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Detection of Porcine Circoviruses in Clinical Specimens Using Multiplex PCR in Hubei, Central China

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Detection of Porcine Circoviruses in Clinical Specimens Using Multiplex PCR in Hubei, Central China Detection of porcine circoviruses in clinical specimens using multiplex PCR in Hubei, central China Keli Yang Corresp., 1 , Zuwu Jiao 1 , Danna Zhou 1 , Rui Guo 1 , Zhengying Duan 1 , Fangyan Yuan 1 , Yongxiang Tian 1 1 Institute of Animal Husbandry and Veterinary, Hubei Academy of Agricultural Sciences, Wuhan, Hubei, China Corresponding Author: Keli Yang Email address: [email protected] In order to detect and simultaneously discriminate PCV1, PCV2 and PCV3, a multiplex PCR assay was developed and used to detect clinical samples in this study. Each of target genes of PCV1, PCV2 and PCV3 was amplified using the designed primers, while no other porcine viruses genes were detected. The limit of detection of the assay was 10 copies/μL of PCV1, PCV2 and PCV3. The tissue samples from eight pig farms were detected using the multiplex PCR assay. The results showed that PCV1, PCV2 and PCV3 are co-circulating in central China. The PCV1, PCV2 and PCV3 singular infection rate was 52.4% (150/286), 61.2% (175/286) and 45.1% (129/286), respectively, while the PCV1 and PCV2 co-infection rate was 11.2% (32/286), the PCV1 and PCV3 co-infection rate was 5.9% (17/286), the PCV2 and PCV3 co-infection rate was 23.4% (67/286), and the PCV1, PCV2 and PCV3 co- infection rate was 1.7% (5/286), respectively, which were 100% consistent with the sequencing method and Real-time PCR methods. It proved that this multiplex PCR assay could be used as a differential diagnostic tool for monitoring and control of PCVs in the field. The results also indicate that the PCVs infection and their co-infection are severe in Hubei Province, Central China. PeerJ Preprints | https://doi.org/10.7287/peerj.preprints.27145v1 | CC BY 4.0 Open Access | rec: 27 Aug 2018, publ: 27 Aug 2018 Detection of porcine circoviruses in clinical specimens using multiplex PCR in Hubei, central China 1 Keli Yang1,2, Zuwu Jiao1, Danna Zhou1,3, Rui Guo1, Zhengying Duan1, 2 Fangyan Yuan1 and Yongxiang Tian1,2 3 1Institute of Animal Husbandry and Veterinary, Hubei Academy of Agricultural Sciences, Wuhan 430064, P. R. 4 China 5 2Key Laboratory of Prevention and Control Agents for Animal Bacteriosis (Ministry of Agriculture), Wuhan 430064, 6 P. R. China 7 3Hubei Key Laboratory of Animal Embryo and Molecular Breeding, Wuhan 430064, P. R. China 8 Corresponding authors 9 Keli Yang, 10 [email protected] 11 Yongxiang Tian, 12 [email protected] 13 14 ABSTRACT 15 In order to detect and simultaneously discriminate PCV1, PCV2 and PCV3, a multiplex PCR 16 assay was developed and used to detect clinical samples in this study. Each of target genes of 17 PCV1, PCV2 and PCV3 was amplified using the designed primers, while no other porcine 18 viruses genes were detected. The limit of detection of the assay was 10 copies/μL of PCV1, 19 PCV2 and PCV3. The tissue samples from eight pig farms were detected using the multiplex 20 PCR assay. The results showed that PCV1, PCV2 and PCV3 are co-circulating in central China. 21 The PCV1, PCV2 and PCV3 singular infection rate was 52.4% (150/286), 61.2% (175/286) and 22 45.1% (129/286), respectively, while the PCV1 and PCV2 co-infection rate was 11.2% (32/286), 23 the PCV1 and PCV3 co-infection rate was 5.9% (17/286), the PCV2 and PCV3 co-infection rate PeerJ Preprints | https://doi.org/10.7287/peerj.preprints.27145v1 | CC BY 4.0 Open Access | rec: 27 Aug 2018, publ: 27 Aug 2018 24 was 23.4% (67/286), and the PCV1, PCV2 and PCV3 co-infection rate was 1.7% (5/286), 25 respectively, which were 100% consistent with the sequencing method and Real-time PCR 26 methods. It proved that this multiplex PCR assay could be used as a differential diagnostic tool 27 for monitoring and control of PCVs in the field. The results also indicate that the PCVs infection 28 and their co-infection are severe in Hubei province, Central China. 29 Keywords Porcine circovirus; PCV1; PCV2; PCV3; viral discrimination; multiplex PCR 30 INTRODUCTION 31 Porcine circoviruses (PCVs), are non-enveloped and circular DNA viruses, which belong to the 32 genus Circovirus, family Circoviridae (Mankertz et al., 2004). At present, PCVs are smallest 33 animal viruses. Two species of circovirus, PCV1 and PCV2, had been proved as infectious to 34 pigs before 2015 (Ku et al., 2017). PCV1 was first isolated and in 1974, which just was a 35 contaminant of the PK-15 cell, and nonpathogenic for pigs (Tischer et al., 1974; 1986). PCV2 36 was first identified from the pigs which was suffering post-weaning multisystemic wasting 37 syndrome (PMWS) in the middle of 1990s (Allan et al., 1998). Pigs infected PCV2 have various 38 clinical diseases, which have made the swine industries