A Duplex SYBR Green I-based Real-time Polymerase Chain Reaction Assay for Rapid Detection of Canine Kobuvirus and Canine Circovirus

Background: Canine Kobuvirus (CaKoV) and Canine Circovirus (CaCV) are viruses that infect dogs causing diarrheal symptoms that are very similar. However, there is no clinical method to detect a co-infection of these two viruses. Results: In this study, a duplex SYBR Green I-based quantitative real-time polymerase chain reaction (PCR) assay for the rapid and simultaneous detection of CaKoV and CaCV was established. CaKoV and CaCV were distinguished by their different melting temperature which was 86 ℃ for CaKoV and 78 ℃ for CaCV. The assay was highly specic, with no cross-reactivity with other common canine viruses and demonstrated high sensitivity. The detection limits of CaKoV and CaCV were 8.924 × 10 1 copies/μL and 3.841 × 10 1 copies/μL, respectively. The highest intra- and inter-assay Ct value variation coecients (CV) of CaKoV were 0.40% and 0.96%, respectively. For CaCV, the highest intra- and inter-assay Ct value variation coecients were 0.26% and 0.70%, respectively. In 57 clinical samples, positive detection rates of CaKoV and CaCV were 8.77% (7/57) and 15.79% (9/57), respectively. The co-infection rate was 7.02% (4/57). Conclusions: The duplex SYBR Green I-based real-time PCR assay established in this study is a fast, ecient, and sensitive method for the simultaneous detection of the two viruses and provides a powerful tool for the rapid detection of CaKoV and CaCV in clinical practice.

1. The study establishes a novel assay for simultaneous detection of canine kobuvirus and canine circovirus.
2. SYBR Green І-based duplex real-time qPCR method was established with high sensitivity, speci city and reliability. 3. The study provides a tool that could be bene cial for clinical diagnostics of canine kobuvirus and canine circovirus infection as well as future research on the virus.

Background
Canine Kobuvirus (CaKoV) is a novel single-strand positive-sense RNA virus belonging to the Picornaviridae virus family [1]., with a genome of 8. 1-8.2 Kb. It contains one 7,332-7,341 nucleotides (NT) open read frame (ORF) that encodes 2,442-2,475 amino acids (AA) [2,3]. CaKoV was identi ed in a feces sample of a diarrhea dog brought in from the United States in 2011 [4], and has since been identi ed in the United Kingdom, Italy, South Korea, Tanzania and Japan [1,[5][6][7]. CaKoV is widespread in China, it was rst detected in 2015 in northeast China [8], and has been subsequently identi ed in southwest and estern China [2,9]. CaKoV exists in a wide range of body parts, including the digestive system, cerebellum, amygdala and liver [10]. However, CaKoV is currently considered a pathogen associated with intestinal diseases; epidemiological investigations have shown that it has been detected both in dogs with diarrhea and in healthy dogs [11], suggesting that there may be a hidden infection of the virus, giving it a large transmission advantage.
Canine Circovirus (CaCV) is a single-stranded, non-capsule DNA virus belonging to the circovirus family [12]. It has two ORFs that encode replicase proteins and capsid proteins, respectively [13]. It was rst reported in the United States in 2012 [14], and has also been reported in China [15]. CaCV infects dogs of different ages, especially young dogs, with high infection rates, and also infects carnivores, such as wolves and badgers [16]. CaCV causes hemorrhagic enteritis in dogs, though this conclusion is controversial [17]. Notably, CaCV may play a role as a co-infectious factor in the development of diarrheal diseases [17].
CaKoV and CaCV are viruses that infect dogs causing diarrheal symptoms that are very similar. However, there is no clinical method to detect a co-infection of these two viruses. Therefore, it is necessary to establish a simple, sensitive and rapid method for simultaneous detection of these two viruses. Fluorescent quantitative polymerase chain reaction (PCR) technology has the advantages of high speci city, high sensitivity and good reproducibility, which results in reduced reaction time and intuitive results. Dual real-time quantitative uorescence PCR is a method of rapid diagnosis that may be used to identify two kinds of viruses based on a difference in the melting temperature (Tm) value caused by the difference in GC content of the ampli ed fragments.
In this study, to effectively detect CaKoV and CaCV in dogs, a duplex SYBR Green I-based quantitative real-time PCR assay for CaKoV and CaCV was established.

Viruses and nucleic acid extraction
The CaKoV RNA and CaCV DNA genomes were extracted from positive fecal samples using the TIANamp Virus DNA/RNA kit (Tiangen, Beijing, China), according to the manufacturer's instructions. Then, the CaKoV RNA genome was converted to cDNA using the PrimeScript™ 1st strand cDNA Synthesis Kit (TaKaRa, Kusatsu, Japan). The following viral strains were used in this study: CaKoV, CaCV, canine astrovirus (CaAstV), canine distempervirus (CDV), canine coronavirus (CCV), and canine parvovirus (CPV).

