Yang Qiankun, Liu Juan, Yu Yanhui, Cao Yutong, Liu Chengwu, Su Haitao, Huang Taojun, Liu Shaoxiong, Yuan Jianqi, Zhao Zhiteng, Li Lu, 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, College of Veterinary Medicine, Jilin University, Changchun 130062, China.
Pingdu People's Hospital, Qingdao 266700, China.
Int J Biol Macromol. 2024 Dec;282(Pt 6):136912. doi: 10.1016/j.ijbiomac.2024.136912. Epub 2024 Nov 7.
Fasciola hepatica is a foodborne zoonotic parasite causing significant economic losses and impacting human and livestock health in resource-limited regions. We developed a rapid, reliable, and sensitive detection method combining recombinase polymerase amplification (RPA) with CRISPR/Cas12a, allowing visualization with the naked eye or a fluorescence reader. Multiple visual methods were used to analyze the assay results. Fluorescence signals were collected using a fluorescence reader or observed under UV or blue light. Lateral flow strips (LFS) were used for visual detection. Among seven primer pairs and three CRISPR RNA (crRNA) screened, F1/R1 and crRNA3 were optimal. The Cas12a reaction buffer was optimized with 50 mM Tris-HCl and 80 mM NaCl, with an RPA reaction time of 20 min. The assay showed high specificity and excellent sensitivity for F. hepatica, detecting 0.122 copies/μL with fluorescence and 8.6 copies/μL with LFS. Testing of 143 sheep and 43 human fecal samples showed 98.39 % consistency with qPCR and nested PCR, with prevalence rates of 52.45 % and 18.6 % in sheep and humans, respectively. Our assay offers substantial potential for point-of-care testing in resource-limited areas, addressing the need for rapid and accurate diagnosis of F. hepatica.
肝片吸虫是一种食源性人畜共患寄生虫，在资源有限的地区造成了重大经济损失，并影响人类和牲畜健康。我们开发了一种将重组酶聚合酶扩增（RPA）与CRISPR/Cas12a相结合的快速、可靠且灵敏的检测方法，可通过肉眼或荧光读数仪进行可视化检测。使用了多种可视化方法来分析检测结果。使用荧光读数仪收集荧光信号，或在紫外线或蓝光下观察。使用侧向流动试纸条（LFS）进行可视化检测。在筛选的7对引物和3种CRISPR RNA（crRNA）中，F1/R1和crRNA3是最佳的。使用50 mM Tris-HCl和80 mM NaCl优化了Cas12a反应缓冲液，RPA反应时间为20分钟。该检测方法对肝片吸虫具有高特异性和出色的灵敏度，荧光检测的灵敏度为0.122拷贝/μL，LFS检测的灵敏度为8.6拷贝/μL。对143份绵羊粪便样本和43份人类粪便样本的检测显示，与定量PCR和巢式PCR的一致性为98.39%，绵羊和人类的患病率分别为52.45%和18.6%。我们的检测方法在资源有限的地区进行即时检测具有巨大潜力，满足了对肝片吸虫进行快速准确诊断的需求。