huge economic losses all over the world 39 (Opriessnig et al., 2007). In 2016, a novel circovirus, called PCV type 3 (PCV3), was isolated 40 from diseased pigs in the USA (Phan et al., 2016; Palinski et al., 2017). Subsequently, several 41 outbreaks of it were reported from the United Kingdom of Great Britain (Collins et al., 2017), 42 Poland (Stadejek et al 2017), Italy, Denmark, Spain (Franzo et al., 2018), Korean (Kwon et al., 43 2017), Brazil (Tochetto et al., 2018), and China (Fan et al., 2017; Ku et al., 2017; Zhai et al., 44 2017; Zheng et al., 2017). PCV1 and PCV3 had been confirmed as potential pathogen associated 45 with many kinds of clinical symptoms, which are similar as PCV2 infection (Saha et al., 2011; 46 Palinski et al., 2017; Kwon et al., 2017; Ku et al., 2017; Franzo et al., 2018). And now, PCV3 47 has been found in about 20 provinces in China (Fig. 1). 48 Both PCV1 and PCV2 infections are common in pig herds all over the world (Beach et al., 49 2010), and PCV3 is the third porcine circovirus type found in swine, which is circulating in the 50 swine population (Palinski et al., 2017). The clinical manifestations of PCV3 are similar to those PeerJ Preprints | https://doi.org/10.7287/peerj.preprints.27145v1 | CC BY 4.0 Open Access | rec: 27 Aug 2018, publ: 27 Aug 2018 51 of PCV2 and to co-infection with PCV1, PCV2 and PCV3 in pig herds in the several countries 52 described above. The co-infection of PCV3 with PCV2 was reported in clinical samples of 53 diseased pigs in Hubei province (Ku et al. 2017). And co-infection of PCV2 with PCV1 was 54 found in Hubei province. However, PCV1, PCV2 and PCV3 or their co-infections were tested 55 separately using different methods in the previous reports. Considering the similarities between 56 the clinical manifestations associated with PCV3 and PCV2, and the high impact of PCV2 and 57 PCV3 on the economy of pig industry, it is necessary to develop a rapid, convenient, sensitive, 58 and specific diagnostic approach to discriminate PCV1, PCV2 and PCV3 infection. 59 However, there is no rapid, convenient and specific diagnostic assay capable of 60 differentiating PCV1, PCV2 and PCV3 infection. Therefore, to understand the current 61 epidemiology of PCVs in Hubei province, a rapid, simple, specific and sensitive multiplex PCR 62 assay was developed to detect and discriminate PCV1, PCV2 and PCV3. The accuracy and 63 applicability of the multiplex PCR were evaluated for detection of PCVs DNA in clinical 64 samples collected from the eight pig farms in Hubei province (Fig. 1) where co-infection of 65 PCVs was reported (Ku et al. 2017). The objective of the present study was to investigate the 66 epidemiological characteristics of PCVs in the Hubei province using the developed multiplex 67 PCR. 68 MATERIALS AND METHODS 69 Cells and viruses 70 PK15 cells were cultured in DMEM supplemented with 10% fetal bovine serum (Gibco) at 37 71 °C in an incubator with humidified 5% CO2. PCV1, PCV2, PCV3 and other viruses, including 72 classical swine fever virus (CSFV), porcine reproductive and respiratory syndrome virus 73 (PRRSV), pseudorabies virus (PRV), porcine parvovirus (PPV), rotavirus (RV), Japanese 74 encephalitis virus (JEV), porcine epidemic diarrhea virus (PEDV), porcine deltacoronavirus 75 (PDCoV), stored at Key Laboratory of Prevention and Control Agents for Animal Bacteriosis 76 (Ministry of Agriculture), were used to verify the specificity of the developed multiplex PCR 77 assay. And the viruses PCV1, PCV2, PCV3 were also used as positive control viruses. Viral PeerJ Preprints | https://doi.org/10.7287/peerj.preprints.27145v1 | CC BY 4.0 Open Access | rec: 27 Aug 2018, publ: 27 Aug 2018 78 genomic DNA or RNA for the specificity of the proposed multiplex PCR assay were extracted 79 according to our previous study (Yang et al., 2017). 80 Primers design 81 The primers were designed based on PCV1, PCV2, PCV3 sequences in GenBank database 82 (Table 1). The PCR-amplified PCV1, PCV2 and PCV3 products were 310, 505 and 1021 bp, 83 respectively. The primers were synthesized by Sangon Biotech Co., Ltd. (Shanghai, China). 84 DNA extraction 85 Viral DNA samples for proposed multiplex PCR were extracted using the Viral DNA/RNA 86 Miniprep Kit (Axygen Scientific, CA, USA) from the tissue samples. Total DNA was eluted 87 with 30 μL of diethyl pyrocarbonate-treated water, used immediately or stored at -80 °C. 88 Optimization of the Multiplex PCR 89 In order to obtain the best reaction parameters, the multiplex PCR was optimized by varying 90 single parameters while other parameters were maintained. The optimization was performed in a 91 50 µL PCR reaction mixture as follows: 10 × PCR buffer 5.0 µL, 10 mM dNTPs 2-4 µL, each 10 92 µM primer (Table 1) 0.5-1 µL, Taq DNA polymerase (5U/µL) (TaKaRa, Dalian, China) 0.5-1 93 µL, the DNA template 3.0-5.0 µL, and added distilled water to 50 µL.
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