In this study,we developed a simple and rapid method for in situ detection of koi herpesvirus(KHV)by combing the advantages of recombinase-aid amplification(RAA)and lateral flow dipstick(LFD)technology,so-called RAA-LFD.According to RAA primer design principle,5 pairs of primers were designed within the highly conserved region of KHV Sph gene,and the No.5 primers were selected as the optimal ones,which were labeled with biotin and FAM at the 5'-end of the reverse-primer and probe,respectively.The reaction time and temperature of RAA were optimized by using the square matrix method,and the results showed that RAA-LFD could effectively amplify the target gene fragment of KHV at a constant temperature of 34℃-40℃for 10 minutes,which allowed an on-site observation.Therefore,a RAA-LFD detection method for KHV was preliminarily established.The specificity test results showed that RAA-LFD just specifically reacted with the nucleic acid of HKV,and had no cross-reaction with that of other poultry pathogens such as cyprini edema virus(CEV),carassius carassius hematopoietic organ necrosis virus(CHNV),epidemic hematopoietic organ necrosis virus(EHNV)and frog rainbow virus(BIV).The RAA-LFD had a comparable sensitivity to the industry conventional PCR,with a minimum detection limit of 50 copies/μL for the plasmid pEASY-KHV samples.The repeatability test results showed that 5 groups of independent pEASY-KHV samples were detected as KHV positive by the RAA-LFD,even though the samples were placed at room temperature for 7 days,indicating that the RAA-LFD has good repeatability.The results from 60 clinical samples detected by both RAA-LFD and conventional PCR showed 100%coincidence between the two methods,including the coincidence rate of negative and positive and the total coincidence.Taken together,the RAA-LFD approach established in this study showed highly specificity,sensitivity and repeatability,which can be applied for rapid clinical diagnosis of KHV.
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