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Concurrent infections of pseudorabies virus and porcine bocavirus in China detected by duplex nanoPCR
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Contents lists available at ScienceDirect
Journal of Virological Methods journal homepage: www.elsevier.com/locate/jviromet
Short communication
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Concurrent infections of pseudorabies virus and porcine bocavirus in China detected by duplex nanoPCR
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Yakun Luo a,b , Lin Liang a , Ling Zhou a , Kai Zhao c,∗∗ , Shangjin Cui a,b,∗ a
Institute of Animal Sciences (IAS), Chinese Academy of Agricultural Sciences, Beijing 100094, China State Key Laboratory of Veterinary Biotechnology, Harbin Veterinary Research Institute of Chinese Academy of Agricultural Sciences, 427 Maduan Street, Nangang District, Harbin 150001, Heilongjiang, China c College of Life Science, Heilongjiang University, Harbin 150080, China b
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a b s t r a c t
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Article history: Received 26 August 2014 Received in revised form 16 March 2015 Accepted 16 March 2015 Available online xxx
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Keywords: Duplex nanoPCR gE NS1 PBoV PRV
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1. Introduction
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Nanoparticle-assisted polymerase chain reaction (nanoPCR) is a novel method for the simple, rapid, and specific amplification of DNA and has been used to detect viruses. A duplex nanoPCR molecular detection system was developed to detect pseudorabies virus (PRV) and porcine bocavirus (PBoV). Primers were selected to target conserved regions within the PRV gE gene and the PBoV NS1 gene. Under optimized nanoPCR reaction conditions, two specific fragments of 316 bp (PRV) and 996 bp (PBoV) were amplified by the duplex nanoPCR with a detection limit of 6 copies for PRV and 95 copies for PBoV; no fragments were amplified when other porcine viruses were used as template. When used to test 550 clinical samples, the duplex nanoPRC assay and a conventional duplex PCR assay provided very similar results (98.1% consistency); single PRV infections, single PBoV infections, and concurrent PRV and PBoV infections were detected in 37%, 15%, and 9% of the samples, respectively. The results indicate that the novel duplex nanoPCR assay is useful for the rapid detection of PRV and PBoV in pigs. © 2015 Elsevier B.V. All rights reserved.
Pseudorabies virus (PRV), also known as Aujeszky’s virus, belongs to the subfamily Alphaherpesvirinae of the family Her26 pesviridae. Acute PRV infection is characterized by high fever, 27 extreme itching, and encephalomyelitis. PRV can cause disease 28 in swine, bovine, sheep, and wild animals (Eraldo et al., 2012). 29 Porcine pseudorabies has become a serious global disease of pigs. 30 Because PRV replication requires the gE gene (Enquist et al., 31 1999), the absence of gE gene is associated with reduced PRV 32 pathogenicity. The gE gene can be used to determine whether ani33 mals are infected with attenuated vaccine strains or wild virus 34 strains. 35 Porcine bocavirus (PBoV) belongs to the family Parvoviri36 37Q3 dae (Huang et al., 2014; Li et al., 2011a,b). PBoV was first described in 2009 when it was isolated from feces of pigs with 38 25
∗ Corresponding author at: Chinese Academy of Agricultural Sciences, Institute of Animal Science, Yuanmingyuan West Road 2, Hai Dian District, Beijing 100193, Q2 China. Tel.: +86 185 18437100; fax: +86 451 51997166. ∗∗ Corresponding author. Tel.: +86 451 86608586; fax: +86 451 86609016. E-mail addresses: [email protected] (K. Zhao), [email protected] (S. Cui).
postweaning multisystemic wasting syndrome (PMWS) in Sweden (Blomstrom et al., 2009). PBoV is a small, single-stranded DNA virus whose genome includes non-structural proteins (NS1 and NP1) and VP1/VP2 structural proteins (Wang et al., 2014). The NS1 protein is an important multifunctional phosphoprotein for parvoviruses (Sun et al., 2009). In addition, the role of the NS1 protein is similar among different paroviruses (Wang et al., 2014). Nanoparticle-assisted polymerase chain reaction (nanoPCR) is an advanced form of PCR in which solid gold nanometal particles (1–100 nm) form colloidal nanofluids that increase thermal conductivity (Shen et al., 2009; Zhang et al., 2005). Therefore, PCR assays with nanofluids reach the target temperature more quickly than PCR assays with original liquids. This reduces the time at non-target temperatures and thereby reduces non-specific amplification and increases specific amplification (Li and Rothberg, 2004; Ma et al., 2013). Since 2010, PRV and PBoV have been commonly associated with increased mortality in pigs in China. In this study, a duplex nanoPCR was developed for PRV and PBoV and the PRV gE gene and the PBoV NS1 gene were chosen as targets. The established duplex nanoPCR assay will be useful to investigate the molecular epidemiology of these viruses.
http://dx.doi.org/10.1016/j.jviromet.2015.03.016 0166-0934/© 2015 Elsevier B.V. All rights reserved.
Please cite this article in press as: Luo, Y., et al., Concurrent infections of pseudorabies virus and porcine bocavirus in China detected by duplex nanoPCR. J. Virol. Methods (2015), http://dx.doi.org/10.1016/j.jviromet.2015.03.016
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a final extension at 72 ◦ C for 10 min. The amplified products were analyzed by electrophoresis on 1.5% agarose gels.
