Swift Detection of Theileria Annulata: A One-Pot Sherlock Assay Approach

Abstract

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 Theileria 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. We established this method having good specificity and no-cross reactivity, moreover it’s sensitivity was 1 x 103 copies/μL. Otherwise, in order to improve the practicality, we used the lysis buffer to handle the blood of the infected animal to detect the T. annulata. The findings of this study presented that T. annulata can be detected from the positive blood via one-pot SHERLOCK assay. This study has preliminarily solved the problem of genome extraction in SHERLOCK field applications. Although this method has a decrease in sensitivity but it reduced the number of operational steps and the probability of aerosol contamination. This method has the potential to be developed into a commercial detection method.

Keywords: Theileria annulata, one-pot, SHERLOCK, on-site, detection method