Simultaneous detection of classical swine fever virus and porcine circovirus 3 by SYBR green I-based duplex real-time fluorescence quantitative PCR

https://doi.org/10.1016/j.mcp.2020.101524Get rights and content

Highlights

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    The SYBR Green І-based duplex real-time PCR assay was developed.

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    CSFV and PCV3 were simultaneously detected in one sample.

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    The detection limits were 23 copies/μL and 36 copies/μL for CSFV and PCV3, respectively.

Abstract

In the present study, the SYBR green I-based duplex quantitative polymerase chain reaction (qPCR) was developed for simultaneous detection of classical swine fever virus (CSFV) and porcine circovirus 3 (PCV3). The assay was used to detect both CSFV and PCV3 in one sample by their distinct melting temperatures (melting peaks at 87°C for CSFV and 81.5 °C for PCV3), and no specific fluorescence signals were detected for other non-targeted porcine pathogens. The assay had a high degree of linearity (R2 > 0.998) with the detection limits of 23 copies/μL for CSFV and 36 copies/μL for PCV3, and exhibited high repeatability and reproducibility with a low coefficient of variation below 2.0% in both intra- and inter-assay. In this study, 130 clinical samples collected from sick pigs in the field were tested by this assay with the positive rates of 9.23% (12/130) for CSFV and 21.54% (28/130) for PCV3 respectively, and the positive rate of CSFV and PCV3 co-infection was 6.92% (9/130). Our results showed that the developed method was a reliable diagnostic tool to monitor and survey CSFV, PCV3 and CSFV/PCV3 co-infection in the field.

Introduction

Classical swine fever virus (CSFV) is a small, enveloped, single-stranded RNA virus with a genome of approximately 12.3 kb in length and belongs to the genus Pestivirus of the family Flaviviridae [1]. CSFV is the causative agent of classical swine fever (CSF), which leads to severe economic losses due to the mortality, reduced growth, and reproductive problems in the affected pigs [2]. CSF was first reported in 1833 in the United States, and then spread worldwide [3], and has been listed as one of the notifiable diseases by World Organization for Animal Health (OIE). Although it was successfully controlled in some countries of the European Union, CSF is still a major threat to the pig industry of developing countries, including China [4].

Porcine circovirus 3 (PCV3) is a non-enveloped, single-stranded circular DNA virus, and belongs to the genus Circovirus of the family Circoviridae [5]. As a newly emerging virus, PCV3 was originally identified in the United States in 2015, from the pigs suffering from cardiac and multi-organ inflammation, porcine dermatitis and nephropathy syndrome (PDNS), and reproductive failure [6,7]. Since then, PCV3 has been detected in many countries, including Japan, South Korea, China, Poland, Sweden, Russia, Thailand, Brazil, Denmark, Italy, Hungarian and Spain [[8], [9], [10], [11], [12], [13], [14], [15], [16]].

Previous studies showed CSFV and PCV3 could cause some similar clinical signs in pigs such as reproductive failure, respiratory disease complex and diarrhea [6,17,18], and the co-infection of the two viruses had been confirmed in pig farms [13,19,20]. Sun et al. had detected 200 clinical samples from 1990 to 1999, and revealed that co-infection of PCV3/CSFV was quite common [13]. Furthermore, Chen et al. reported co-infection of CSFV and PCV3 might lead to the complexity of infection status and more difficulties in preventing and controlling the diseases [21]. Therefore, it was critical for the swine industry to prevent and control the diseases by real-time monitoring the co-infection of the two viruses. However, there was no specific assay with the capability simultaneously to detect CSFV and PCV3 in the field. Here, a SYBR green I-based duplex quantitative polymerase chain reaction (qPCR) was developed for simultaneous detection of CSFV and PCV3, and used as an efficient detection tool for epidemiological investigations of CSFV and PCV3 infections.

Section snippets

Virus and clinical samples

CSFV named Qixian strain was provided by the Key Laboratory for Animal-derived Food Safety of Henan Province in China, and cultivated in swine testicular cells (ST cells). PCV3-positive clinical tissue sample was collected and identified as described previously [22]. Porcine circovirus type 2 (PCV2, DF-2 strain), porcine pseudorabies virus (PRV, HN2012 strain), porcine parvovirus (PPV, HNeK strain), porcine reproductive and respiratory syndrome virus (PRRSV, HeN-21 strain) and porcine epidemic

Optimization of duplex qPCR

For the singleplex qPCR, the melting curves displayed a melting temperature (Tm) of 87 °C for CSFV (Fig. 1A) and 81.5 °C for PCV3 (Fig. 1B), respectively. For the duplex qPCR, the primer concentration and the annealing temperature were optimized to generate the highest fluorescence and the lowest threshold cycle. The results showed the final optimized primer concentration was determined as 0.25 μM for each primer of CSFV and PCV3, and the optimal denature temperature was 58 °C for both CSFV and

Discussion

PCV3 was first described in the US in 2015 associated with different diseases, such as cardiac and multi-systemic syndrome inflammation, PDNS, reproductive disorders, porcine respiratory disease complex, and diarrhea, in infected pigs [6,7,17,18,25]. Subsequent reports have identified the co-infection of PCV3 with CSFV in pig farms [13,19,20], suggesting the co-infection of two viruses could aggravate the infection status and make it harder to control the diseases. Therefore, it was necessary

CRediT authorship contribution statement

Hui-Hua Zheng: Formal analysis, Writing - original draft, Data curation, Methodology. Shu-Jian Zhang: Formal analysis, Visualization, Investigation. Jian-Tao Cui: Formal analysis, Supervision. Jia Zhang: Formal analysis, Validation. Leyi Wang: Formal analysis, Writing - review & editing. Fang Liu: Funding acquisition, Writing - review & editing. Hong-Ying Chen: Conceptualization, Resources, Funding acquisition, Writing - review & editing, Writing - original draft.

Declaration of competing interest

The authors declare that they have no conflict of interest.

Acknowledgement

This work was funded by the Henan Province Education Department Science and Technology Specific Projects (18A230007), Fang Liu received a China Scholarship Council (CSC; Beijing) to train at the University of Illinois at Urbana-Champaign.

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    Hui-Hua Zheng, Shu-Jian Zhang and Jian-Tao Cui contributed equally to this work.

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