Development And Application Of Detection Kits By Insulated Isothermal Fluorescence RT-PCR Of BVDV1

Bovine viral diarrhea virus(BVDV)is an important pathogen that causes bovine respiratory syndrome,reproductive disease,diarrhea/mucosal disease,immunosuppression and persistent infection,which has caused huge losses to the global cattle industry.PCR detection technology is the mainstream means to detect BVDV at present.Although many BVDV PCR detection methods established at present,there are no PCR detection methods and kits suitable for clinical detection of BVDV,which restricts the rapid diagnosis and quarantine of BVDV.The purpose of the study is to develop a kit for detecting BVDV1 based on insulated isothermal RT-PCR(iiRT-PCR)technology,and to use the kit for BVDV1 field diagnosis and epidemiological investigation.And achieved the following results: 1.Development and application of BVDV1 isothermally isolated fluorescent RT-PCR detection kitIn order to develop a fluorescence RT-PCR detection kit for on-site detection of BVDV1,a total of 54 BVDV1,43 BVDV2,and 11 BVDV3 5?UTR sequences downloaded from GenBank were analyzed and the specific detection primers(BVDV1 iiF: 5'-AAGCCTCGAGATGCCA-CG-3',BVDV1 iiR: 5'-GCAGCACCCTATCAGGCTGT-3')and probes(BVDV1 iiPb: 5'-FAM-CCCACAGCACATCTTAA-MGB-3')was designed of BVDV1.Through the optimization of the system,a BVDV1 constant temperature isolated fluorescent RT-PCR detection method was established.The stability and specificity of the method was good.It could be detected in the BVDV1 reference strains,BVDV1 a,BVDV1b,BVDV1 c,and BVDV1 d.No pathogens were detected in BVDV2 and other unrelated pathogens.The detection limit is 52.7 copies· ?L-1,and the sensitivity is high.60 diarrhea stool samples and 60 serum samples were tested with this method and the RT-PCR method recommended by SNT1905-2007 and the fluorescence quantitative RT-PCR methods reported in the two literatures.The results showed that this method was superior to the other three methods.The reagents optimized for this method were quantitatively lyophilized and co-assembled with the positive plasmid template and the lysate Buffer into a 50 ?L system test kit,which can meet the requirements of one 50 ?L system or two 25 ?L systems,reducing the number of sample addition steps and the reaction time and the pollution during the operation,and has the advantage of being able to store and transport at room temperature.In combination with the PetNAD nucleic acid extraction kit and the POCKIT hand-held nucleic acid analyzer,the on-site diagnosis of BVDV1 was realized,and it takes only 1 h from the extraction of nucleic acid to the detection result.Using the BVDV1 iiRT-PCR detection kit developed in this experiment,on-site detection and diagnosis of diseases characterized by diarrhoea in two ranches in Ruer Township and Anqu Town in Aba were detected,and BVDV1 was detected in two ranches.A total of 29 commercially available bovine semen samples from 9 provinces/municipalities in China and 20 fetal bovine serum samples from 4 brands from 2016 to 2017 were detected,and the detection rate of BVDV1 was 34.48%.75% indicates that the rate of BVDV1 in commercial bovine serum concentrate and fetal bovine serum circulating in Sichuan Province is relatively high.2.Epidemiological investigation of pathogenic yak BVDV1 in Northwest SichuanThe northwest Sichuan grassland is one of the main areas for yak farming in China.Yak diarrhea is a common and frequently occurring disease in this area.In order to investigate the prevalence of BVDV1 in diarrhea samples of yak from the northwestern Sichuan and the core population of the Maiwa yak,448 diarrhea samples in 25 pastures in the northwestern Sichuan during 2015-2017 and 627 serum samples in 3 pastures in the core population of the Maiwa yak were collected.The epidemiological investigation of BVDV1 pathogens was conducted using the developed BVDV1 iiRT-PCR kit,and the mixed infection of BVDV1 with bovine rotavirus(BRV)and bovine coronavirus(BCoV).The results showed that the average detection rate of BVDV1 was 29.01% in the diarrhea samples from 2015 to 2017,and the detection rates of BVDV1 in 2015,2016,and 2017 were 27.47%,30%,and 29.11% respectively.The investigation of mixed infections of BRV and BCoV in 130 BVDV1 positive samples showed that the mixed infection rates of BVDV1 with BRV and BCoV were 90% and 75.38% respectively.The results of this experiment indicate that the mixed infection of BVDV1,BRV,and BCoV in the grasslands of northwestern Sichuan is serious.