Multiplex PCR methods for detection of several viruses associated with canine respiratory and enteric diseases

Viral respiratory and intestinal infections are the most common causes of canine viral illness. Infection with multiple pathogens occurs in many cases. Rapid diagnosis of these multiple infections is important for providing timely and effective treatment. To improve diagnosis, in this study, two new multiplex polymerase chain reactions (mPCRs) were developed for simultaneous detection of canine respiratory viruses (CRV) and canine enteric viruses (CEV) using two separate primer mixes. The viruses included canine adenovirus type 2 (CAV-2), canine distemper virus (CDV), canine influenza virus (CIV), canine parainfluenza virus (CPIV), canine circovirus (CanineCV), canine coronavirus (CCoV) and canine parvovirus (CPV). The sensitivity of the mPCR results showed that the detection limit of both mPCR methods was 1×104 viral copies. Twenty nasal swabs (NS) and 20 anal swabs (AS) collected from dogs with symptoms of respiratory disease or enteric disease were evaluated using the novel mPCR methods as a clinical test. The mPCR protocols, when applied to these respiratory specimens and intestinal samples, could detect 7 viruses simultaneously, allowing rapid investigation of CRV (CAV-2, CDV, CIV and CPIV) and CEV (CAV-2, CanineCV, CCoV and CPV) status and prompt evaluation of coinfection. Our study provides an effective and accurate tool for rapid differential diagnosis and epidemiological surveillance in dogs.


Introduction
Pet dogs play an important role in humans' daily lives. Recently, the emergence of new pathogens and the continuous circulation of common etiological agents in dog populations have complicated canine diseases [1]. Among these diseases, canine infectious respiratory diseases (CIRD) and viral enteritis pose notable threats to dog health. a1111111111 a1111111111 a1111111111 a1111111111 a1111111111

Specific primer design
Primer pairs for the detection of CPV and H3N2 CIV were adopted from previous studies [30,31]. To design primers for viral conserved regions, several sequences of CDV, CPIV, Cani-neCV and CCoV were compared by multiple alignments using BioEdit Sequence Alignment Editor Version 7.0.9.0 (Ibis Biosciences, Carlsbad, CA, U.S.A.), and primers specific to highly conserved regions of these viruses were designed using Primer Premier 5.0 software (Premier Biosoft International). Primers for the detection of CAV-2 were designed by reference to previous studies with some modification [32]. All primers used for mPCRs were required to have similar annealing temperatures, lack dimmers or hairpin structures, and used degenerate bases as little as possible to avoid mismatches. The primers ( Table 1) were synthesized by Tianyihuiyuan Gene Technology Co., Ltd. (Guangzhou, China).

Viral nucleic acid extraction and reverse transcription
Total nucleic acids (TNAs) from the positive controls and twenty nasal swabs (NS) and 20 anal swabs (AS) for clinical samples were extracted using the RaPure Viral RNA/DNA Kit (Magen, Guangzhou, China) according to the manufacturer's protocol. The extracted TNA was divided into two parts; one was used for the amplification of the DNA viruses (CAV-2, CanineCV and CPV) and the other for the reverse transcription (RT) and amplification of the RNA viruses (CDV, CIV, CPIV and CCoV). The RNA extracted from the positive controls and clinical samples was reverse transcribed to cDNA using random primer (TaKaRa, Dalian, China) and Moloney murine leukemia virus (M-MLV) reverse transcriptase (TaKaRa, Dalian, China) according to the manufacturer's instructions. The cDNA and DNA were stored at -20˚C until PCR amplification.

Standard plasmid preparations
The extracted DNA viruses (CAV-2, CanineCV and CPV) and cDNA of RNA viruses (CDV, CIV, CPIV and CCoV) were used in simplex PCR amplification. The reaction for each virus was performed in a 20-μl volume that contained 10 μl of 2 × Taq Master Mix (Vazyme Biotech, Nanjing, China), 1 μl each of the various templates described above, and 0.5 μl each of the forward primer (10 mM) and reverse primer (10 mM) and 8 μl double distilled water (ddH 2 O). The procedure for simplex PCR was as follows: 95˚C for 5 min, followed by 35 cycles of 95˚C for 30 s, 57˚C for 30 s, and 72˚C for 60 s, with a final extension at 72˚C for 8 min. The PCR products were evaluated by 1.5% agarose gel electrophoresis.

