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

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.