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 °C 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.

Key words: Porcine parvovirus, Loop?mediated isothermal amplification, CRISPR/Cas12a, Nucleic acid extraction?free, Rapid visual detection

摘要： 为提升猪细小病毒（PPV）临床防控能力，选取PPV保守NS1 基因作为靶序列设计环介导等温扩增（LAMP）特异性引物及配套的单链向导RNA（sgRNA），建立融合LAMP与CRISPR/Cas12a酶切反应的可视化现场快速检测体系，并使用26份临床猪血清样本进行检测。结果表明，该体系无需核酸提取纯化，猪血清样本经5 min煮沸预处理便可直接作为扩增模板；经反应条件优化筛选得到识别效率最优的sgRNA2；确定LAMP扩增参数为64 ℃恒温孵育35 min，LbCas12a酶切适宜反应时长10~15 min，体系阳性荧光信号可在自然光、蓝光、紫外光下实现肉眼可视化判读。特异性验证结果显示，该方法仅对PPV产生阳性信号，最低检测限达1.183×10^⁻² copies/μL，检测灵敏度较qPCR与PCR-CRISPR/Cas12a均提升5个数量级。26份临床猪血清样本检测结果证实，LAMP-CRISPR/Cas12a与PCR-CRISPR/Cas12a两种体系检测结果一致，共检出12份阳性样本。综上，建立的免核酸提取LAMP-CRISPR/Cas12a检测体系兼具高灵敏度、高特异性、操作简便、适配现场检测、用时较短等特点，可为规模化猪场PPV大批量临床快速筛查提供简便可靠的技术支撑。

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