State Key Laboratory for Animal Disease Control and Prevention, Key Laboratory of Veterinary Parasitology of Gansu Province, National Para-reference Laboratory for Animal Echinococcosis, Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences (CAAS), Lanzhou, China.
Jiangsu Co-Innovation Center for the Prevention and Control of Important Animal Infectious Disease and Zoonoses, Yangzhou University, Yangzhou, China.
FASEB J. 2024 May 31;38(10):e23708. doi: 10.1096/fj.202302449R.
Metacestodiasis is an infectious disease caused by the larval stage of cestode parasites. This disease poses a serious health hazard to wildlife, livestock, and humans, and it incurs substantial economic losses by impacting the safety of the livestock industry, the quality of meat production, and public health security. Unfortunately, there is currently no available molecular diagnostic method capable of distinguishing cysticercus- and Echinococcus-derived microRNAs (miRNAs) from other helminthes and hosts in the plasma of metacestode-infected animals. This study aims to develop a specific, sensitive, and cost-efficient molecular diagnostic method for cysticercosis and echinococcosis, particularly for early detection. The study developed a rolling circular amplification (RCA)-assisted CRISPR/Cas9 detection method based on parasite-derived miRNA let-7-5p. Using a series of dilutions of the let-7 standard, the limit of detection (LOD) of the qPCR, RCA, and RCA-assisted CRISPR/Cas9 methods was compared. The specificity of qPCR and CRISPR/Cas9 was evaluated using four artificially synthesized let-7 standards from different species. A total of 151 plasma samples were used to evaluate the diagnostic performance. Additionally, the study also assessed the correlation between plasma levels of let-7-5p, the number of Taenia pisiformis cysticerci, and the weight of Echinococcus multilocularis cysts. The results demonstrated that the RCA-assisted CRISPR/Cas9 assay could significantly distinguish let-7 from cestodes and other species, achieving a LOD of 10 aM; the diagnostic sensitivity and specificity for rabbit cysticercosis and mouse E. multilocularis were 100% and 97.67%, and 100% and 100%, respectively. Notably, let-7-5p gradually increased in the plasma of T. pisiformis-infected rabbits from 15 days post infection (dpi), peaked at 60 dpi, and persisted until 120 dpi. In E. multilocularis-infected mice, let-7-5p gradually increased from 15 dpi and persisted until 90 dpi. Furthermore, the expression of let-7-5p positively correlated with the number of cysticerci and cyst weight. These results indicated that the let-7-5p-based RCA-assisted CRISPR/Cas9 assay is a sensitive and specific detection method that can be used as a universal diagnostic method for metacestodiasis, particularly for early diagnosis (15 dpi).
包虫蚴病是一种由绦虫幼虫引起的传染病。这种疾病对野生动物、家畜和人类的健康构成严重威胁，通过影响家畜业的安全、肉类生产的质量和公共卫生安全，造成了巨大的经济损失。不幸的是，目前尚无可用的分子诊断方法能够区分囊尾蚴和细粒棘球蚴衍生的 microRNAs（miRNAs）与其他寄生虫和动物宿主的血浆中的微 RNA。本研究旨在开发一种用于囊虫病和包虫病的特异性、敏感性和具有成本效益的分子诊断方法，特别是用于早期检测。本研究基于寄生虫衍生的 miRNA let-7-5p 开发了一种滚环扩增（RCA）辅助的 CRISPR/Cas9 检测方法。使用一系列稀释的 let-7 标准品，比较 qPCR、RCA 和 RCA 辅助的 CRISPR/Cas9 方法的检测限（LOD）。使用四种来自不同物种的人工合成的 let-7 标准品评估 qPCR 和 CRISPR/Cas9 的特异性。总共使用 151 个血浆样本评估诊断性能。此外，还评估了血浆中 let-7-5p 水平与带绦虫囊尾蚴数量和泡状棘球蚴囊肿重量之间的相关性。结果表明，RCA 辅助的 CRISPR/Cas9 检测方法能够显著区分 let-7 与绦虫和其他物种，达到 10 aM 的 LOD；兔囊尾蚴病和小鼠泡状棘球蚴病的诊断敏感性和特异性分别为 100%和 97.67%，100%和 100%。值得注意的是，从感染后 15 天（dpi）开始，T. pisiformis 感染兔的血浆中 let-7-5p 逐渐增加，在 60 dpi 时达到峰值，并持续至 120 dpi。在 E. multilocularis 感染的小鼠中，let-7-5p 从 15 dpi 开始逐渐增加，并持续至 90 dpi。此外，let-7-5p 的表达与囊尾蚴数量和囊肿重量呈正相关。这些结果表明，基于 let-7-5p 的 RCA 辅助的 CRISPR/Cas9 检测方法是一种敏感且特异性的检测方法，可作为包虫蚴病的通用诊断方法，特别是用于早期诊断（15 dpi）。
