Development of a recombinase-aided amplification assay for rapid and sensitive detection of porcine circovirus 3

Highlights

• First description of an RAA assay to detect PCV3.

• The PCV3 RAA assay is a sensitive, specific, and rapid method.

• This new method can be used for epidemiological studies of PCV3.

• The new method costs less than real-time PCR.

Abstract

Porcine circovirus type 3 (PCV3) is a novel member of the genus Circovirus, first detected in the United States in 2016, with subsequent reports in many countries. PCV3 infections have caused serious economic losses in the pig industry. Alternative rapid and sensitive assays for PCV3 detection are needed for clinical diagnosis, especially in laboratories not equipped with more sophisticated equipment. Here, a real-time recombinase-aided amplification assay (RAA) was developed for PCV3 detection. Specific primers and probes targeting the conserved region of the capsid gene of PCV3 were designed. The assay was performed at 39 °C for 30 min using specialized equipment. Furthermore, 36 clinical samples were used to evaluate the RAA. The analytical sensitivity of the RAA for PCV3 was 38 copies per reaction at 95% probability level, using a probit regression model. There was no cross-reactivity with other DNA viruses belonging to the Circoviridae and Parvoviridae families. The detection rate agreed with that obtained by an established real-time PCR assay with a kappa value of 1.0. Our results demonstrated that this new RAA could be used for the rapid, accurate, and sensitive detection of PCV3.

Introduction

Porcine circovirus (PCV) is a member of the genus Circovirus of the family Circoviridae. This non-enveloped icosahedral virus, the smallest known mammalian virus, has an approximately 2.0-kb single-stranded circular DNA genome. The genome encodes two major open reading frames (ORF1 and ORF2). ORF2 is highly conserved and relatively stable and comprises the cap gene encoding cap protein, which is associated with virus invasion into host cells and stimulation of the host immune response (Bexton et al., 2015; Ellis, 2014; Shen et al., 2018). Two Circovirus species have been reported in pigs prior to 2016: PCV type 1 (PCV1) and 2 (PCV2). PCV1 was first reported as a contaminant in PK-15 cell cultures and is nonpathogenic to pigs. PCV2 is the primary cause of porcine circovirus-associated diseases and has spread rapidly in swine, causing serious economic losses to the industry worldwide (Ellis, 2014; Palinski et al., 2017; Phan et al., 2016).

In 2016, a novel Circovirus was reported in the United States and named PCV type 3 (PCV3) (Phan et al., 2016). It is associated with porcine dermatitis, nephropathy syndrome (PDNS), reproductive failure, cardiac inflammation, and multisystemic inflammation, which may cause a large number of deaths in the herd, leading to huge economic losses in the swine industry. PCV3 has been reported in Italy, Korea, Denmark, Spain, UK, Japan, and China (Chen et al., 2017; Feng et al., 2019; Franzo et al., 2018; Hayashi et al., 2018; Ku et al., 2017; Kwon et al., 2017). It commonly circulates in swine populations in many countries, with the potential for cross-border transmission (Feng et al., 2019; Franzo et al., 2019; Hayashi et al., 2018; Kwon et al., 2017; Phan et al., 2016; Shen et al., 2018; Wen et al., 2018). As PCV2 and PCV3 infections cause similar clinical symptoms (Zhao et al., 2019), rapid, sensitive, and specific detection methods to distinguish PCV3 from other viral infections are of utmost importance for disease control. Current routine laboratory methods for PCV3 detection include polymerase chain reaction (PCR), enzyme-linked immunosorbent assay, SYBR Green I real-time PCR, and TaqMan real-time PCR. However, these approaches have multiple drawbacks, including high costs, significant time requirements, and expensive equipment, making them impractical for wide application (Zhao et al., 2011). Compared with traditional methods, isothermal amplification technology, including loop-mediated isothermal amplification (LAMP), is accurate, rapid, and cost-effective (Park et al., 2018; Zheng et al., 2018).

In this study, we describe a novel, isothermal, recombinase-aided amplification (RAA) method that uses special enzymes and proteins to achieve exponential DNA amplification at 39 °C in 20–30 min. The reaction mixture included single-stranded DNA-binding protein (SSB), recombinase UvsX, and DNA polymerase. The RAA could also be combined with a fluorescent probe system for real-time detection or naked-eye detection with a lateral flow dipstick. This general assay has been used to detect multiple bacterial and viral pathogens and is a promising advance in clinical diagnosis and field surveillance. In this study, an RAA method targeting the capsid gene of PCV3 was successfully established and its performance was evaluated using clinical specimens.