Establishment Of Visualized Loop-Mediated Isothermal Amplification Method For The Detection Of IBRV,BRSV And M.Bovis

Infectious bovine rhinotracheitis virus,mycoplasma bovis and bovine respiratory syncytial virus,as common causes of bovine respiratory diseases,can be collectively referred to as bovine respiratory disease complex,which seriously threatens the health of the cattle industry.The commonly used PCR,q PCR,pathogen isolation and identification methods for clinical sample detection have reliable results and strong specificity.However,it is difficult for the above methods to be applied to the grass-roots units such as livestock farms because they do not have corresponding instruments and equipment.In order to solve this problem,this study adopted the Loop-mediated isothermal amplification method.In addition,acid-base dyes neutral red or cresol red were pre-added into the reaction solution,and a new type of visual(RT)LAMP method which can be directly observed and judged by naked eye was established.The visual(RT)LAMP detection methods of bovine infectious bovine rhinotracheitis virus(IBRV),bovine respiratory syncytial virus(BRSV)and Mycoplasma bovis(M.bovis)were established by using the above method,which provided convenience for the field detection of bovine respiratory diseases.The visualization(RT)LAMP method of IBRV,BRSV and M.bovis was established successfully.Given that IBRV and M.bovis genomes are DNA and BRSV are RNA viruses,corresponding DNA sequences and c DNA sequences are used as templates respectively.Several pairs of specific primers were designed according to the conserved region sequences of g B gene of IBRV,N gene of BRSV and opp D/F gene of M.bovis,respectively.Multiple pairs of primers,reaction time,reaction temperature and various color developing agents were optimized and screened.The visualization(RT)LAMP method was highly specific and did not cross-react with other respiratory pathogens in cattle and sheep.The minimum detections of IBRV,M.bovis and BRSV target genes were 2.74×10~1copies/μL,2.54×10~1copies/μL,2.17×10~1copies/μL,respectively,and compared with the(RT)q PCR method recommended by WOAH et al.The detection limits of(RT)q PCR were10~2 copies/μL(IBRV),1.91×10~1copies/μL(M.bovis)and 10~3copies/μL(BRSV).The present method was applied to 39 clinical samples collected from cattle farms in Hebei Province,and the results showed that all three pathogens were detected and mixed infection existed.The positive rates of visualization(RT)LAMP were 12.8%(IBRV),15.4%(BRSV)and 12.8%(M.bovis),respectively,which were basically consistent with the results of comparison method.This study successfully established a field detection method for three pathogens of bovine respiratory diseases,which can be applied to the rapid detection of a large number of clinical samples,providing a convenient detection method for the prevention and control of bovine respiratory diseases in the cattle industry.