Table 1 Sequences of the primers used in this study. Virus
Gene
Primer
Primer sequence (5 –3 )
Fragment size
PRV
gE
E1 E2
F.ACGAGCCCCGCTTCCACGCG R.CACCGGTCCCCGAGCAGCGG
316 bp
PBoV
NS1
S1 S2
F.CATCCTTTAGTCAATGCGAGAA R.CGGTAACAGCATAGAGTCCC
996 bp
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2. Materials and methods
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2.1. Viruses and recombinant plasmids
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The recombinant plasmids pET30a-PRV-gE and pUC57-PBoVNS1 and additional viruses including porcine parvovirus (PPV, strain BQ-C), porcine circovirus type 2 (PCV2, strain SH), porcine reproductive and respiratory syndrome virus (PRRSV, strain HB), classic swine fever virus (CSFV), porcine Teschovirus (PTV), and African swine fever virus (ASFV) were obtained from the Harbin Veterinary Research Institute of Chinese Academy of Agricultural Science. The nanoPCR Kit (NPK02) was purchased from GREDBIO (Weihai, China). The recombinant plasmids pET30a-PRV-gE and pUC57-PBoV-NS1 were amplified in Escherichia coli DH5␣ and purified with the AxyPrepTM Plasmid Midi and Maxi Plasmid Kits (AXYGEN Biotechnology Company, Hangzhou, China). The plasmids were stored at −20 ◦ C.
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2.2. Primer design
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The full-length genes of PRV and PBoV (Table 1) were chosen according to published sequences available in GenBank (GenBank accession numbers NC 006151 and HM053694). The primers were designed with Primer Premier 5.0 software. All of the primers were synthesized by BoShi Biotech (Harbin, China), and their sequences primers are listed in Table 1.
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2.3. Sample preparation
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Animal experiments were approved by the Harbin Veterinary Research Institute of the Chinese Academy of Agricultural Sciences, and animal experiments were performed in accordance with animal ethics guidelines and approved protocols. The Animal Ethics Committee approval number was Heilongjiang-SQ 2013-2046. The samples were ground in a mortar and then repeatedly frozen and thawed. DNA was recovered from 200 L of the homogenized samples according to the instructions of the TIANamp Virus genomic DNA/RNA kit (Beijing Tiangen Biotech Company, Beijing, China). The extracted DNA was stored at −20 ◦ C before it was subjected to conventional duplex PCR and to the novel duplex nanoPCR as described in the following sections. In addition, identified PRV and PBoV bands by duplex nanoPCR in the first 54 samples were checked by sequencing.
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2.4. Establishment of the conventional duplex PCR assay
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The conventional duplex PCR assay was performed in a 20 L system that included 2.4 L of template (for comparison with the duplex nanoPCR and for positive controls); 1.2 L of each of the recombinant plasmids pET30a-PRV-gE and pUC57-PBoV-NS1; (pET30a-PRV-gE and pUC57-PBoV-NS1); 1.0 L each of primers E1 and E2 and 0.8 L each of primers S1 and S2 (Table 1); 2.0 L of dNTPs; 1.0 L of KOD FX Neo (1 U/L) (TOYOBO Biotechnology Company, Shanghai, China); 5.0 L of 2× PCR buffer for KOD FX Neo (TOYOBO Biotechnology Company, Shanghai, China); and ddH2 O up to 20 L. PCR reaction conditions were: 94 ◦ C for 5 min; followed by 30 cycles of 94 ◦ C for 40 s, 58 ◦ C for 40 s, and 72 ◦ C for 60 s; and
2.5. Establishment of the duplex nanoPCR assay 2.5.1. Optimization of the duplex nanoPCR assay The duplex nanoPCR assay is based on the conventional duplex PCR assay. The buffers were prepared as described (Zhang et al., 2005). Experiments were performed to optimize the annealing temperature and primer volume for the duplex nanoPCR assay. The duplex nanoPCR assay was performed in a 20 L system. Each of the recombinant plasmids (pET30a-PRV-gE and pUC57-PBoV-NS1) was tested at volumes ranging from 0.2 L to 1.4 L (Table 1) in increments of 0.2 L. Each pair of forward and reverse primers (E1 and E2, and S1 and S2 at 10 M) was tested at volumes ranging from 0.2 L to 1.2 L (Table 1) in increments of 0.2 L. The reaction volume also contained 0.5 L of Taq DNA polymerase (5 U/L) (GRED, Shandong, China), 10 L of 2× nanoPCR Buffer (GRED, Shandong, China), and ddH2 O up to 20 L. PCR reaction conditions were: 94 ◦ C for 5 min; followed by 30 cycles of 94 ◦ C for 40 s, annealing temperatures from 50 ◦ C to 60 ◦ C for 30 s, and 72 ◦ C for 60 s; and a final extension at 72 ◦ C for 10 min. Amplified products were analyzed by electrophoresis on 1.5% agarose gels. The optimization was conducted in the following steps. First, the optimal annealing temperature was determined using primer concentrations and template concentrations as indicated for the conventional duplex PCR. The optimal annealing temperature was then used to determine the optimal primer concentrations with the template concentrations as indicated for the conventional duplex PCR. Finally, the optimal template concentrations were determined using the previously determined optimal annealing temperature and primer concentrations. 2.5.2. Sensitivity of the duplex nanoPCR assay To determine the sensitivity of the duplex nanoPCR assay, pET30a-PRV-gE and pUC57-PBoV-NS1 were purified with the AxyPrepTM Plasmid Midi and Maxi Plasmid Kits (AXYGEN Biotechnology Company, Hangzhou, China) and quantified using UV spectroscopy (6.44 × 1010 copies/L of pET30a-PRV-gE and 9.51 × 1010 copies/L of pUC57-PBoV-NS1). When 10-fold serial dilutions were prepared, the concentration ranged from 6.44 × 1010 to 6 × 100 copies/L for pET30a-PRV-gE and from 9.51 × 1010 to 9 × 100 copies/L for pUC57-PBoV-NS1. Each dilution was tested by the duplex nanoPCR assay and the conventional duplex PCR assay, and ddH2 O was used as a negative control. Amplified products were analyzed by electrophoresis on 1.5% agarose gels. 2.5.3. Specificity of the duplex nanoPCR assay DNA or cDNA of the following viruses and bacterium were separately subjected to the duplex nanoPCR and the conventional duplex PCR: PPV, PCV2, PRRSV, CSFV, PTV, ASFV, and Escherichia coli. The recombinant plasmids pET30a-PRV-gE and pUC57-PBoV-NS1 were used as positive controls. PCR products were analyzed by electrophoresis on 1.5% agarose gels.
