# Sensitive, specific, and rapid on-site detection of calf diarrhea pathogens using the RPA-CRISPR/Cas 12a assay

## TL;DR

A new rapid and sensitive test using RPA-CRISPR/Cas12a can detect four calf diarrhea pathogens on-site in under 50 minutes.

## Contribution

A novel RPA-CRISPR/Cas12a assay for on-site detection of multiple calf diarrhea pathogens with high sensitivity and specificity.

## Key findings

- The RPA-CRISPR/Cas12a assay detects BVDV, BCoV, BRV, and ETEC at concentrations as low as 10 copies/μL.
- The assay is specific and does not produce false positives from other pathogens.
- The test is completed in under 50 minutes in a single test tube without intermediate steps.

## Abstract

Calf diarrhea is a common gastrointestinal disease that usually occurs within one month of birth. The disease causes the greatest economic losses to the cattle industry. Currently, a variety of diagnostic methods have been developed for calf diarrhea infections. However, existing methods are still unsatisfactory in terms of sensitivity, specificity, simplicity, cost, and speed.To provide a more sensitive, specific, simpler, and faster detection method, we recently developed an RPA-CRISPR/Cas12a assay that can detect BVDV, BCoV, BRV, and ETEC infections in cattle on-site. Testing for each pathogen is performed in a single test tube, without the need to open the tube in the middle, and can be completed in under 50 minutes.The RPA-CRISPR/Cas12a assay can detect BVDV, BCoV, BRV, and ETEC at concentrations of at least 10 copies/μL. The RPA-CRISPR/Cas12a assay does not produce false-positive results due to the presence of other pathogens. The sensitivity of BCoV, BRV, and ETEC in the RPA-CRISPR/Cas12a quadruple assay is equivalent to that of single qPCR. The sensitivity of BVDV in the quadruple assay is slightly lower than that of the single qPCR method.Due to its sensitivity, specificity, simplicity, and rapidity, the RPA-CRISPR/Cas12a assay is more practical for on-site detection of cattle diarrhea pathogens than any existing detection method.