Development of a recombinase polymerase amplification isothermal assay for rapid visual and lateral flow detection of...

Porcine parvoviruses (PPVs) are globally recognized as significant contributors to reproductive failure in swine, primarily due to their association with fetal death. Infection in pregnant sows can lead to severe reproductive disorders including stillbirth, mummification, embryonic death and infertility. A recombinase polymerase amplification assay targeting the variable region of the outer capsid protein gene of the PPV-7 genome was developed and systematically optimized under a range of reaction conditions. The assay showed optimal amplification at a constant temperature of 35.00 ˚C for 25 min, using 0.72 µM of each forward and reverse primer and 14.00 mM magnesium acetate. It demonstrated high sensitivity, reliably detecting as few as 2,050 copies of viral nucleic acids in both the conventional and fluorescent dye-based formats. The assay also showed high specificity, exhibiting no cross-reactivity with other common porcine pathogens such as porcine sapelovirus, porcine circovirus and classical swine fever virus. Of the 167 field samples tested, 23 were positive for PPV-7, corresponding to a positivity rate of 13.77%. Operating at a low and constant temperature, the assay eliminates the need for advanced laboratory equipment, making it highly suitable for pen-side application in field settings. In conclusion, this novel assay demonstrated strong potential for field-based detection of PPV-7 circulating within the swine population of Haryana, India, marking the first report of its kind from this region. Further validation using samples from clinically affected herds will strengthen its diagnostic applicability.