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3. Results
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3.1. Optimization of the duplex nanoPCR assay
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The recombinant plasmids pUC57-PBoV-NS1 and pET30a-PBoVNS1 were used as templates in the optimization experiments. The tested annealing temperatures ranged from 50 to 60 ◦ C, and amplification results were best with an annealing temperature of 55 ◦ C (data not shown). An annealing temperature of 55 ◦ C was used to determine the optimal primer concentrations. The tested volume of primers
Please cite this article in press as: Luo, Y., et al., Concurrent infections of pseudorabies virus and porcine bocavirus in China detected by duplex nanoPCR. J. Virol. Methods (2015), http://dx.doi.org/10.1016/j.jviromet.2015.03.016
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Fig. 1. Sensitivity of the conventional duplex PCR assay (A) and the duplex nanoPCR assay (B) using different PRV and PBoV plasmid dilutions. Lane M: DL2000 marker; lanes 1–11: pET30a-PRV-gE concentrations ranging from 9.51 × 1010 copies/L to 9.51 × 100 copies/L and pUC57-PBoV-NS1 concentrations ranging from 6.44 × 1010 copies/L to 6.44 × 100 copies/L; lane 12: negative control.
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(combinations of E1 and E2 and S1 and S2) ranged from 0.4 L to 1.2 L in increments of 0.2 L, and amplification results were best with 0.8 L of both E1 and E2 and with 1.0 L of both S1 and S2 (data not shown). The optimal annealing temperature and primer volumes as indicated in the previous paragraphs were used to determine
the optimal template volume. Equal volumes of the templates (pET30a-PRV-gE and pUC57-PBoV-NS1) were mixed to provide total volumes of 0.2 L to 1.4 L in increments of 0.2 L. Amplification results were best with 0.8 L of pET30a-PRV-gE and 1.0 L of pUC57-PBoV-NS1 (data not shown).
Fig. 2. Specificity of the duplex nanoPCR assay (A) and the duplex conventional PCR assay (B). Lane M: DL2000 marker; lane 1: PRVl; lane 2: PBoV; lane 3: PCV2; lane 4: PPV; lane 5: CSFV; lane 6: PTV; lane 7: PRRSV; lane 8: ASFV; lane 9: E. coli; lane 10: PRV and PBoV; lane 11: negative control.
Please cite this article in press as: Luo, Y., et al., Concurrent infections of pseudorabies virus and porcine bocavirus in China detected by duplex nanoPCR. J. Virol. Methods (2015), http://dx.doi.org/10.1016/j.jviromet.2015.03.016
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Table 2 Detection rates of PRV and PBoV in samples from 550 diseased Chinese pigs by conventional duplex PCR and by duplex nanoPCR. Geographic area
Viruses detected
No. of positive samples detected by duplex nanoPCR
No. of positive samples detected by conventional duplex PCR
% of positive samples with the duplex nanoPCR
% of positive samples with the conventional duplex PCR
22
PRV PBoV PRV + PBoV
6 3 2
6 2 1
27.3 13.6 9.1
27.3 9.1 4.5
Liaoning
7
PRV PBoV PRV + PBoV
2 1 1
2 1 1
28.6 14.3 14.3
28.6 14.3 14.3
Jilin
6
PRV PBoV PRV + PBoV
3 2 1
3 2 1
50 33.3 16.7
50 33.3 16.7
13
PRV PBoV PRV + PBoV
5 2 1
5 2 1
38.5 15.4 7.7
38.5 15.4 7.7
Henan
1
PRV PBoV PRV + PBoV
1 0 0
1 0 0
100 0 0
100 0 0
Tianjin
1
PRV PBoV PRV + PBoV
1 0 0
1 0 0
100 0 0
100 0 0
Beijing
1
PRV PBoV PRV + PBoV
1 0 0
1 0 0
100 0 0
100 0 0
Anhui
3
PRV PBoV PRV + PBoV
1 0 0
1 0 0
33 0 0
33 0 0
Others
496
PRV PBoV PRV + PBoV
185 75 57
183 72 55
37.2 15.1 11.5
36.9 14.5 11.1
Total
550
PRV PBoV PRV + PBoV
205 83 62
203 79 59
37.3 15.1 11.3
36.9 14.4 10.7
Heilongjiang
Hebei
182 183 184 185 186 187 188 189
190
191 192 193 194 195 196 197 198 199 200 201 202
203
204 205 206
No. of samples
Based on the optimized conditions, the duplex nanoPCR assay was performed in a 20-L reaction mixture that included: 0.8 L of the PRV and 1.0 L of the PBoV extracted DNA or standard plasmid; 10 L of 2× nano-buffer; 0.8 L of both E1 and E2 and 1.0 L of both S1 and S2 (10 M); 1.0 L of Taq DNA polymerase (5 U/L); and ddH2 O up to 20 L. PCR reaction conditions were: 94 ◦ C for 5 min; 30 cycle of 94 ◦ C for 40 s, 55 ◦ C for 40 s, and 72 ◦ C for 60 s; and a final extension at 72 ◦ C for 10 min. 3.2. Sensitivity of the duplex nanoPCR assay The sensitivities of the duplex nanoPCR assay and the conventional duplex PCR assay were compared by using serial dilutions of the pUC57-PBoV-NS1 and pET30a-PRV-gE plasmids as templates. For pUC57-PBoV-NS1, the detection limit was 9.51 × 103 copies/L with the conventional duplex PCR assay (Fig. 1A) and 9.5 × 101 copies/L with the duplex nanoPCR assay (Fig. 1B). For pET30a-PRV-gE, the detection limit was 6.44 × 102 copies/L with the conventional duplex PCR assay (Fig. 1A) and 6 × 100 copies/L with the duplex nanoPCR assay (Fig. 1B). These results indicated that the duplex nanoPCR assay was 100–1000 times more sensitive than the conventional duplex PCR assay. 