畜牧兽医学报2026，Vol.57Issue(8)：4626-4635,10.DOI:10.11843/j.issn.0366-6964.2026.08.046
猫传染性腹膜炎病毒CRISPR-Cas13a检测方法的建立及应用
Establishment and Application of CRISPR-Cas13a Detection Method for Cat Infectious Peritonitis Virus
摘要
Abstract
Feline infectious peritonitis(FIP)caused by feline infectious peritonitis virus(FIPV)is highly lethal,posing a serious threat to the health of cat populations,especially kittens and young cats.To develop a FIPV diagnostic method with high sensitivity,low cost and easy operation,this study combined RT-RPA with the CRISPR detection system.Based on laboratory isolation,sequencing and NCBI database alignment,the N gene sequence of FIPV strains was selected and cloned into the pUC57 vector to construct a positive control plasmid;specific RPA primers and reaction temperature were screened,CRISPR reaction parameters were optimized,and the specificity and sensitivity of the method were evaluated;LwCas13a protein was induced,expressed and purified by the Escherichia coli(E.coli)prokaryotic expression system.The active LwCas13a protein was successfully obtained;the F1R1 primer pair showed the optimal amplification efficiency,and the established FIPV-Cas13a detection method could specifically recognize FIPV nucleic acid without cross-reaction;the optimal reaction conditions were 37℃,crRNA dosage of 7.5 ng,LwCas13a protein dosage of 250 ng,the fluorescence was stable after 40 min of reaction,the limit of detection reached 101 copies·μL-1,and visual detection could be achieved by combining with test strips.The detection of 38 clinical samples of anal swabs showed that the positive detection rates of both the fluorescence method and the test strip method were 50%(19/38),which was highly consistent with the industry standard RT-PCR at 42%(16/38).In conclusion,the FIPV-Cas13a detection system provides a robust and visual alternative detection method,which is suitable for on-site FIPV monitoring.关键词
CRISPR/Cas系统/Cas13a蛋白/猫传染性腹膜炎(FIP)/可视化检测Key words
CRISPR/Cas system/LwCas13a protein/feline infectious peritonitis/visual inspection分类
农业科技引用本文复制引用
曾永萍,廖珊,刘骁..猫传染性腹膜炎病毒CRISPR-Cas13a检测方法的建立及应用[J].畜牧兽医学报,2026,57(8):4626-4635,10.基金项目
重庆基础研究与前沿技术项目(cstc2020jcyj-msxm1458) （cstc2020jcyj-msxm1458）
四川省科技计划项目(2022yfn0069 （）
2022yfq0023 （）
2021zdzx0010) （）
四川省"十四五"规划项目(2021zdzx0010) （2021zdzx0010）
国家蚕基因组生物学重点实验室开放课题(sklsgb-orp202104) （sklsgb-orp202104）
重庆市畜牧科学院市级财政科研项目资助(26508C) （26508C）
