Development and Clinical Validation of a Potential Penside Colorimetric Loop-Mediated Isothermal Amplification Assay of Porcine Circovirus Type 3.

Jie Zhang,Qian Hou,Yaozhong Ding,Xiaoyun Li,Danian Chen,Katarzyna Podgorska,Yunwen Ou,Yanjun Li,Alexei D Zaberezhny,Weibing Zhang,Yang Wang,Miaomiao Li,Luoyi Zhou,Anna Szczotka-Bochniarz,Yongsheng Liu

doi:10.3389/fmicb.2021.758064

Abstract

Porcine circovirus type 3 (PCV3), a novel circovirus, imposes great burdens on the global pig industry. The penside tests for detecting PCV3 are critical for assessing the epidemiological status and working out disease prevention and control programs due to the unavailability of a commercial vaccine. A one-step molecular assay based on visual loop-mediated isothermal amplification (vLAMP) was developed for simple and rapid detection of PCV3. We compared its sensitivity and specificity with TaqMan quantitative real-time polymerase chain reaction (qPCR) and applied the developed assay in the epidemiological study of (n = 407) pooled swine sera collected from almost the entire mainland China during the years 2017–2018. We also explored the feasibility of the vLAMP assay for detecting raw samples without a prior DNA isolation step to expand its application capability. Results showed that the vLAMP assay could reliably detect the PCV3 cap gene with a detection limit of 10 DNA copies equal to that of the Taqman qPCR assay. In the epidemiological study, the PCV3 positive detection rate for 407 swine pooled sera detected by the vLAMP assay was 37.35% (152/407), whereas it was 39.01% (159/407) for Taqman qPCR. For the detection method without genome extraction, the results kept satisfactory specificity (100%) but displayed lower sensitivity (100% for CT < 32), indicating the direct detection is not sensitive enough to discriminate the samples with low viral loads. The one-step vLAMP is a convenient, rapid, and cost-effective diagnostic for penside detection and will enable the epidemiological surveillance of PCV3, which has widely spread in mainland China.

Highlights

In 2016, a novel porcine circovirus [named porcine circovirus type 3 (PCV3)] distantly related to the known circoviruses was discovered to be associated with porcine dermatitis and nephropathy syndrome, reproductive failure (Palinski et al, 2017), and cardiac and multisystemic inflammation (Phan et al, 2016) in the United States
The phylogenetic analysis showed that the nucleotide homology of Porcine circovirus type 3 (PCV3) strain was high (>97%), which indicated the visual loop-mediated isothermal amplification (vLAMP) primers designed here could theoretically target the vast majority of PCV3 in the world
All results showed that the PCV3 vLAMP primers could effectively amplify the target gene

Summary

Introduction

In 2016, a novel porcine circovirus [named porcine circovirus type 3 (PCV3)] distantly related to the known circoviruses was discovered to be associated with porcine dermatitis and nephropathy syndrome, reproductive failure (Palinski et al, 2017), and cardiac and multisystemic inflammation (Phan et al, 2016) in the United States. PCV3 was later proven to be widely prevalent in the swine population worldwide (Ku et al, 2017; Stadejek et al, 2017; Wang et al, 2017; Hayashi et al, 2018; Yuzhakov et al, 2018; Zoltán et al, 2019). This newly discovered DNA virus became an important threat to the healthy development of the pig industry. As prevention strategies only targeting symptomatic animals are not enough to prevent the virus spread, the ability to rapidly, reliably, and cost-effectively diagnose PCV3 is crucial

Methods

Results

Conclusion