3.3. Specificity of the duplex nanoPCR assay Neither the optimized duplex nanoPCR assay nor the conventional duplex PCR assay amplified products when nontarget DNA or cDNA was used as template (Fig. 2). Both assays amplified
products when PRV, PBoV, or PRV + PBoV were used as template. These results indicated that these assays are highly specific. 3.4. Detection of PRV and PBoV DNA in clinical samples with the duplex nanoPCR assay A total of 550 samples were processed by the duplex nanoPCR assay and the conventional duplex PCR assay. According to the results of the duplex nanoPCR assay, 37.3% of the samples were positive for PRV, 15.1% were positive for PBoV, and 11.3% were positive for both viruses (Table 2). Similar results were obtained with the conventional duplex PCR assay, i.e., there was 98.1% agreement between the two assays (Table 2). Sequencing of the amplicons obtained with the first 54 samples confirmed that the duplex nanoPCR assay correctly identified PRV and PBoV (data not shown). 4. Discussion Infection by PRV can result in neurological symptoms and death in young piglets, respiratory disorders in older pigs, and abortion in pregnant swine (Ducatelle et al., 1982). As of 2011, PRV had been detected in more than 23 provinces in China, and virulent strains of PRV have been occasionally isolated from stillbirths and mummified piglets on large-scale swine farms (Zhu et al., 2011; Zhai et al., 2010). Pigs with PMWS in Sweden were determined to be infected by a virus that was initially designated as porcine boca-like virus (PBo-likeV) (Blomstrom et al., 2009). Subsequently, PBo-likeV was detected in symptomatic pigs in China and was PBoV (Zhai et al., 2010; Zeng et al., 2011). The PBoV has also been identified in healthy
Please cite this article in press as: Luo, Y., et al., Concurrent infections of pseudorabies virus and porcine bocavirus in China detected by duplex nanoPCR. J. Virol. Methods (2015), http://dx.doi.org/10.1016/j.jviromet.2015.03.016
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pigs in China (Zhang et al., 2011) and was detected in 38.7% of pigs with PMWS (Zhai et al., 2010). PBoV is now considered a threat to the pig industry in China. Assays that detect only one virus, either PRV or PBoV, have been described and include conventional PCR, real-time PCR, LAMP, and nanoPCR (Wang et al., 2014; Ma et al., 2013). Conventional PCR is relatively slow and insensitive, real-time PCR requires expensive equipment, and LAMP samples are easily contaminated (Wang et al., 2014). A duplex nanoPCR detection method, that targets the gE gene of PRV and the NS1 gene of PBoV, was therefore established and optimized in this study. The new method is highly specific. Although results were similar for the conventional duplex PCR and the duplex nanoPCR, the later assay was more sensitive. As noted in the Section 3, the duplex nanoPCR detected PRV infection, PBoV infection, and concurrent PRV and PBoV infection in relatively high percentages of the investigated 550 clinical samples from Heilongjiang, Jilin, Liaoning, and other Chinese provinces. These results indicate that the duplex nanoPCR assay will be useful for the detection of single or concurrent PRV and PBoV infections in clinical samples. In addition, the data suggest that concurrent infection by PRV and PBoV is widespread among pigs in Heilongjiang, Jilin, Liaoning, and other provinces in China. 5. Conclusion
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In conclusion, a rapid, sensitive, and specific duplex nanoPCR assay is described. The new assay can quickly and simultaneously detect PRV and PBoV and will be useful for diagnosing, studying, and in future perhaps also controlling PRV and PBoV.
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Li et al. (2012).
