Preparation Of Nucleic Acid Reference Material For Bluetongue Virus Type 17 And Establishment Of BTV QRT-PCR Assay

Bluetongue(BT)is an arbovirus disease caused by Bluetongue virus(BTV)transmitted by blood bank midges.Cattle,sheep and other ruminants are susceptible to it.At present,there is no effective treatment method.The World Organization for Animal Health(WOAH)lists BT as a statutory notifiable multi-animal disease,and China lists it as a Class II animal disease.In view of the lack of literature on BTV nucleic acid reference material and the lack of nucleic acid reference material such as whole strain in BTV nucleic acid detection,it is impossible to conduct sample quality control and other problems.The preparation of BTV-17 nucleic acid reference material and the establishment of BTV real-time quantitative RT-PCR(qRT-PCR)with internal parameters were studied.1.Preparation,calibration and application of BTV-17 nucleic acid standard materialIn this study,BTV-17 strain was proliferated,inactivated and lyophilized,and BTV-17suspension was tested by BTV qRT-PCR for concentration detection,initial uniformity detection and inactivation detection,and BTV-17 nucleic acid reference material was prepared.Physical character test,inactivation test,mycoplasma test and purity test of BTV-17 nucleic acid reference material were carried out.The homogeneity,short-term stability and long-term stability of random samples were investigated.The BTV-17 nucleic acid reference material is yellow,loose solid.After three generations of blind transmission of the inactivated BTV-17 into BHK-21 cells,there was no cytolesion in the cells of each generation,and the amount of virus in each generation decreased by qRT-PCR,indicating that BTV-17 had been inactivated.Mycoplasma test was performed and the results were negative.The pure test showed no amplification curve except BTV-17.10 bottles of reference materials were randomly selected for uniformity test,and the results showed that the samples were uniform and stable.BTV-17 nucleic acid reference material can be stored for 9 days at-20℃,4℃,25℃and 37℃.There is no significant difference at-80℃,-20℃,4℃,25℃,all can be stored for 6 months.BTV-17 nucleic acid reference material was set by using multiple laboratories to set values,cooperating with three laboratories to set values,and statistical analysis was conducted on the data of multiple laboratories.The uniformity,stability and uncertainty in the setting process were statistically analyzed.The standard value of BTV-17 copies was 9.15×10~6copies/μL,and the extended standard uncertainty(k=2)was 5.55×10~5copies/μL.The final determination result of BTV-17 was(9.15±0.56)×10~6copies/μL.2.Establishment of qRT-PCR method for Bluetongue disease containing internal reference geneBased on the primers and probes of the qRT-PCR method for detection of BTV NS3in the Manual of Diagnostic Tests and Vaccines for Terrestrial Animals(2022)of the World Organization for Animal Health,a pair of reference gene GAPDH primers and probes were designed.Standard p UC57-BTV-NS3 and p UC57-GAPDH were used to construct standard curves of NS3 gene and reference gene GAPDH in qRT-PCR,respectively.QRT-PCR method for BTV detection using GAPDH as reference gene was established by optimizing the reaction conditions.The results showed that established method has no cross-reaction with Epizootic haemorrhagic disease Virus,Bovine viral diarrhea virus,Foot-and-mouth disease virus,Peste des petits ruminants virus,Orf virus,Goatpox virus and Vesicular stomatitis virus,and has good specificity.The minimum detections of BTV and GAPDH positive plasmid standard copies were 6.685 copies/μL and 1.9×10~2copies/μL,respectively.The coefficient of variation of BTV and GAPDH in the intra and interlot reproducibility tests were below 1.2%and 0.8%,respectively,showing good reproducibility.The established BTV qRT-PCR method was used to detect 88 sheep serum samples stored in the laboratory,and the positive rate was 4.55%.Compared with the nested PCR for detecting BTV recommended by WOAH,the results showed that the detection rate of the two methods was 100%,but the qRT-PCR method was more convenient to operate.The results are accurate and more suitable for the detection of a large number of clinical samples.The qRT-PCR method established in this study for simultaneously detecting BTV and internal reference gene GAPDH has high sensitivity,strong specificity and high repeatability.In summary,BTV-17 nucleic acid reference material was prepared in this study,and the reference material was calibrated collaboratively to provide reliable reference value.A BTV qRT-PCR detection method containing internal reference genes was established,and the internal reference genes were added for sample quality control,which improved the accuracy of detection.This study is beneficial to improve the BTV nucleic acid detection system and promote the standardization of BT detection methods.