Preparation of standard plasmids
The 3D of CaKoV and the Rep of CaCV were used as template DNA. Target fragments have been PCRampli ed using primers shown in Table 1 (CaKoV-F/CaKoV-R and CaCV-F/CaCV-R). Products were cloned into the pMD-19T vector in accordance with the manufacturer's protocol and transformed into Escherichia coli DH5α cells. Recombinant plasmids were extracted using the EasyPure Plasmid MiniPrep kit (TransGen Biotech, Beijing, China) and named pMD-19T-CaKoV and pMD-19T-CaCV, respectively. General Biosystems (Chuzhou, China) sequenced the recombinant plasmids. The concentrations of the recombinant plasmids were determined using an ND-2000 spectrophotometer (Thermo Scienti c, Dreieich, Germany) and the copy number was calculated according to the formula: (plasmid concentration [ng] × 6.02 × 10 23 )/(genome length × 10 9 × 660 Da/bp). Next, the standard plasmids were serially diluted 10-fold and stored at -20°C for further use. Table 1 The primers used in this study.

Primer name
Sequences CaCV-SYBR-R CAAGACAGATCATCATCAAGA 2.3 Primers design MN449341 was used as a reference sequence for CaKoV and MN863535 was used as a reference sequence for CaCV. Two speci c primers were designed using Primer Premier 5 for the 3D and Rep genes of CaKoV (CaKoV-SYBR-F and CaKoV-SYBR-R) and CaCV (CaCV-SYBR-F and CaCV-SYBR-R), respectively. The primers for conventional PCR were designed using the same conserved region. All primer sequences used in this study are listed in Table 1. General Biosystems Company (Chuzhou, China) synthesized all primers.

Optimization of the duplex real-time PCR assay
Standard plasmids of CaKoV and CaCV were used as templates to optimize the duplex real-time PCR assay. The reaction system including the parameters of PCR reaction, concentrations of primers and reagent was optimized, in order to get the best detection results.

Standard curves for CaKoV and CaCV
The 10-fold serially diluted recombinant plasmid standards of CaKoV and CaCV were used as templates with each concentration repeated thrice. According to the optimized reaction system, duplex SYBR Green I-based real-time PCR assay was performed to obtain standard curves for CaKoV and CaCV.
Standard plasmid concentrations for CaKoV and CaCV ranging from 10 1 to 10 8 copies/µL were applied to the optimized duplex real-time PCR assay to determine the minimum detection limit of this method.

Speci city analysis
The speci city of the duplex qPCR assay was evaluated using several viruses which are common in dogs. The DNA concentrations of CaCV and CPV and the cDNA concentrations of CaKoV, CaAstV, CDV, and CCV were 1 × 10 7 copies/µL. A negative control (ddH 2 O) was also included. After each ampli cation, 2% agarose gel electrophoresis was performed to verify whether the expected product was obtained.

Reproducibility analysis
Three different concentrations of 10 7 , 10 5 and 10 3 were elected to assess the reproducibility. Each dilution was performed three parallel tests under the same conditions at equal intervals. The Ct value variation coe cients (CV) were calculated to determine intra-and inter-assay variation.

Detection of clinical samples
A total of 57 unknown clinical samples from diarrheic dogs were collected and then nucleic acid of the virus was extracted using the DNA/RNA kit. After all products were reverse transcribed, the duplex SYBR Green I-based real-time PCR assay was used to detect the target viruses. And the same samples were also tested using conventional PCR.

Optimization of the duplex SYBR Green I-based realtime PCR assay
The total volume of the optimized duplex real-time PCR system was 40µL, including 20µL of SuperReal PreMix Plus, 0.6µL of forward and reserve primes for CaKoV, 0.4µL of forward and reserve primes for CaCV, 1µL of each template, and ddH 2 O. The conditions were 95 •C for 15 min, followed by 40 cycles of 95 •C for 10 s and 60 •C for 30 s. As shown in Fig. 1a-b, the Tms were 86 •C for CaKoV and 78 •C for CaCV. And as shown in Fig. 2, the duplex melting curve showed the same T m as the single experiment.

Sensitivity analysis
As shown in Fig. 4a-b, 10 folded dilution of CaKoV and CaCV were used as templates from 10 8 to 10 1 . The ampli cation curve showed that the lowest sensitivity detected by the duplex real-time PCR assay was 10 1 copies/µL.

Speci city analysis
As shown in Fig. 5, CaKoV, CaCV, CaAstV, CDV, CCV and CPV templates were ampli ed using the newly developed duplex real-time PCR assay, and ddH 2 O was used as a negative control. There were no speci c melting peaks for other viruses and negative control as well as the target viruses could be distinguished.

