Su Shuxiao, Zhao Shuaiyang, Bai Ling, Obaid Muhammad Kashif, Zhu Haohan, Luo Jin, Liu Junlong, Luo Jianxun, Yin Hong, Guan Guiquan
State Key Laboratory for Animal Disease Control and Prevention, Key Laboratory of Veterinary Parasitology of Gansu Province Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Xujiaping 1, Lanzhou, Gansu 730046, PR China.
State Key Laboratory for Animal Disease Control and Prevention, Key Laboratory of Veterinary Parasitology of Gansu Province Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Xujiaping 1, Lanzhou, Gansu 730046, PR China.
Vet Parasitol. 2025 Jul;337:110508. doi: 10.1016/j.vetpar.2025.110508. Epub 2025 May 22.
The protozoan pathogen, Theileria annulata, is transmitted by ticks which is very detrimental to various breeds of cattle, leading to the development of theileriosis. Early diagnosis and control of the disease is a crucial aspect of preventing and treating any infection. In order to detect T. annulata on-site, we selected sporozoite surface antigen gene (SPAG) as the target gene to design Recombinase Polymerase Amplification (RPA) primers and crRNA to build a one-pot SHERLOCK method. The results showed the assay could specifically detect T. annulata infection, but have no-cross reaction with the genomic DNA of T. annulata, Theileria sinensis, Theileria orientalis, Babesia bigemina and Babesia bovis. For its sensitivity, the assay could detect 1 × 10 copies/μL at least. In comparison with the published PCR assay, the relative specificity and sensitivity of the one-pot SHERLOCK assay was 100 % and 100 % respectively, and the coincidence rate was 100 %. Meanwhile, to improve the practicality, we used the lysis buffer to handle the blood of the infected animal to detect the T. annulata. The findings presented that T. annulata can be detected by using the one-pot SHERLOCK assay, which had preliminarily solved the problem of genome extraction in SHERLOCK field applications. Although this assay has a decrease in sensitivity but it reduced the number of operational steps and the probability of aerosol contamination. This assay has the potential to be developed into a commercial detection method and provide good support for the prevention and control of theileriosis.
原生动物病原体环形泰勒虫通过蜱传播，对各种牛品种危害极大，会引发泰勒虫病。该疾病的早期诊断和控制是预防和治疗任何感染的关键环节。为了现场检测环形泰勒虫，我们选择子孢子表面抗原基因（SPAG）作为靶基因，设计重组酶聚合酶扩增（RPA）引物和crRNA，构建了一锅式SHERLOCK方法。结果表明，该检测方法能够特异性地检测环形泰勒虫感染，与环形泰勒虫、中华泰勒虫、东方泰勒虫、双芽巴贝斯虫和牛巴贝斯虫的基因组DNA无交叉反应。在灵敏度方面，该检测方法至少能检测到1×10拷贝/μL。与已发表的PCR检测方法相比，一锅式SHERLOCK检测方法的相对特异性和灵敏度分别为100%和100%，符合率为100%。同时，为提高实用性，我们使用裂解缓冲液处理感染动物的血液来检测环形泰勒虫。研究结果表明，使用一锅式SHERLOCK检测方法可以检测到环形泰勒虫，初步解决了SHERLOCK现场应用中的基因组提取问题。虽然该检测方法灵敏度有所下降，但减少了操作步骤和气溶胶污染的可能性。该检测方法有潜力发展成为一种商业检测方法，为泰勒虫病的防控提供有力支持。