Acknowledgments
This work was supported by a grant from the project of Q6 Excellent Youth Foundation of Heilongjiang Scientific Committee 264 (no. JC201216). This work was partly supported by the Agricultural 265 Science and Technology Innovation Program (ASTIP-IAS15). 266 263
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Contents lists available at ScienceDirect
Journal of Virological Methods journal homepage: www.elsevier.com/locate/jviromet
Short communication
1
Concurrent infections of pseudorabies virus and porcine bocavirus in China detected by duplex nanoPCR
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Q1
Yakun Luo a,b , Lin Liang a , Ling Zhou a , Kai Zhao c,∗∗ , Shangjin Cui a,b,∗ a
Institute of Animal Sciences (IAS), Chinese Academy of Agricultural Sciences, Beijing 100094, China State Key Laboratory of Veterinary Biotechnology, Harbin Veterinary Research Institute of Chinese Academy of Agricultural Sciences, 427 Maduan Street, Nangang District, Harbin 150001, Heilongjiang, China c College of Life Science, Heilongjiang University, Harbin 150080, China b
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a b s t r a c t
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Article history: Received 26 August 2014 Received in revised form 16 March 2015 Accepted 16 March 2015 Available online xxx
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Keywords: Duplex nanoPCR gE NS1 PBoV PRV
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1. Introduction
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Nanoparticle-assisted polymerase chain reaction (nanoPCR) is a novel method for the simple, rapid, and specific amplification of DNA and has been used to detect viruses. A duplex nanoPCR molecular detection system was developed to detect pseudorabies virus (PRV) and porcine bocavirus (PBoV). Primers were selected to target conserved regions within the PRV gE gene and the PBoV NS1 gene. Under optimized nanoPCR reaction conditions, two specific fragments of 316 bp (PRV) and 996 bp (PBoV) were amplified by the duplex nanoPCR with a detection limit of 6 copies for PRV and 95 copies for PBoV; no fragments were amplified when other porcine viruses were used as template. When used to test 550 clinical samples, the duplex nanoPRC assay and a conventional duplex PCR assay provided very similar results (98.1% consistency); single PRV infections, single PBoV infections, and concurrent PRV and PBoV infections were detected in 37%, 15%, and 9% of the samples, respectively. The results indicate that the novel duplex nanoPCR assay is useful for the rapid detection of PRV and PBoV in pigs. © 2015 Elsevier B.V. All rights reserved.
Pseudorabies virus (PRV), also known as Aujeszky’s virus, belongs to the subfamily Alphaherpesvirinae of the family Her26 pesviridae. Acute PRV infection is characterized by high fever, 27 extreme itching, and encephalomyelitis. PRV can cause disease 28 in swine, bovine, sheep, and wild animals (Eraldo et al., 2012). 29 Porcine pseudorabies has become a serious global disease of pigs. 30 Because PRV replication requires the gE gene (Enquist et al., 31 1999), the absence of gE gene is associated with reduced PRV 32 pathogenicity. The gE gene can be used to determine whether ani33 mals are infected with attenuated vaccine strains or wild virus 34 strains. 35 Porcine bocavirus (PBoV) belongs to the family Parvoviri36 37Q3 dae (Huang et al., 2014; Li et al., 2011a,b). PBoV was first described in 2009 when it was isolated from feces of pigs with 38 25
∗ Corresponding author at: Chinese Academy of Agricultural Sciences, Institute of Animal Science, Yuanmingyuan West Road 2, Hai Dian District, Beijing 100193, Q2 China. Tel.: +86 185 18437100; fax: +86 451 51997166. ∗∗ Corresponding author. Tel.: +86 451 86608586; fax: +86 451 86609016. E-mail addresses: [email protected] (K. Zhao), [email protected] (S. Cui).
postweaning multisystemic wasting syndrome (PMWS) in Sweden (Blomstrom et al., 2009). PBoV is a small, single-stranded DNA virus whose genome includes non-structural proteins (NS1 and NP1) and VP1/VP2 structural proteins (Wang et al., 2014). The NS1 protein is an important multifunctional phosphoprotein for parvoviruses (Sun et al., 2009). In addition, the role of the NS1 protein is similar among different paroviruses (Wang et al., 2014). Nanoparticle-assisted polymerase chain reaction (nanoPCR) is an advanced form of PCR in which solid gold nanometal particles (1–100 nm) form colloidal nanofluids that increase thermal conductivity (Shen et al., 2009; Zhang et al., 2005). Therefore, PCR assays with nanofluids reach the target temperature more quickly than PCR assays with original liquids. This reduces the time at non-target temperatures and thereby reduces non-specific amplification and increases specific amplification (Li and Rothberg, 2004; Ma et al., 2013). Since 2010, PRV and PBoV have been commonly associated with increased mortality in pigs in China. In this study, a duplex nanoPCR was developed for PRV and PBoV and the PRV gE gene and the PBoV NS1 gene were chosen as targets. The established duplex nanoPCR assay will be useful to investigate the molecular epidemiology of these viruses.
http://dx.doi.org/10.1016/j.jviromet.2015.03.016 0166-0934/© 2015 Elsevier B.V. All rights reserved.
Please cite this article in press as: Luo, Y., et al., Concurrent infections of pseudorabies virus and porcine bocavirus in China detected by duplex nanoPCR. J. Virol. Methods (2015), http://dx.doi.org/10.1016/j.jviromet.2015.03.016
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a final extension at 72 ◦ C for 10 min. The amplified products were analyzed by electrophoresis on 1.5% agarose gels.