Repeatability analysis
As shown in Table 2 and Table 3, the intra-and inter-assay CVs for CaKoV and CaCV were small. The intra-assay CV values for CaKoV and CaCV ranged from 0.21-0.34% and 0.04-0.26%, respectively and the inter-assay CV values for CaKoV and CaCV ranged from 0.31-0.96% and 0.16-0.70%, respectively.

Clinical sample analysis
As shown in Table 4, 57 unknown clinical samples from diarrheic dogs were used in the duplex SYBR Green I-based real-time PCR and conventional PCR assays. When using the duplex real-time PCR method, the positive rates of CaKoV and CaCV were 8.77% (5/57) and 14.04% (8/57), respectively, and the coinfection rate was 7.02% (4/57). However, positive rates of CaKoV, CaCV, and co-infection by the conventional PCR method were 5.26% (3/57), 10.53% (6/57), and 3.51% (2/57), respectively. The main clinical symptom of canine gastroenteritis is diarrhea, and the common viruses that cause diarrhea in dogs are CPV, CDV and CCV [19][20][21]. In addition, there are two new viruses, CaKoV and CaCV, which can also cause diarrhea in dogs[8, 13,22]. CaKoV and CaCV infection is a serious threat to the health of dogs, the mortality rate is as high as 50-100%, which has caused huge economic losses to the canine industry [23]. CaKoV has been detected in cattle, sheep, pigs, and dogs in different countries [24][25][26][27]. Since circovirus was rst identi ed in the United States in 2012 [13], it has been reported in Italy, Germany, China, Thailand and other countries [15,22,[28][29][30]. Because the clinical symptoms of the two viruses are very similar, it is di cult to differentiate and diagnose the two viruses [17,23]. Therefore, a test that simultaneously detects and identi es these two viruses is required.
Viruses are detected in a variety of ways, such as loop-mediated isothermal ampli cation, enzyme-linked immunosorbent assays, and indirect immuno uorescence assays [31][32][33][34]. Compared to these traditional detection methods, real-time uorescence quantitative PCR has great advantages in terms of sensitivity and speci city [33,35], and detects virus even at lower concentrations [36]. In addition, compared with TaqMan-based real-time PCR, the SYBR Green I-based real-time PCR method is cheaper and easier [12]. Compared to a single assay, a dual assay was designed with the same sensitivity, but with the ability to detect multiple viruses. Therefore, a duplex SYBR Green I-based real-time PCR was developed that simultaneously detected CaKoV and CaCV.
In this study, a duplex SYBR Green I-based real-time PCR assay was successfully established. Two speci c primers were designed based on the conserved regions, 3D for CaKoV and Rep for CaCV. The method distinguished the two viruses by their different Tm values, which were 86°C for CaKoV and 78°C for CaCV. The detection limits of CaKoV and CaCV were 8.924 × 10 1 copies/µL and 3.841 × 10 1 copies/ µL, respectively, demonstrating the high sensitivity of the method. Moreover, when this method was used to detect other viruses, such as CaAstV, CDV, CCV, and CPV, there was no obvious melting curve, indicating that the assay had good speci city. The intra -and inter-assay CV values were low, demonstrating that the experiment could be repeated with a high degree of reproducibility. Furthermore, the duplex SYBR Green Ibased real-time PCR and conventional PCR assays were used to detect viruses within clinical samples.
The positive infection rate of the former was signi cantly higher than that of the latter, indicating that the duplex SYBR Green I-based real-time PCR assay was more suitable for the detection of these two viruses.

Conclusions
In conclusion, a duplex SYBR Green I-based real-time PCR assay for the detection of CaKoV and CaCV was successfully established. This assay is rapid, has high sensitivity, high speci city, a simple operation, and low price. In addition, this method will facilitate the detection and differential diagnosis of these two viruses and has important signi cance for their study and prevention.

Declarations
Ethics approval and consent to participate All experiments were compliant with the ethical standards of Anhui Agricultural 220 University (Permit number: SYXK 2016-007).

Consent for publication
Not applicable.

Availability of data and materials
Availability of data and materials All data generated or analyzed in this study can be obtained within the tables and gures of the manuscript.

Con icts of interest/Competing interests
The authors declare no con ict of interest.

Funding
This study was supported by the Ningbo Health Branding Subject Fund (No. ppxk2018-10).
Author contributions YP and JC conceived of the study, carried out the experiment and drafted the manuscript, contributed equally to this work. JW and YXW participated in the data collection and analysis. JZ, JY, and FX participated in statistical analysis. YW conceived of the study, revising the manuscript critically. All authors have read and approved the nal manuscript.