河南农业科学2026，Vol.55Issue(9)：148-161,14.DOI:10.15933/j.cnki.1004-3268.2026.09.014
免核酸提取猪细小病毒LAMP-CRISPR/Cas12a可视化检测方法的建立
Establishment of Visual Detection Method for Porcine Parvovirus Using LAMP-CRISPR/Cas12a without Nucleic Acid Extraction
摘要
Abstract
To improve the clinical prevention and control ability of porcine parvovirus(PPV),the conserved NS1 gene of PPV was selected as the target sequence to design specific primers for loop-mediated isothermal amplification(LAMP)and the corresponding single guide RNA(sgRNA).A visual on-site rapid detection system integrating LAMP and CRISPR/Cas12a cleavage reaction was established,and 26 clinical porcine serum samples were used for detection.The results showed that the system did not require nucleic acid extraction and purification,and the pig serum sample could be directly used as an amplification template after boiling pretreatment for 5 min.The sgRNA2 with the best recognition efficiency was obtained by optimizing the reaction conditions.The LAMP amplification parameters were determined to be incubation at 64 ℃ for 35 min,and the appropriate reaction time for LbCas12a digestion was 10-15 min.The positive fluorescence signal of the system can be visually interpreted by the naked eye under natural light,blue light and ultraviolet light.The specificity verification results showed that the method only produced positive signals for PPV,and the minimum detection limit was 1.183×10-2 copies/μL.The detection sensitivity was all 5 orders of magnitude higher than those of qPCR and PCR-CRISPR/Cas12a.The test results of 26 clinical pig serum samples confirmed that the results of LAMP-CRISPR/Cas12a and PCR-CRISPR/Cas12a were consistent,and a total of 12 positive samples were detected.In summary,the established nucleic acid extraction-free LAMP-CRISPR/Cas12a detection system has the characteristics of high sensitivity,high specificity,simple operation,suitability for on-site detection,and short reaction time,which can provide simple and reliable technical support for rapid clinical screening of PPV in large-scale pig farms.关键词
猪细小病毒/环介导等温扩增/CRISPR/Cas12a/免核酸提取/快速可视化检测Key words
Porcine parvovirus/Loop-mediated isothermal amplification/CRISPR/Cas12a/Nucleic acid extraction-free/Rapid visual detection分类
农业科技引用本文复制引用
李聪聪,张乐乐,徐秋良..免核酸提取猪细小病毒LAMP-CRISPR/Cas12a可视化检测方法的建立[J].河南农业科学,2026,55(9):148-161,14.基金项目
国家重点研发计划项目(2021YFD1301200) （2021YFD1301200）
河南省揭榜挂帅项目(261000110600) （261000110600）
河南省高校青年骨干教师计划项目(2024GGJS152) （2024GGJS152）
河南省国际科技合作重点项目(251111520200) （251111520200）
河南省科技攻关项目(222102110232,252102110070) （222102110232,252102110070）
