Establishment Of A Rapid Detection Method For Bovine Viral Diarrhea Virus Nucleic Acid

Bovine viral diarrhea(BVD)is an infectious disease caused by bovine viral diarrhea virus(BVDV)infection in cattle.After infection,it will not only produce severe clinical symptoms,but also reduce the immunity of animals and increase the risk of infection by other pathogens,leading to an increase in the morbidity and mortality of diseased animals,which seriously hindered the healthy development of the cattle industry.The comprehensive prevention and control of BVD relies on its rapid,specific and convenient diagnosis,and the rapid detection of BVDV nucleic acid is the key technology for diagnosing its infection.Loop-mediated isothermal amplification(LAMP)and recombinase polymerase amplification(RPA)are two new nucleic acid isothermal amplification technologies.The technology does not require special equipment,has advantage of simple operation,short reaction time,high sensitivity and strong specificity.So,it is very suitable for field application.Therefore,on the basis of referring to the existing detection methods and analyzing the BVDV genome,this study established RPA and LAMP detection methods,and then selected a visualization method more suitable for the rapid detection of BVDV nucleic acid in the field,in order to provide technology for the diagnosis and prevention of BVD.The results of the study are as follows:1.Based on the conserved 5’-UTR sequence of BVDV genome,a LAMP detection method for BVDV was established,and the optimal reaction time was 50 min,the optimal reaction temperature was 62℃ and the reaction system was determined.The specificity results showed that the method could distinguish BVDV strains from other strains associated with diarrhea with good specificity.The sensitivity results showed that the lowest detection limit of LAMP was 1.9×102copies/μL,and the sensitivity was 10 times higher than that of PCR.2.Based on the conserved sequence of 5’-UTR of BVDV genome,a basic RPA detection method for BVDV was established,and the optimal reaction time was 15min and the optimal reaction temperature was 37℃.Using the positive plasmid standard pUC57-BVDV 1254 as a template for gradient dilution,the minimum detection limit of basic RPA was determined to be 1.9×102copies/μL,which was consistent with the minimum detection limit of LAMP.The results showed that the method has good sensitivity,specificity and reproducibility.3.The LFD-RPA detection method of BVDV is established on the basis of basic RPA.The optimum reaction time was determined to be 15 min,and the optimum reaction temperature was 35℃.Using cDNA as the template for gradient dilution,the minimum detection amount was 5.8×10-2ng/μL;the positive plasmid standard pUC57-BVDV1254 was used as the template for gradient dilution,and the minimum detection limit was determined to be 1.9×102copies/μL.The sensitivity was consistent with basic RPA and LAMP.The results showed that the method has good sensitivity,specificity and reproducibility,and can distinguish BVDV from other pathogens associated with diarrhea.4.Using the established BVDV LAMP,basic RPA and LFD-RPA methods and PCR methods to detect 107 clinical serum samples,the positive detection rates of LAMP and PCR were 1.87%(2/107),and the positive detection rate of basic RPA and LFD-RPA were 2.80%(3/107).In conclusion,by analyzing the BVDV genome and selecting highly conserved sequences,the detection methods of BVDV LAMP,basic RPA and LFD-RPA were successfully established.These three isothermal nucleic acid amplification methods have good specificity and sensitivity.Based on the comparison of its rapidity,simplicity and visualization,the LFD-RPA method is more suitable for the detection of BVDV in the field,and can provide technical support for the prevention,control and elimination of BVD.