst DNA polymerase.In addition,the method was optimized by selecting the reaction conditions and reaction system components.The 25μL optimum system of TGEV amplication included d NTP_S0.4 mmol/L,Mg²⁺4 mmol/L,Betaine 0.4 mol/L,10×Bst DNA Polymerase Buffer2.5μL,Bst DNA polymerase 1μL,HNB（5 mmol/L）1μL,appropriate template,and the ratio of internal to external primers 1:6.Using the LAMP primers to amplify the viruses preserved in our laboratory,which including TGEV,PEDV,Po RV,PPV,PCV2 and PRRSV.The results displayed that only TGEV could be detected,which means the specificity of the method is good.At the same time,its sensitivity was evaluated and it was found that the lowest detectable number of p ET-21b-M plasmid copies was 3.7×10³.It was two orders of magnitude higher detected with LAMP than conventional PCR.By adding HNB before the reaction,the color of the positive tube could be visually observed to change from purple to blue,however the color of negative reaction tube did not change.The color difference between negative and positive tubes was obvious.42 clinical samples from swine farms in Shaanxi,Ningxia and Shandong were assayed with the established LAMP method,the results showed that 21 samples were positive.The visualized detection results were completely consistent with the identification of agarose gel electrophoresis.Only 4 positive samples could be detected by regular PCR.The established LAMP detection method of TGEV in this study could be completed in a constant temperature within 1 h,and HNB was added into the system before the reaction,which not only avoids aerosol pollution caused by open cover detection,but also achieves the purpose of visualized diagnosis of pathogens.In generally,this study provides a practical technique for clinical units to diagnose TGE more conveniently and rapidly.