Cao Yutong, Yang Qiankun, Guo Yanbing, Wang Xiaoceng, Li Xin, Zhang Nan, Lu Wenxue, Li Jianhua, Zhang Xichen, Cao Lili, Gong Pengtao
State Key Laboratory for Diagnosis and Treatment of Severe Zoonotic Infectious Diseases, Key Laboratory for Zoonosis Research of the Ministry of Education, Institute of Zoonosis, and College of Veterinary Medicine, Jilin University, Changchun 130062, China.
Jilin Academy of Animal Husbandry and Veterinary Medicine, Changchun 130062, China.
Vet Parasitol. 2025 Feb;334:110421. doi: 10.1016/j.vetpar.2025.110421. Epub 2025 Feb 13.
Haemonchus contortus (H. contortus), a highly pathogenic and blood-feeding nematode, could cause haemonchosis，resulting in tens of billions of dollars in production losses and significantly impacting the development of sheep husbandry. Rapid and accurate detection methods were particularly important for the prevention and control of haemonchosis. In this study, we developed a one-pot effective detection method that integrating recombinase polymerase amplification (RPA) with CRISPR/Cas12a technology based on the conserved region of ITS2 of H. contortus, with readout through fluorescence signals visualized by lateral flow strips (LFS) and observable under UV or blue light. The detection procedure was successfully finished in within 1 h and demonstrated high specificity and sensitivity, with no cross-reactivity detected with nine other common ovine pathogens and a detection limit as low as 0.1 copies/μL for fluorescence and 100 copies/μL for LFS. Validation with 89 sheep fecal samples revealed a 46.07 % positivity rate, fully consistent with quantitative PCR results. In summary, the RPA-CRISPR/Cas12a method for H. contortus detection exhibited the advantages of high specificity, high sensitivity, and low device dependence, portable and visible results. The technique presented significant potential for large-scale clinical application and provided novel point-of-care testing for clinical use in remote rural and resource-constrained areas.
捻转血矛线虫（H. contortus）是一种高致病性吸血线虫，可引发捻转血矛线虫病，导致数百亿美元的生产损失，并对养羊业的发展产生重大影响。快速准确的检测方法对于捻转血矛线虫病的预防和控制尤为重要。在本研究中，我们基于捻转血矛线虫ITS2保守区域，开发了一种将重组酶聚合酶扩增（RPA）与CRISPR/Cas12a技术相结合的一锅法高效检测方法，通过侧向流动试纸条（LFS）可视化荧光信号进行读数，并可在紫外线或蓝光下观察。检测过程在1小时内成功完成，具有高特异性和高灵敏度，与其他九种常见羊病原体无交叉反应，荧光检测限低至0.1拷贝/μL，LFS检测限低至100拷贝/μL。对89份绵羊粪便样本进行验证，阳性率为46.07%，与定量PCR结果完全一致。综上所述，用于检测捻转血矛线虫的RPA-CRISPR/Cas12a方法具有特异性高、灵敏度高、设备依赖性低、结果便携且可见的优点。该技术在大规模临床应用中具有巨大潜力，为偏远农村和资源有限地区的临床即时检测提供了新方法。
