The Establishment Of RT-LAMP Detection Method For PEVD Wild Strain And The Promotion Effect Of Lactate On PEVD Proliferation

Porcine epidemic diarrhea（PED）is caused by Porcine epidemic diarrhea virus（PEDV）infection,which is characterized by vomiting,diarrhea and dehydration,and has the characteristics of rapid transmission and high lethality,and has brought great harm to the pig industry in China.In clinical practice,it was found that simple,rapid and applicable means for field detection of PEDV is of great importance for the prevention and control of PED.In this study,based on the characteristic of partial fragment deletion of ORF3 gene in PEDV CV777 vaccine virus compared with the clinical wild strain of PEDV,we designed Loop-mediated isothermal amplification（LAMP）primers as well as loop primer probes and burst probes for this region,and established the RT-LAMP method for PEDV wild strain.The RT-LAMP method of PEDV wild strain was established with the portable integrated reaction device and visualized to discriminate the reaction results.The RT-LAMP method was optimized in terms of reaction system and reaction temperature,and evaluated for sensitivity,specificity and clinical application.The results showed that 1.6 m M d NTPs,6 m M Mg SO4 and 0.12μM probe were the optimal reaction concentrations,and 65℃was the optimal reaction temperature,and the reaction could be completed within 60 min.The minimum detection line was 2.2×10³copies,and there was no non-specific reaction with other viruses.Meanwhile,the q RT-PCR method was compared with the published q RT-PCR method for detecting PEDV wild strains,and the results were consistent with the q RT-PCR method,with a100%compliance rate.In addition,the method has the characteristics of easy operation,fast reaction time and sensitive detection,and does not require expensive fluorescence quantification instruments,which has a good prospect of promoting clinical application.Since PEDV isolation and culture are difficult and cells are poorly adapted,the study of virus titer boosting technology is crucial in the production process of PEDV vaccine.In this study,based on the characteristics of cellular metabolic changes during virus infection,the effects of cellular metabolites glucose,glutamine and lactate on PEDV proliferation were investigated in order to screen for components that promote PEDV proliferation and lay the foundation for later PEDV vaccine development and process improvement.In this study,PEDV was cultured on Vero cells by using DMEM culture medium lacking glucose and DMEM culture medium supplemented with 20 m M glucose.the effect of glucose on PEDV proliferation was analyzed by Western blot,q RT-PCR and TCID₅₀.Similarly,DMEM cultures deficient in glutamine and DMEM cultures supplemented with 2 m M glutamine were used to verify the effect of glutamine on PEDV.The results showed that glucose and glutamine promoted the proliferation of PEDV.However,no promotion of PEDV proliferation was found when 5,10,20,40 and80 m M of glucose or glutamine were added sequentially to the normal DMEM culture medium.In this study,we further investigated the role of lactate,a common downstream metabolite in glucose metabolism and glutamine metabolism,in the proliferation of PEDV.Firstly,a toxicity analysis assay of lactate on Vero cells was determined.The results showed that lactate at concentrations lower than 23.06 m M did not have a significant effect on Vero cells.Secondly,when PEDV was proliferated using DMEM cultures deficient in glucose and DMEM cultures back supplemented with 20 m M lactate,it was found that lactate significantly promoted PEDV proliferation and additional addition of lactate to normal DMEM cultures still significantly promoted PEDV proliferation.This study provides effective scientific data for the attack of key technologies such as rapid virus detection in PEDV clinical samples in the field and improving the PEDV vaccine process to enhance the number of virus particles.