Development of reverse transcription recombinase polymerase amplification assay for rapid detection of porcine sapelovirus
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Date
2023-04
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LUVAS Hisar
Abstract
Porcine Sapelovirus (PSV) A, a member of newly classified genus Sapelovirus, belongs to
family Picornaviridae. PSV is non-enveloped virus with icosahedral symmetry with diameter of
around ~35nm and the genome is a linear, non-segmented, single-stranded positive-sense RNA. PSV
infection in pigs is mostly asymptomatic, but it can also cause severe neurologic, enteric, and
respiratory symptoms or reproductive failure. Sapelovirus infections have been reported worldwide in
pigs. PSV is considered as the emerging pathogen of porcine species. For the virus detection,
isothermal Recombinase polymerase amplification (RPA) were developed.
The RT-RPA assay was developed using commercially synthesized positive control and primers
based on 5´conserved region of PSV genome. The assay was optimized at low and constant
temperature of 35°C for 20 minutes with forward primer concentration 20pm, reverse primer
concentration 5pm and MgOAc concentration of 14mM. The analytical sensitivity of assay was
determined using serial dilutions of positive control. The limit of detection was up to 283 copies using
agarose gel electrophoresis. The assay was specific to PSV and no cross reactivity was observed with
other related viruses. The assay was validated using field samples followed by visual assay for
detection. A total 85 samples were collected from different organized and unorganized piggery farms
of Haryana. Out of 85 samples 21 samples were found positive for PSV. The positivity rate of PSV was
24.7%. This is the first report of PSV from Haryana suggesting the virus circulation among pig
population. Therefore, the above study concludes that the developed RT-RPA assay is rapid, highly
sensitive and specific and works at low, constant temperature and do not require any special
instrument. Visual dye-based detection makes the developed RPA assay a potential candidate for pointof-care testing (POCT)