Establishment And Comparison Of Loop-mediated Amplification And Real-time Fluorescent PCR For Detection Of African Classical Swine Fever Virus

African swine fever(ASFV)is a kind of virus that infects pigs Since the first case of African swine fever appeared in China in 2018,the epidemic has spread to 31 provinces,seriously affecting the production performance of pigs,restricting economic development,and causing great harm to public health and safety.In 2020,a total of 27 countries and regions in the world will have 2538 domestic pigs and 8188 wild pigs,with a total of 10726 African swine plague cases.In epidemic prevention and control,vaccine immunization is one of the main means to prevent and control animal diseases.However,there is no effective commercial African swine fever virus vaccine in the world.Therefore,detection of African classical swine fever has become particularly important in epidemic air defense.In view of this,this study established ASFV colorimetric loop mediated isothermal amplification detection method,and established ASFV MGB probe method.1.The conserved fragment of African classical swine fever virus b646 l gene was selected as the detection target,and six specific primers were designed for t he six targets of the gene.After optimizing the concentration ratio of internal and external primers,magnesium ion concentration,d NTP concentration and other components in the system,the African classical swine fever virus colorimetric loop mediated is othermal detection method was successfully established.This method was used to detect porcine epidemic diarrhea virus(PEDV),porcine delta coronavirus(pdcov),highly pathogenic porcine reproductive and respiratory syndrome virus(hpprrsv)and pseudorabi es virus(PRRSV),The results showed that the established method had good specificity.The detection limit of 10 copies / ? l can be achieved by using the known concentration of positive reference sample as the reaction template after 10 fold gradient dilu tion.The sensitivity of this method is comparable to that of fluorescent quantitative PCR.2.Similarly,a pair of specific primers and MGB probe were designed with the conserved region of ASFV b646 l gene as the detection target.The 5 'end of the probe was labeled with FAM fluorescent group,and the 3' end was labeled with MGB quenching group.The fluorescence quantitative PCR method for detection of ASFV was established.The results showed that the method had good specificity and the minimum detection limit was 6 The sensitivity of lamp is more sensitive than that of lamp,but lamp is better than lamp in terms of detection cost.3.In this study,different methods were compared to interpret the lamp results.The results were interpreted by using SYBR Green I,cresol red,hydroxy kaempferol blue and calcein respectively.The results showed that the sensitivity was SYBR Green I >cresol red ? hydroxy kaempferol blue > calcein Cresol red method was used in this study.The results showed that the method was stable,and the color of positive samples remained stable after 3 days at 4 ?.