福建农业学报2025，Vol.40Issue(2)：151-157,7.DOI:10.19303/j.issn.1008-0384.2025.02.005
RAA-CRISPR/cas13a(cpf1)-LFD兔出血症2型诊断方法的建立
RAA-CRISPR/Cas13a(cpf1)-LFD Method for Diagnosis of Rabbit Hemorrhagic Disease
摘要
Abstract
[Objective]An urgently needed rapid and simple method for diagnosing the endemic rabbit hemorrhagic disease caused by RHdV-2 identified in China in 2020 was established.[Method]Vector NTI Alignment was used to analyze the genomic sequences of the two serotypes of RHdV,Type 1 and Type 2.The specific and conserved fragment VP60 of RHdV-2 was obtained to design probes and specific primers for the recombinase-aided amplification-lateral flow dipstick(RAA-LFD)assay.Based on the RAA amplification products,a CRISPR-derived RNA was selected with the RAA-CRISPR/Cas13a(Cpf1)-LFD reaction conditions optimized and assay sensitivity and specificity evaluated.Subsequently,in comparison with the RAA previously established in our laboratory,the newly developed assay was used to detect clinical specimens.[Result]A specific and conserved sequence in VP60 of RHdV-2 was found that led to synthesis of the primers for RAA amplification.Using the amplified product as the reaction template,positive sample could be best detected with crRNA1.By adding 10 min to the RAA reaction time,a clear positive result on the test strip was obtained.The sensitivity test using the recombinant plasmid as the template showed the lowest number of detectable plasmid copies to be 6.7×101 copies·μL-1.The new assay was highly specific as it detected only RHdV-2 as positive.And it tested 10 positives on 31 clinical specimens with a detection rate of 30.3%.[Conclusion]The RAA-CRISPR/Cas13a(Cpf1)-LFD method was rapid and simple to operate and highly sensitive and specific in detecting RHdV-2.It significantly improved the diagnostic method of the hemorrhagic disease in rabbits.关键词
重组酶介导的核酸等温扩增/CRISPR-Cas13a系统/侧流层析试纸条/兔出血症病毒2型/诊断方法Key words
recombinase-aided amplification/CRISPR-Cas13a system/lateral flow dipstick/rabbit hemorrhagic disease type 2 virus/diagnostic method分类
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林芸,王媛,刘立荣,陈佳祺,陈蓓蕾,王全溪..RAA-CRISPR/cas13a(cpf1)-LFD兔出血症2型诊断方法的建立[J].福建农业学报,2025,40(2):151-157,7.基金项目
福建省星火计划项目(2020S0005) （2020S0005）
