Novel polymerase spiral reaction assay for the visible molecular detection of porcine circovirus type 3

Abstract

Background Porcine circovirus type 3 (PCV3) is a newly emerging circovirus that might be associated with porcine dermatitis and nephropathy syndrome, reproductive failure, and cardiac and multisystemic inflammation. To aid the prevention and control of the infectious disease caused by PCV3, we developed a novel isothermal amplification assay using polymerase spiral reaction (PSR), which allows the visual detection of preserved strains and clinical samples. Results This assay precisely amplified the PCV3 genome with the use of a water bath at 62 °C for 50 min. The detection limit was found to be 1.13 × 10 2 copies/μL by gel electrophoresis or with the use of a visible dye (an indicator comprising phenol red and cresol red). No cross-reaction with other porcine infectious viruses was observed. The detection results for 23 PCV3-positive samples by PSR were in accordance with loop-mediated isothermal amplification (LAMP) assay. The detection rate of the PSR assay for PCV3 positivity of clinical samples was 68/97, which was higher than LAMP assay (67/97). Conclusions These results indicated that the PSR assay provides an accurate and rapid method for the detection of PCV3 with high sensitivity and specificity. It is particularly suited for use in a simple laboratory setting without a thermal cycler or gel electrophoresis equipment.

What do we know about porcine circovirus 3 (PCV3) diagnosis so far?: A review

“…Further, polymerase spiral reaction (PSR), also an isothermal amplification assay based on PCV3 Cap gene, has been described. The PSR assay, run at 62 • C for 50 min, detected PCV3 at LOD 1.13 × 102 copies/μl with phenol red and cresol red (Ji et al, 2019).…”

First detection of porcine circovirus type 3 in Ukraine

“…The serological tests, developed on the basis of indirect enzyme immunoassay, allowed conducting the retrospective study on PCV-3 in the pig population in China which demonstrated an increase in the number of PCV-3-positive cases from 22.35 to 51.88 % in three years (Deng J et al, 2018), and the examination of the population of sows at the farms of China, the results of which demonstrated the spreading with the seroprevalence more than 50 % (of PCV-3 among both clinically healthy sows and the animals with reproductive failures (Zhang S et al, 2019). Some studies showed, that (Q) PCR is more effective for PCV-3 diagnostics in compare to serological methods or in situ hybridization (Kedkovid R et al 2018 At present, PCV-3 detection is mostly based on the use of molecular genetic methods using different PCR formats (Rodrigues ILF et al, 2018;Fux R et al, 2018;Liu Y et al, 2019;Dal Santo AC et al, 2020) and amplifi cation technologies (Ji J et al, 2019;Ye X et al, 2018;Zheng S et al, 2018).…”

Single Multiple Cross Displacement Amplification for Rapid and Real-Time Detection of Porcine Circovirus 3

“…Moreover, the reaction system can be carried out at a constant temperature of 60 • C and completed within 30 min, and the results can be visually judged by the real-time detection system or using a smartphone-based cassette (19). The test can save 20 min compared with novel polymerase spiral reaction (20), 40 min compared with real-time loop-mediated isothermal amplification (15), and at least 60 min compared with qPCR or PCR (13,14).…”

“…The analysis showed that this method had good specificity, with no cross false reaction with other porcine viral pathogens. Most isothermal amplification techniques use color change (4,20) or turbidity (17) to interpret the results, but P-S-MCDA monitors the amplification of the target gene in real time quantitatively by the real-time PCR detection system. Therefore, the P-S-MCDA not only avoids the error of visual interpretation of the results but also is capable of quantitative analysis of target gene sequences.…”