Establishment of mPCR methods
The mPCRs were optimized separately for CRV and CEV. Two mixtures containing standard plasmid were used as templates to optimize the annealing temperature (Ta). The primer concentrations used in this study were 10 mM. The reaction for detection of CRV was performed in a 20-μl volume that contained 10 μl of 2× Taq  , and ddH 2 O. Gradient mPCR was performed for each mixture to optimize the reaction with gradient annealing temperatures (Ta) ranging from 55˚C to 65˚C. Both reaction procedures were as follows: initial denaturation at 97˚C for 5 min; 30 cycles of denaturation at 97˚C for 30 s, varied Ta for 45 s and 72˚C for 1 min; and a final extension at 72˚C for 8 min. 10 μl of mPCR product loans were evaluated by 1.5% agarose gel electrophoresis.

Specificity of mPCR methods
To evaluate the specificity of the mPCRs, we performed specificity assays on CRV and CEV with CRV-and CEV-specific primers, respectively. Similar procedures were used to detect possible cross-reaction of CRV and CEV primers with RNA/DNA extracted from MDCK cells or from other pathogens (RABV, E. coli and Salmonella enterica). The nucleic acid extraction products of the MDCK cells, E. coli and Salmonella enterica were used directly as PCR templates. In contrast, the viral RNA extraction products of RABV required RT prior to use as templates. Both the individual plasmid and premixed plasmids were tested separately in this assay. The empty pMD-18T vector was used as a negative control.

Sensitivity of mPCR methods
The sensitivity of the mPCR assays was also examined. The concentrations of the seven plasmids (pMD-CAV-2, pMD-CDV, pMD-CIV, pMD-CPIV, pMD-CanineCV, pMD-CCoV and pMD-CPV) were measured using a NanoDrop 2000 UV-vis spectrophotometer (Thermo Scientific, Wilmington, USA). Copy number was calculated by the following Table 1. Primers used for PCR amplification of CRV and CEV.

Reproducibility of mPCR methods
To verify the stability of the assay, the established mPCR methods were performed as three independent mPCR assays by using three different PCR instruments at different times. The plasmid mixtures for CRV and CEV were used as templates after dilution from 1×10 5 copies/ μl to 1×10 3 copies/μl.

Evaluation of clinical samples
In the period of February to May 2018, 20 NS and 20 AS were randomly collected from dogs with symptoms of respiratory disease or enteric disease at different animal hospitals in Guangdong Province, China. In this study, NS and AS were selected because they were easy to be collected from animals and can reduce the pain of animals compared with blood samples or other animal samples. Clinical samples were collected with the consent of the animal owners. All swabs were placed in 1 ml ice-cold Dulbecco's modified Eagle medium (DMEM) containing 10,000 U/ ml each of penicillin and streptomycin and centrifuged at 12,000 rpm for 10 min at 4˚C to remove impurities. Then, all samples were stored at -80˚C until RNA/DNA extraction. Each swab sample was extracted with a volume of 200 μl according to the manufacturer's protocol. The template was a mixture of 1 μl DNA extracted from each sample and 1 μl cDNA obtained by RT. Finally, each mixed template from the swab samples was subjected to the mPCR methods.

Ethics statement
All procedures in the sample collection met the requirements and were approved by the Experimental Animal Welfare Ethics Committee of the South China Agricultural University.

Establishment of mPCR methods
The optimal reaction conditions were explored by adjusting the annealing temperature, primer concentration, extension time, and cycle number. Premixed plasmids of CRV and CEV were used as templates to verify the mPCRs, and the products were visualized by 1.5% agarose gel electrophoresis. Ta was evaluated using a gradient, and the optimum Ta values were 59.4˚C and 58.2˚C, which yielded no primer dimers or nonspecific amplicons for CRV ( Fig 1A) or CEV (Fig 1B).

Specificity of mPCR methods
The specificity of the mPCRs was evaluated using other pathogens (RABV, E. coli and Salmonella enterica) and MDCK cells as templates. No specific amplicons showed in the lanes representing negative controls, E. coli, Salmonella enterica, RABV and MDCK cells, whereas the lanes for detection of CRV and CEV showed amplified bands in both CRV mPCR (Fig 2A) and CEV mPCR (Fig 2B).

