基于RAA-CRISPR/Cas12a建立牛病毒性腹泻病毒的可视化快速检测方法及其初步应用  

Establishment and application of visualization detection technology for bovine viral diarrhea virus based on RAA-CRISPR/Cas12a

作　　者：热则古丽·艾科拜尔 张亚平 刘浩然 杨莉 马雪连 姚刚[1,2] 史慧君 付强[1,2] REZECULI Aikebaier;ZHANG Yaping;LIU Haoran;YANG Li;MA Xuelan;YAO Gang;SHI Huijun;FU Qiang(College of Veterinary Medicine,Xinjiang Agricultural University,Urumqi 830000;Xinjiang A utonomous Region Key Laboratory of New Drug Research and Development for herbivores,Urumqi 830000)

机构地区：[1]新疆农业大学动物医学学院,新疆乌鲁木齐830000 [2]新疆草食动物新药研究与创制自治区重点实验室,新疆乌鲁木齐830000

出　　处：《中国兽医科学》2025年第3期321-329,共9页Chinese Veterinary Science

基　　金：新疆维吾尔自治区杰出青年基金项目(2022D01E15);新疆维吾尔自治区“天山英才”青年科技拔尖人才专项(2022TSYCCX0049);新疆维吾尔自治区重大科技专项(2023A02007-2)。

摘　　要：为建立一种基于重组酶介导的扩增技术(RAA)和CRISPR/Cas12a相结合的牛病毒性腹泻病毒(bovine viral diarrhea virus,BVDV)可视化检测方法。参考GenBank中BVDV 5’UTR基因设计RAA反应的特异性引物和探针,进一步优化RAA-CRISPR/Cas12a反应条件,通过测定灵敏度、特异性和重复性来评估RAA-CRISPR/Cas12a检测方法 。分别使用PCR和RAA-CRISPR/Cas12a对251份犊牛腹泻样品进行了BVDV阳性检测。结果表明,RAA-CRISPR/Cas12a检测方法检测BVDV的最低检测限为3 copies/μL,检测时间为30 min。在对比不同拷贝数阳性样品进行多批次检验后,批内和批间变异系数皆小于5%。用该方法在检测其他牛常见病原微生物时结果均呈阴性,特异性良好。在临床样本检测中,BVDV的PCR阳性检出率为6.37%;同时使用RAA-CRISPR/Cas12a检测时,阳性检出率为29.48%,阳性检出率为PCR的4.63倍。本研究建立了基于RAA-CRISPR/Cas12a的BVDV可视化快速检测方法,对犊牛腹泻的防控具有重要意义。To establish a visual detection method for bovine viral diarrhea virus(BVDV)based on the combination of recombinant enzyme mediated amplification(RAA)and CRISPR/Cas12a.By referring to the BVDV 5'UTR gene in the GenBank database,specific primers and probes for the RAA reaction were de-signed to further optimize the reaction conditions of RAA-CRISPR/Cas12a,and the detection method of RAA-CRISPR/Cas12a was evaluated by measuring sensitivity,specificity,and repeatability.BVDV was de-tected in 251 calf diarrhea samples by PCR and RAA-CRISPR/Cas12a,respectively.The results indicated that for the RAA-CRISPR/Cas12a detection method,the minimum detection limit of BVDV was 3 copies/μL and the detection time was 30 min.After multiple batches of positive samples with different copy num-bers were tested,the coefficients of variation intra-and inter-batches were both less than 5%and and the results of this method were negative in the detection of other common pathogens of cattle with good specificity.In clinical samples,the PCR positive rate of BVDV was 6.37%.When RAA-CRISPR/Cas12a was utilized simultaneously,the positive detection rate was 29.48%,and the positive detection rate reached 4.63 times.In this study,a rapid visual detection method for BVDV based on RAA-CRISPR/Cas12a was established,which is of great significance for the prevention and control of diarrhea in calves.

关 键 词：牛病毒性腹泻病毒 重组酶介导等温核酸扩增技术 CRISPR/Cas12a 检测 

分 类 号：S852.659.6[农业科学—基础兽医学]