中国兽医科学2024，Vol.54Issue(6)：728-734,7.DOI:10.16656/j.issn.1673-4696.2024.0116
猪圆环病毒2型LAMP-CRISPR/Cas12a检测方法的建立与应用
Establishment and application of a method for the detection of porcine circovirus type 2 LAMP-CRISPR/Cas12a
摘要
Abstract
This study aimed to establish a rapid,accurate,and user-friendly detection method for porcine circovirus 2(PCV2).Initially,targeting the ORF2 gene of PCV2,four LAMP and five CRISPR-specific primers were designed and the optimal concentrations of components in the LAMP reaction system and CRISPR detection system were optimized.Following validation of specificity,sensitivity,repeatabili-ty,and compliance,the rapid detection method for PCV2,LAMP-CRISPR/Cas12a,was successfully devel-oped.The results demonstrated that the method's minimum detection threshold was close to 1 copy of plasmid DNA,showing significantly higher sensitivity compared to the PCR method commonly used in our laboratory.Under the same conditions,only PCV2 yielded positive results,while all others tested nega-tive,indicating excellent specificity.The method exhibited good repeatability,with an intra-batch coefficient of variation below 5％and an inter-batch coefficient of variation below 10％.It showed high agreement with qPCR and allowed for visual observation of reaction products under blue light.These findings suggest that the established LAMP-CRISPR/Cas12a system is suitable for the rapid detec-tion of PCV2.关键词
猪圆环病毒2型/CRISPR/Cas12a检测/环介导等温扩增Key words
porcine circovirus type 2/CRISPR/Cas12a detection/loop-mediated isothermal amplification分类
农业科技引用本文复制引用
毛首会,杜国玉,刘晓波,吴锦艳,兰喜,何继军,尚佑军,王桂琴,刘永杰..猪圆环病毒2型LAMP-CRISPR/Cas12a检测方法的建立与应用[J].中国兽医科学,2024,54(6):728-734,7.基金项目
国家生猪技术创新中心先导科技项目(NCTIP-XD/C03) （NCTIP-XD/C03）