Reproducibility of mPCR methods
To evaluate the reproducibility of the assay, the detection mPCRs for both CRV and CEV were performed as three independent mPCR assays by using three different PCR instruments at different times. Three premixed plasmids for CRV ( Fig 5A) and CEV (Fig 5B), with different dilutions, could be amplified under different conditions and showed similar results among the assays.

Evaluation of clinical samples
The mPCRs for detection of CRV ( Fig 6A) and CEV (Fig 6B) were tested on 20 NS and 20 AS clinical samples, respectively, and the statistics are shown in Table 2.   treatment initiation. A few methods for detecting virus-induced respiratory or enteric disease have been developed [4,27,28,34], but no previous study had developed a systematic way to detect both CRV and CEV in dogs. Here, we developed two mPCR methods for detection of the most frequently coinfected viruses; these methods could be performed to diagnose dogs according to their clinical symptoms.
Primer design is the first and most important step in the process of establishing a detection method, and the following conditions must be satisfied: primers were designed to bind to conserved sequence regions, to have similar annealing temperatures, and to lack dimers or hairpin structures [28]. In these novel mPCR methods, the primer combination produced amplicons that were easy to distinguish from each other, the primer annealing temperatures were similar, and degenerate bases were required only infrequently. The specificity, sensitivity and reproducibility tests all showed good results.
The mPCR methods were tested on 20 NS and 20 AS samples collected from dogs with symptoms of respiratory disease or enteric disease. The ratio of positive samples to total samples was 80% (16/20) for CRV detection and 85% (17/20) for CEV detection. Because the sample number was insufficient, these results were not statistically significant. However, CPV and CDV clearly remain two of the more serious and epidemic diseases in dogs in worldwide at present [35][36][37][38]. Epidemiological monitoring of CPV is particularly important because CPV evolves at a rapid rate, similar to that of Porcine Circovirus 3 [39,40]. Because a small number of dogs were negative for the viruses tested by the CRV or CEV detection assays, although they suffered respiratory illness or intestinal problems, we suggest that some viruses with low prevalence and pathogenic bacteria may also cause disease in dogs [2,41]. A variety of pathogenic bacteria are often present along with viruses in canine infections [42,43], and thus, it is essential to expand the coverage of mPCR detection in the future. For example, CIRD also include CHV-1, canine reovirus, and Bordetella bronchiseptica and so on. At the same time, other pathogens causing serious zoonotic diseases, such as pseudorabies virus, should also be monitored in future [44,45].
https://doi.org/10.1371/journal.pone.0213295.g005 pathogenic mechanism of CanineCV is unclear, epidemiological testing is important for future research. CanineCV was not detected from the AS clinical samples; perhaps the limited source of these clinical samples was responsible for this result. We didn't get a lot of clinical samples because it was not easy to get disease samples. CAV-2 mostly replicates in the lower respiratory tract and was detected in the NS samples [2]; however, the CAV-2 primer pair used in this study was probably able to amplify the CAV-1 DNA virus despite the optimization performed [32]. Notably, the live vaccine strains used may have an unavoidable impact on disease detection using the methods developed in this study. Additionally, discriminating between wildtype infections and vaccines is important [34], and therefore, a trend exists toward later development of broad-spectrum and accurate mPCR detection methods. Sometimes, cross contamination may lead to experimental failure. It is worth noting that PCR pretreatment and posttreatmen performed in different isolation zones can effectively avoid pollution. Besides, regular air spray cleaning will also play a role.
In conclusion, these newly established mPCR methods provide an efficient, sensitive, specific and low-cost testing tool for the detection of CRV (CAV-2, CDV, CIV and CPIV) and CEV (CAV-2, CanineCV, CCoV and CPV). The use of Taq Master Mix makes the detection process more convenient and reduces the chance of contamination during the process of sample addition; PCRs can be initiated by simply adding enzyme, ddH 2 O, premixed primers, and template, and thus, this method is superior to other mPCR detection methods. Here, detection of CanineCV was added to mPCR for the first time, making this method suitable for the further study of coinfection by CanineCV and other pathogens. This study provides a novel tool for systematic clinical diagnosis and laboratory epidemiological surveillance of CRV and CEV among dogs.