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

Jun Ji,Chaoliang Leng,Lunguang Yao,Zhili Li,Kejing Zuo,Xin Xu,Xueyu Wang,Yunchao Kan,Yingzuo Bi

doi:10.1186/s12917-019-2072-9

Abstract

BackgroundPorcine 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.ResultsThis 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 × 102 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).ConclusionsThese 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.

Highlights

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
Porcine circovirus type 3 (PCV3) is a newly emerging virus associated with porcine dermatitis and nephropathy syndrome, reproductive failure, and multiorgan inflammation, even though its causative role in any of these syndromes has yet to be demonstrated [1,2,3]
Optimal reaction temperature and time for the PCV3 polymerase spiral reaction (PSR) assay In the agarose gel electrophoresis analyses, a little difference displayed between the reactions from 60 °C to 65 °C; the PSR products at 62 °C displayed the brightest bands

Summary

Introduction

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. The virus was first reported in pig farms in the USA in 2016 through metagenomic sequencing and was identified to be a new member of the genus Circovirus of the family Circoviridae [4] This genus includes porcine circovirus type 1, which has no clinical manifestations, and porcine circovirus type 2 (PCV2), which has proved to be a significant economic threat for the pig Conventional serological methods have been used to identify PCV3. Molecular methods based on polymerase chain reaction (PCR) assays, both routine and quantitative, have been developed to monitor and detect PCV3 rapidly and [16,17,18] These methods require sophisticated thermal cyclers, thereby limiting their usefulness for the practical point-of-care testing of clinical samples.

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Conclusion