Table 1 Sequences of the primers used in this study. Virus
Gene
Primer
Primer sequence (5 –3 )
Fragment size
PRV
gE
E1 E2
F.ACGAGCCCCGCTTCCACGCG R.CACCGGTCCCCGAGCAGCGG
316 bp
PBoV
NS1
S1 S2
F.CATCCTTTAGTCAATGCGAGAA R.CGGTAACAGCATAGAGTCCC
996 bp
62
2. Materials and methods
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2.1. Viruses and recombinant plasmids
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The recombinant plasmids pET30a-PRV-gE and pUC57-PBoVNS1 and additional viruses including porcine parvovirus (PPV, strain BQ-C), porcine circovirus type 2 (PCV2, strain SH), porcine reproductive and respiratory syndrome virus (PRRSV, strain HB), classic swine fever virus (CSFV), porcine Teschovirus (PTV), and African swine fever virus (ASFV) were obtained from the Harbin Veterinary Research Institute of Chinese Academy of Agricultural Science. The nanoPCR Kit (NPK02) was purchased from GREDBIO (Weihai, China). The recombinant plasmids pET30a-PRV-gE and pUC57-PBoV-NS1 were amplified in Escherichia coli DH5␣ and purified with the AxyPrepTM Plasmid Midi and Maxi Plasmid Kits (AXYGEN Biotechnology Company, Hangzhou, China). The plasmids were stored at −20 ◦ C.
77
2.2. Primer design
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83
The full-length genes of PRV and PBoV (Table 1) were chosen according to published sequences available in GenBank (GenBank accession numbers NC 006151 and HM053694). The primers were designed with Primer Premier 5.0 software. All of the primers were synthesized by BoShi Biotech (Harbin, China), and their sequences primers are listed in Table 1.
84
2.3. Sample preparation
78 79 80 81 82
98
Animal experiments were approved by the Harbin Veterinary Research Institute of the Chinese Academy of Agricultural Sciences, and animal experiments were performed in accordance with animal ethics guidelines and approved protocols. The Animal Ethics Committee approval number was Heilongjiang-SQ 2013-2046. The samples were ground in a mortar and then repeatedly frozen and thawed. DNA was recovered from 200 L of the homogenized samples according to the instructions of the TIANamp Virus genomic DNA/RNA kit (Beijing Tiangen Biotech Company, Beijing, China). The extracted DNA was stored at −20 ◦ C before it was subjected to conventional duplex PCR and to the novel duplex nanoPCR as described in the following sections. In addition, identified PRV and PBoV bands by duplex nanoPCR in the first 54 samples were checked by sequencing.
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2.4. Establishment of the conventional duplex PCR assay
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The conventional duplex PCR assay was performed in a 20 L system that included 2.4 L of template (for comparison with the duplex nanoPCR and for positive controls); 1.2 L of each of the recombinant plasmids pET30a-PRV-gE and pUC57-PBoV-NS1; (pET30a-PRV-gE and pUC57-PBoV-NS1); 1.0 L each of primers E1 and E2 and 0.8 L each of primers S1 and S2 (Table 1); 2.0 L of dNTPs; 1.0 L of KOD FX Neo (1 U/L) (TOYOBO Biotechnology Company, Shanghai, China); 5.0 L of 2× PCR buffer for KOD FX Neo (TOYOBO Biotechnology Company, Shanghai, China); and ddH2 O up to 20 L. PCR reaction conditions were: 94 ◦ C for 5 min; followed by 30 cycles of 94 ◦ C for 40 s, 58 ◦ C for 40 s, and 72 ◦ C for 60 s; and
2.5. Establishment of the duplex nanoPCR assay 2.5.1. Optimization of the duplex nanoPCR assay The duplex nanoPCR assay is based on the conventional duplex PCR assay. The buffers were prepared as described (Zhang et al., 2005). Experiments were performed to optimize the annealing temperature and primer volume for the duplex nanoPCR assay. The duplex nanoPCR assay was performed in a 20 L system. Each of the recombinant plasmids (pET30a-PRV-gE and pUC57-PBoV-NS1) was tested at volumes ranging from 0.2 L to 1.4 L (Table 1) in increments of 0.2 L. Each pair of forward and reverse primers (E1 and E2, and S1 and S2 at 10 M) was tested at volumes ranging from 0.2 L to 1.2 L (Table 1) in increments of 0.2 L. The reaction volume also contained 0.5 L of Taq DNA polymerase (5 U/L) (GRED, Shandong, China), 10 L of 2× nanoPCR Buffer (GRED, Shandong, China), and ddH2 O up to 20 L. PCR reaction conditions were: 94 ◦ C for 5 min; followed by 30 cycles of 94 ◦ C for 40 s, annealing temperatures from 50 ◦ C to 60 ◦ C for 30 s, and 72 ◦ C for 60 s; and a final extension at 72 ◦ C for 10 min. Amplified products were analyzed by electrophoresis on 1.5% agarose gels. The optimization was conducted in the following steps. First, the optimal annealing temperature was determined using primer concentrations and template concentrations as indicated for the conventional duplex PCR. The optimal annealing temperature was then used to determine the optimal primer concentrations with the template concentrations as indicated for the conventional duplex PCR. Finally, the optimal template concentrations were determined using the previously determined optimal annealing temperature and primer concentrations. 2.5.2. Sensitivity of the duplex nanoPCR assay To determine the sensitivity of the duplex nanoPCR assay, pET30a-PRV-gE and pUC57-PBoV-NS1 were purified with the AxyPrepTM Plasmid Midi and Maxi Plasmid Kits (AXYGEN Biotechnology Company, Hangzhou, China) and quantified using UV spectroscopy (6.44 × 1010 copies/L of pET30a-PRV-gE and 9.51 × 1010 copies/L of pUC57-PBoV-NS1). When 10-fold serial dilutions were prepared, the concentration ranged from 6.44 × 1010 to 6 × 100 copies/L for pET30a-PRV-gE and from 9.51 × 1010 to 9 × 100 copies/L for pUC57-PBoV-NS1. Each dilution was tested by the duplex nanoPCR assay and the conventional duplex PCR assay, and ddH2 O was used as a negative control. Amplified products were analyzed by electrophoresis on 1.5% agarose gels. 2.5.3. Specificity of the duplex nanoPCR assay DNA or cDNA of the following viruses and bacterium were separately subjected to the duplex nanoPCR and the conventional duplex PCR: PPV, PCV2, PRRSV, CSFV, PTV, ASFV, and Escherichia coli. The recombinant plasmids pET30a-PRV-gE and pUC57-PBoV-NS1 were used as positive controls. PCR products were analyzed by electrophoresis on 1.5% agarose gels.
