Establishment Of A Loop-mediated Isothermal Amplification (LAMP) For Rapid Detection Of ORF Virus

In the present study,the VIR gene amplification of ORFV was carried out by pair of outer primers that were designed according to the VIR gene sequence of ORFV released in GenBank.After the correct band was recycled according to the results of the UV gel imager,the cloned partial gene of VIR target sequence was then loaded into the T vector to provide positive material for subsequent series of test experiments.Using two pairs of primers,the VIR gene amplification of ORFV was carried out.At last,through the optimized LAMP reaction conditions,we established a method to detect ORFV with LAMP.In this study,the detection of ORFV was carried out by using optimized LAMP.The negative positive difference was tested by chromogenic reaction,and then the specific and the sensitivity experiment were conducted.The results showed that using the optimized LAMP,the positive group in the color yellow was separated from the negative group in the color orange.Detection of the amplified plasmid and positive control samples showed positive bands.But there were no positive bands in samples of goat pox virus,sheep pox virus,infectious bovine rhinotracheitis virus and bovine parainfluenza type 3 virus.The sensitivity of the optimized LAMP to detect ORFV was 1000 times higher than that of the traditional PCR.The detection results of 13 clinical samples showed that the detection rate of ORFV was 100% using optimized LAMP,which was consistent with the results of conventional PCR.In conclusion,the results showed that the optimized LAMP has the advantages of specificity,high sensitivity and convenience for ORFV detection,so it is more suitable for high efficiency on-site detection,which will buy more time for subsequent work.