猫杯状病毒RAA-CRISPR/Cas12a-LFS检测方法的建立及初步应用  

Establishment and preliminary application of RAA-CRISPR/Cas12a-LFS assay for detection of feline calicivirus

作　　者：周红蕾[1] 程淑琴 李佳鹏 刘涵 卓国荣[1] 张蕾[1] ZHOU Hong-lei;CHENG Shu-qin;LI Jia-peng;LIU Han;ZHUO Guo-rong;ZHANG Lei(Jiangsu Agri-animal Husbandry Vocational College,Taizhou 225300,China;College of Veterinary Medicine,Jilin University,Changchun 130062,China)

机构地区：[1]江苏农牧科技职业学院,江苏泰州225300 [2]吉林大学动物医学学院,吉林长春130062

出　　处：《中国预防兽医学报》2023年第5期494-501,共8页Chinese Journal of Preventive Veterinary Medicine

基　　金：江苏农牧科技职业学院项目(NSF2021ZR11);国家自然科学基金青年基金项目(32002157)。

摘　　要：为建立猫杯状病毒(FCV)基于重组酶介导的等温核酸扩增(RAA)结合CRISPR-Cas12a系统及侧向流层析试纸条(LFS)(RAA-CRISPR/Cas12a-LFS)的快速检测的方法,本研究基于FCV衣壳蛋白(VP1)基因序列保守区设计RAA引物、4对针对VP1靶标的扩增引物、4对VP1-cr RNA扩增引物和1条报告探针。利用4对VP1-crRNA扩增引物经退火和T7 RNA聚合酶转录合成4条靶标crRNA:VP1-1/2/3/4-crRNA,利用4对VP1靶标扩增引物经退火合成4条靶标双链DNA:VP1-1/2/3/4,经EnGen Lba Cas12a (Cpf1)核酸酶试剂的切割反应和Cas12a试纸条检测,根据出现条带的强弱筛选最佳VP1-crRNA。结果显示,VP1-2-crRNA对应的T线条带最强且C线不完全切割的条带最弱。因此选择VP1-2-crRNA进行后续试验。分别以FCV VR-782株及6株分离的FCV RNA为模板,利用RAA引物扩增其VP1基因,并通过琼脂糖凝胶电泳检测并评估RAA引物的扩增效果;利用Cas12a (Cpf1)核酸酶,以上述RAA扩增的VP1基因(来自FCV VR-782株RNA)为模板,在37℃恒温水浴30 min进行CRISPR/Cas12a-LFS检测,评估RAA产物、Cas12a、VP1-2-crRNA和ss DNA报告基团对CRISPR/Cas12a-LFS检测体系的重要性。RAA扩增效果的评估结果显示,从FCV VR-782株及6株分离的FCV RNA中均扩增到282 bp的VP1基因目的条带。重要性评估结果显示,只有上述4种试剂全部存在时CRISPR/Cas12a-LFS检测体系才能有效扩增。利用该方法检测FCV、猫疱疹病毒I型、支气管败血波氏菌、猫细小病毒、沙门氏菌、金黄色葡萄球菌、F81细胞,结果显示,该方法仅能够特异性检测FCV,而其他病原的检测结果 均为阴性,且分别在42℃30 min内和37℃40 min内完成对FCV的RAA扩增及CRISPR/Cas12a-LFS检测;利用该方法检测10倍倍比稀释的重组质粒标准品p MD18T-FCV-VP1以评估其敏感性,结果显示,该方法的检测限为37.5拷贝/μL,与本研究室建立的Taq Man荧光定量PCR方法的敏感性相当;分别利用同一批次和3批次配置的RAA-CRISPR/CTo establish a method for rapid detection of feline calicivirus(FCV) based on lateral flow chromatography strip(LFS),recombinant enzyme-mediated isothermal nucleic acid amplification(RAA) combined with CRISPR-Cas12a system(RAA-CRISPR/Cas12a-LFS),this study designed RAA primers,four amplification primers targeting VP1,four VP1-crRNA amplification primers,and one reporter probe based on the conserved region of the FCV capsid protein(VP1) gene.Four pairs of VP1-crRNA amplification primers were annealed and transcribed by T7 RNA polymerase to synthesize four target crRNAs:VP1-1-crRNA-VP1-4-crRNA;four pairs of VP1 target amplification primers were annealed to synthesize four target doublestranded DNA:VP1-1-VP1-4,after the cleavage reaction of EnGen Lba Cas12a(Cpf1) nuclease reagent and the detection of Cas12/13 strip,the best VP1-crRNA was screened according to the intensity of the bands.The VP1 gene of FCV RNA isolated from FCV VR-782 strains and the other six strains was amplified by RAA primer,and the amplification effect of RAA primer was detected and evaluated by agar-gel electrophoresis.Using Cas12a(Cpf1) nuclease,the VP1 gene(from FCV VR-782 strain RNA)amplified by RAA was used as the template,and the product was subjected to a constant temperature water bath at 37℃ for 30min for CRISPR/Cas12A-LFS detection.To evaluate the importance of RAA products,Cas12a,VP1-2-crRNA,and ss DNA reporter groups for CRISPR/Cas12A-LFS assay systems.The screening results of VP1-crRNA showed that the T-line band corresponding to VP1-2-crRNA was the strongest and the band with incomplete C-line cutting was the weakest.The results of RAA amplification showed that the VP1 gene target band of 282bp could be amplified from FCV VR-782 strains and FCV RNA isolated from six strains.The importance evaluation results showed that the CRISPR/Cas12a-LFS assay system could be amplified effectively when the four above reagents were present.FCV,feline herpesvirus type I,bordetella bronchosepticus,feline parvovirus,Salmonella,Staphylococcus aureu

关 键 词：猫杯状病毒 CRISPR-Cas12a系统 侧向流层析试纸条 现场检测 

分 类 号：S852.65[农业科学—基础兽医学]