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3. Results
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3.1. Optimization of the duplex nanoPCR assay
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The recombinant plasmids pUC57-PBoV-NS1 and pET30a-PBoVNS1 were used as templates in the optimization experiments. The tested annealing temperatures ranged from 50 to 60 ◦ C, and amplification results were best with an annealing temperature of 55 ◦ C (data not shown). An annealing temperature of 55 ◦ C was used to determine the optimal primer concentrations. The tested volume of primers
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Fig. 1. Sensitivity of the conventional duplex PCR assay (A) and the duplex nanoPCR assay (B) using different PRV and PBoV plasmid dilutions. Lane M: DL2000 marker; lanes 1–11: pET30a-PRV-gE concentrations ranging from 9.51 × 1010 copies/L to 9.51 × 100 copies/L and pUC57-PBoV-NS1 concentrations ranging from 6.44 × 1010 copies/L to 6.44 × 100 copies/L; lane 12: negative control.
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(combinations of E1 and E2 and S1 and S2) ranged from 0.4 L to 1.2 L in increments of 0.2 L, and amplification results were best with 0.8 L of both E1 and E2 and with 1.0 L of both S1 and S2 (data not shown). The optimal annealing temperature and primer volumes as indicated in the previous paragraphs were used to determine
the optimal template volume. Equal volumes of the templates (pET30a-PRV-gE and pUC57-PBoV-NS1) were mixed to provide total volumes of 0.2 L to 1.4 L in increments of 0.2 L. Amplification results were best with 0.8 L of pET30a-PRV-gE and 1.0 L of pUC57-PBoV-NS1 (data not shown).
Fig. 2. Specificity of the duplex nanoPCR assay (A) and the duplex conventional PCR assay (B). Lane M: DL2000 marker; lane 1: PRVl; lane 2: PBoV; lane 3: PCV2; lane 4: PPV; lane 5: CSFV; lane 6: PTV; lane 7: PRRSV; lane 8: ASFV; lane 9: E. coli; lane 10: PRV and PBoV; lane 11: negative control.
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Table 2 Detection rates of PRV and PBoV in samples from 550 diseased Chinese pigs by conventional duplex PCR and by duplex nanoPCR. Geographic area
Viruses detected
No. of positive samples detected by duplex nanoPCR
No. of positive samples detected by conventional duplex PCR
% of positive samples with the duplex nanoPCR
% of positive samples with the conventional duplex PCR
22
PRV PBoV PRV + PBoV
6 3 2
6 2 1
27.3 13.6 9.1
27.3 9.1 4.5
Liaoning
7
PRV PBoV PRV + PBoV
2 1 1
2 1 1
28.6 14.3 14.3
28.6 14.3 14.3
Jilin
6
PRV PBoV PRV + PBoV
3 2 1
3 2 1
50 33.3 16.7
50 33.3 16.7
13
PRV PBoV PRV + PBoV
5 2 1
5 2 1
38.5 15.4 7.7
38.5 15.4 7.7
Henan
1
PRV PBoV PRV + PBoV
1 0 0
1 0 0
100 0 0
100 0 0
Tianjin
1
PRV PBoV PRV + PBoV
1 0 0
1 0 0
100 0 0
100 0 0
Beijing
1
PRV PBoV PRV + PBoV
1 0 0
1 0 0
100 0 0
100 0 0
Anhui
3
PRV PBoV PRV + PBoV
1 0 0
1 0 0
33 0 0
33 0 0
Others
496
PRV PBoV PRV + PBoV
185 75 57
183 72 55
37.2 15.1 11.5
36.9 14.5 11.1
Total
550
PRV PBoV PRV + PBoV
205 83 62
203 79 59
37.3 15.1 11.3
36.9 14.4 10.7
Heilongjiang
Hebei
182 183 184 185 186 187 188 189
190
191 192 193 194 195 196 197 198 199 200 201 202
203
204 205 206
No. of samples
Based on the optimized conditions, the duplex nanoPCR assay was performed in a 20-L reaction mixture that included: 0.8 L of the PRV and 1.0 L of the PBoV extracted DNA or standard plasmid; 10 L of 2× nano-buffer; 0.8 L of both E1 and E2 and 1.0 L of both S1 and S2 (10 M); 1.0 L of Taq DNA polymerase (5 U/L); and ddH2 O up to 20 L. PCR reaction conditions were: 94 ◦ C for 5 min; 30 cycle of 94 ◦ C for 40 s, 55 ◦ C for 40 s, and 72 ◦ C for 60 s; and a final extension at 72 ◦ C for 10 min. 3.2. Sensitivity of the duplex nanoPCR assay The sensitivities of the duplex nanoPCR assay and the conventional duplex PCR assay were compared by using serial dilutions of the pUC57-PBoV-NS1 and pET30a-PRV-gE plasmids as templates. For pUC57-PBoV-NS1, the detection limit was 9.51 × 103 copies/L with the conventional duplex PCR assay (Fig. 1A) and 9.5 × 101 copies/L with the duplex nanoPCR assay (Fig. 1B). For pET30a-PRV-gE, the detection limit was 6.44 × 102 copies/L with the conventional duplex PCR assay (Fig. 1A) and 6 × 100 copies/L with the duplex nanoPCR assay (Fig. 1B). These results indicated that the duplex nanoPCR assay was 100–1000 times more sensitive than the conventional duplex PCR assay. 3.3. Specificity of the duplex nanoPCR assay Neither the optimized duplex nanoPCR assay nor the conventional duplex PCR assay amplified products when nontarget DNA or cDNA was used as template (Fig. 2). Both assays amplified
