Swift detection of Theileria annulata: A one-pot SHERLOCK Assay approach.

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 × 103 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.