Novel CRISPR/Cas13- based assay for diagnosis of avian infectious bronchitis

Abstract

Infectious bronchitis is an acute respiratory disease of poultry associated with reduced egg production and heavy economic losses in chicken flocks. Rabid and accurate detection of IB virus (IBV) is essential for controlling and preventing the infection. In this study, we developed a rapid, accurate, and instrument less assay to detect IBV. For the first time, reverse transcription-Recombinase polymerase amplification (RT-RPA) coupled with CRISPR/Cas13 (SHERLOCK) was used to rapidly visualize IBV. The novel assay was tested in timing, sensitivity, and specificity. The spike gene (S gene) was used as a target gene for detecting the virus. Three samples were used to optimize the assay; sample form confirmed infected chickens with IB, positive sample (full synthesis of S gene), and negative sample from free IB infected chickens. The results show that the Sherlock-based Cas13 platform is a highly specificity and sensitivity assay for detecting infectious bronchitis virus. The assay detected ten copies per µL of the input RNA. No false positives or cross-reactions were seen when bovine coronavirus (BCV) was used instead of IBV in the tested sample. Readout of the results needs just fifty minutes, including RNA extraction. Furthermore, No instrument was used, and amplification of the virus's nucleic acid was performed at room temperature. Sherlock-based Cas13 should clinically use for rapid diagnosis of infectious bronchitis in chickens. However, further studies and experiments are needed to perform the assay at the sample base without extraction of RNA.