# Isothermal nucleic acid amplification assays for the detection of porcine stool-associated RNA virus

## TL;DR

This paper introduces two fast and sensitive methods for detecting a new virus in pig stool, which could help monitor and manage swine health.

## Contribution

The study develops and optimizes RPA and PSR assays for rapid and specific detection of posavirus in pig stool samples.

## Key findings

- RPA assay detected posavirus with a limit of 5.34 × 10⁶ copies.
- PSR assay showed higher sensitivity at 6.5 × 10³ copies.
- Only three out of 132 field samples tested positive for posavirus.

## Abstract

Pigs are a vital component of agricultural economies and a major source of livestock worldwide. The Porcine Stool-Associated virus (Posavirus), a newly identified member of the Picornavirales order, has been associated with enteric infections in swine. Recombinase Polymerase Amplification (RPA) and Polymerase Spiral Reaction (PSR), two isothermal amplification methods, were developed and optimized in this study to identify the posavirus in pig stool samples quickly and effectively. Primers that target the posavirus’s polyprotein region were designed for both RPA and PSR assays, and reaction parameters were optimized. Sensitivity assessments revealed that the RPA assay had a detection limit of 5.34 × 106copies, while the PSR assay has higher sensitivity at 6.5 × 103copies. Both assays showed high specificity for the posavirus, with no cross-reactivity. An evaluation of 132 field samples revealed that only three samples were positive for posavirus, highlighting the need for continued surveillance. This study reported the successfully development and optimisation of RPA and PSR assays as dependable and easily accessible diagnostic methods for posavirus detection. Their speed, sensitivity, and specificity make them adapted for use in a range of field and laboratory scenarios.

The online version contains supplementary material available at 10.1038/s41598-025-22146-4.