鸡传染性贫血病毒CRISPR/Cas13a检测方法的建立及初步应用

Establishment and preliminary application of the CRISPR/Cas13adetection method for chicken infectious anemia virus

为了快速、准确和简便地检测鸡传染性贫血病毒(chicken infectious anemia virus,CIAV),本研究参照GenBank中CIAV的VP2基因设计了特异的RPA引物和用于LwCas13a反应系统的crRNA,通过优化反应条件建立了检测CIAV的CRISPR/Cas13a方法,并验证了该方法的特异性、敏感性和复合性。结果显示,该法对感染鸡的其他常见病毒的检测均为阴性,特异性良好无交叉反应;灵敏度为50copies/μL;与PCR的检测结果有很好的符合率。本研究建立的CRISPR/Cas13a检测方法配合试纸条显色在1.5h内可对CIAV进行快速的鉴别诊断,为该病的诊断及防控净化提供了强有力的技术支撑。

In order to detect chicken infectious anemia virus(CIAV)quickly,accurately and simply,the specific RPA primers and crRNA used in the LwCas13areaction system were designed with reference to the VP2gene of CIAV in GenBank,and the CRISPR/Cas13amethod for detecting CIAV was established by optimizing the reaction conditions,then the specificity,sensitivity and complexity of the method were verified.The results showed that the detection of other common viruses infecting chickens by this method was negative,with good specificity and no cross-reaction.The sensitivity was 50copies/μL.It had a good coincidence rate with the detection results of PCR.The CRISPR/Cas13adetection method established in this study,combined with the color development of the test strip,can quickly differentiate the diagnosis of CIAV within 1.5h,which provides a strong technical support for the diagnosis,prevention and purification of this disease.