非洲猪瘟病毒的RPA-CRISPR-Cas12a快速检测方法的建立

Establishment of RPA-CRISPR-Cas12a rapid detection method for African swine fever virus

为了建立一种特异性好、灵敏度高的非洲猪瘟病毒(ASFV)快速检测方法,试验针对ASFV B646基因保守片段设计并合成特异性crRNA(crRNA1、crRNA2、crRNA3),并根据crRNA所在的基因序列区域设计重组酶聚合酶扩增(RPA)引物(RPA1-F/RPA1-R、RPA2F/RPA2-R),通过筛选最佳RPA引物-crRNA序列组合和crRNA浓度建立ASFV的RPA-CRISPR-Cas12a快速检测方法,在评估该方法的特异性和灵敏度后对ASFV阳性临床样本进行验证。结果表明:最佳RPA引物-crRNA序列组合为RPA2-crRNA3,最佳crRNA浓度为5μmol/L;该方法仅能检测出ASFV,不能检测出猪瘟病毒(CSFV)、猪传染性胃肠炎病毒(TGEV)、猪繁殖与呼吸综合征病毒(PRRSV)、猪圆环病毒2型(PCV-2),特异性良好;最低检测病毒浓度为5.8 copies/μL,灵敏度较高;应用该方法对20份ASFV阳性临床样本进行检测,结果均为阳性,符合率为100%。说明成功建立了ASFV的RPA-CRISPR-Cas12a快速检测方法,该方法特异性好、灵敏度高,可用于ASFV的现场快速检测。

In order to establish a rapid detection method for African swine fever virus(ASFV)with good specificity and high sensitivity,in this experiment,design and synthesis of specific crRNA(crRNA1,crRNA2,crRNA3)for conserved fragments of ASFV B646 gene were carried out,RPA primers(RPA1-F/RPA1-R,RPA2F/RPA2-R)were designed according to the gene sequence region in which the crRNA was located,and the RPA-CRISPR-Cas12a rapid detection method for ASFV was established by screening the best RPA primer-crRNA sequence combination and crRNA concentration.The specificity and sensitivity of the method were evaluated and then the clinical samples were used in verification.The results showed that the optimal RPA primer-crRNA sequence combination was RPA2-crRNA3,and the crRNA concentration was 5μmol/L.This method could only detect ASFV,but could not detect Classical swine fever virus(CSFV),Transmissible gastroenteritis virus(TGEV),Porcine reproductive and respiratory syndrome virus(PRRSV),Porcine circovirus type 2(PCV-2),with good specificity.The lowest detection concentration was 5.8 copies/μL,and the sensitivity was high.This method was used to test the DNA of 20 ASFVpositive clinical samples,and the results were all positive,and the compliance rate was 100%.The results suggested that the RPA-CRISPRCas12a rapid detection method for ASFV had been successfully established,with good specificity and high sensitivity,which can be used for the on-siterapiddetection of ASFV.