# Establishment of a droplet digital PCR detection method for Vp4 gene of PoRV

## TL;DR

A new droplet digital PCR method for detecting PoRV in piglets was developed, offering higher sensitivity than existing tests.

## Contribution

A probe-based DDPCR method targeting the Vp4 gene of PoRV was established with significantly improved detection sensitivity.

## Key findings

- The DDPCR assay achieved a detection limit of 0.21 copies/μL, 100 times more sensitive than RT-qPCR.
- Four RT-qPCR-negative samples tested positive using DDPCR, confirming its higher sensitivity.
- Optimal DDPCR performance was achieved at 400:400 nM primer:probe concentration and 57 °C annealing temperature.

## Abstract

Diarrhea outbreaks in pigs occur most frequently during winter, porcine rotavirus (PoRV) is one of the important diarrheal diseases. Droplet digital polymerase chain reaction (DDPCR), a detection method that can perform absolute quantification of genes. The study aimed to diagnose PoRV infection using a probe-based DDPCR. 10 2-day-old piglets with mild diarrhea were obtained from a commercial pig farm. No PoRV was detected in the anal swab using colloidal gold test strips. To verify the results of the colloidal gold test strips, RT-qPCR was performed, which identified PoRV in six piglets. Given the limited sensitivity of colloidal gold test strips and RT-qPCR, we developed a DDPCR assay targeting the PoRV Vp4 gene for enhanced detection. The DDPCR assay demonstrated optimal performance at a primer:probe concentration of 400:400 nM and an annealing temperature of 57 °C. It achieved a minimum detection limit of 0.21 copies/μL, the detection sensitivity has been enhanced by 100 times compared to RT-qPCR. Using the established DDPCR detection method, the four samples that tested negative by RT-qPCR were re-tested, and all were found to be PoRV-positive, indicating that the sensitivity of DDPCR was higher than that of RT-qPCR. This study highlights its potential as a valuable tool for early clinical diagnosis and disease control in piglets.