products when PRV, PBoV, or PRV + PBoV were used as template. These results indicated that these assays are highly specific. 3.4. Detection of PRV and PBoV DNA in clinical samples with the duplex nanoPCR assay A total of 550 samples were processed by the duplex nanoPCR assay and the conventional duplex PCR assay. According to the results of the duplex nanoPCR assay, 37.3% of the samples were positive for PRV, 15.1% were positive for PBoV, and 11.3% were positive for both viruses (Table 2). Similar results were obtained with the conventional duplex PCR assay, i.e., there was 98.1% agreement between the two assays (Table 2). Sequencing of the amplicons obtained with the first 54 samples confirmed that the duplex nanoPCR assay correctly identified PRV and PBoV (data not shown). 4. Discussion Infection by PRV can result in neurological symptoms and death in young piglets, respiratory disorders in older pigs, and abortion in pregnant swine (Ducatelle et al., 1982). As of 2011, PRV had been detected in more than 23 provinces in China, and virulent strains of PRV have been occasionally isolated from stillbirths and mummified piglets on large-scale swine farms (Zhu et al., 2011; Zhai et al., 2010). Pigs with PMWS in Sweden were determined to be infected by a virus that was initially designated as porcine boca-like virus (PBo-likeV) (Blomstrom et al., 2009). Subsequently, PBo-likeV was detected in symptomatic pigs in China and was PBoV (Zhai et al., 2010; Zeng et al., 2011). The PBoV has also been identified in healthy
Please cite this article in press as: Luo, Y., et al., Concurrent infections of pseudorabies virus and porcine bocavirus in China detected by duplex nanoPCR. J. Virol. Methods (2015), http://dx.doi.org/10.1016/j.jviromet.2015.03.016
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pigs in China (Zhang et al., 2011) and was detected in 38.7% of pigs with PMWS (Zhai et al., 2010). PBoV is now considered a threat to the pig industry in China. Assays that detect only one virus, either PRV or PBoV, have been described and include conventional PCR, real-time PCR, LAMP, and nanoPCR (Wang et al., 2014; Ma et al., 2013). Conventional PCR is relatively slow and insensitive, real-time PCR requires expensive equipment, and LAMP samples are easily contaminated (Wang et al., 2014). A duplex nanoPCR detection method, that targets the gE gene of PRV and the NS1 gene of PBoV, was therefore established and optimized in this study. The new method is highly specific. Although results were similar for the conventional duplex PCR and the duplex nanoPCR, the later assay was more sensitive. As noted in the Section 3, the duplex nanoPCR detected PRV infection, PBoV infection, and concurrent PRV and PBoV infection in relatively high percentages of the investigated 550 clinical samples from Heilongjiang, Jilin, Liaoning, and other Chinese provinces. These results indicate that the duplex nanoPCR assay will be useful for the detection of single or concurrent PRV and PBoV infections in clinical samples. In addition, the data suggest that concurrent infection by PRV and PBoV is widespread among pigs in Heilongjiang, Jilin, Liaoning, and other provinces in China. 5. Conclusion
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In conclusion, a rapid, sensitive, and specific duplex nanoPCR assay is described. The new assay can quickly and simultaneously detect PRV and PBoV and will be useful for diagnosing, studying, and in future perhaps also controlling PRV and PBoV.
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Li et al. (2012).
Acknowledgments
This work was supported by a grant from the project of Q6 Excellent Youth Foundation of Heilongjiang Scientific Committee 264 (no. JC201216). This work was partly supported by the Agricultural 265 Science and Technology Innovation Program (ASTIP-IAS15). 266 263
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