Establishment Of QuantStudio^TM 3D Digital PCR Method For Detection Of Equine Herpesvirus Type 1

Equine herpesvirus1(EHV-1)is the main cause of equine rhinopneumonitis(ER).ER is an acute,febrile infectious disease in horses,which can cause abortion of mares and newborn foals.Death or neurological dysfunction.At present,there are many researches on the detection of EHV-1 by Quantitative real-time PCR(qPCR)method.However,in qPCR detection,non-specificamplification may occur in clinical samples,resulting in the inability to determine negative positive results.Relying on the standard curve and Ct value,the detection sensitivity is limited.In the early stage of ER infection,the virus content is low.Therefore,the detection sensitivity of EHV-1 needs to be improved.It is used for the early detection of EHV-1 infection in equines and the early diagnosis and prevention of ER.In this study,3D digital PCR(3D-dPCR)technology was used to design primers and TaqMan probes using the EHV-1 glycoprotein B gene ORF33 to optimize the primer probe concentration and annealing temperature in the 3D-dPCR reaction system.Specificity and repeatability analysis,establish 3D-dPCR detection method of EHV-1;and use primers andprobes of EHV-1 in OIE Terrestrial Animal Handbook to optimize primer probe concentration and annealing temperature in qPCR reaction system.Methods Sensitivity,specificity,and reproducibility were analyzed.A qPCR method was established and the results of the two methods were compared.The results show that the optimal concentration of primers and probes for the 3D-dPCR method established in this study is 0.40 ?mol/L,0.40 ?mol/L,and the optimal annealing temperature is 60?.The absolute quantitative curve of this method has R2=0.9985,which has good performance.Compared with the qPCR method,the sensitivity is an order of magnitude higher,and the minimum detection limit is 5.83 copies/?L;the coefficients of variation of the repeatability test results within batches are less than 3.2%;There was no cross reaction with equine herpes virus type 4,Theileria equi and the nucleic acid of Equine Viral Arteritis;by testing 3D-dPCR on 123 clinical samples,the results showed that the positive detection rate of the 3D-dPCR method was 66.7%,the positive detection rate of the qPCR method was 64.2%,and the 3D-dPCR This method has a higher positive detection rate than the qPCR method.The 3D-dPCR method is consistent with the results of real-time PCR for sampleswith higher viruscontent,and is more sensitive to samples with lower virus content,which can effectively detect suspicious samples.It shows that the established 3D-dPCR method has higher sensitivity,specificity and reproducibility,and can be used foraccurate quantitative detection of EHV-1.New technologies for diagnosis,prevention and control.