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Simultaneous detection of porcine parvovirus and porcine circovirus type 2 by duplex real-time PCR and amplicon melting curve analysis using SYBR Green
Journal of Virological Methods 187 (2013) 15–19
Contents lists available at SciVerse ScienceDirect
Journal of Virological Methods journal homepage: www.elsevier.com/locate/jviromet
Simultaneous detection of porcine parvovirus and porcine circovirus type 2 by duplex real-time PCR and amplicon melting curve analysis using SYBR Green Lan-lan Zheng a,1 , Ya-bin Wang b,1 , Ming-feng Li a , Hong-ying Chen b , Xian-po Guo b , Jing-wei Geng a , Zhen-ya Wang b , Zhan-yong Wei a,∗ , Bao-an Cui b,∗ a b
The College of Animal Science and Veterinary Medicine, Henan Agricultural University, Zhengzhou, Henan 450002, China Key Laboratory for Animal-derived Food Safety of Henan Province, Zhengzhou, Henan 450002, China
a b s t r a c t Article history: Received 10 November 2011 Received in revised form 20 June 2012 Accepted 27 June 2012 Available online 4 July 2012 Keywords: Porcine parvovirus (PPV) Porcine circovirus type 2 (PCV-2) Duplex real-time PCR SYBR-Green
The development of a SYBR Green-based duplex real-time PCR is described for simultaneous detection of porcine parvovirus (PPV) and porcine circovirus type 2 (PCV-2) genomes. Viral genomes were identified in the same sample by their distinctive melting temperature (Tm ) which is 77.5 ◦ C for PPV VP2 313 bp amplicon and 82.3 ◦ C for PCV-2 ORF2 171 bp amplicon, respectively. The detection limit of the method was 0.01 TCID50 /mL for PPV and PCV-2, about 10 times more sensitive than conventional PCR. In addition, PPV and PCV-2 viral load were measured in 126 field samples, confirming the sensitivity and specificity, and the result showed that 70/126 samples were positive for PPV and 92/126 samples were positive for PCV2 by the duplex real-time PCR. This method may be a useful alternative rapid and reliable method for the detection of PPV/PCV-2 co-infection. © 2012 Elsevier B.V. All rights reserved.
1. Introduction Porcine circovirus (PCV) is a small, nonenveloped, singlestranded DNA virus with a circular genome and belongs to the Circoviridae family. In the late 1990s, PCV-2 was associated with the post-weaning multisystemic wasting syndrome (PMWS) which is characterized by progressive weight loss, respiratory signs, and jaundice. Depletion of lymphocytes in the lymphoid follicles and their replacement by macrophages are the hallmark lesions observed in this syndrome (Allan et al., 1999; Meehan et al., 1998). Porcine parvovirus (PPV) is a small non-enveloped single-stranded DNA virus of Parvoviridae, and is the major causative virus of the syndrome of reproductive failure in swine, which includes stillbirths and infertility (Dunne et al., 1965; Mengeling and Cutlip, 1976). Co-infection with PCV-2 and PPV has been demonstrated in a significant proportion of field cases of PMWS in pigs in many countries (Choi and Chae, 2000; Ellis et al., 2000; Kim and Chae, 2001; Kim et al., 2002; Yue et al., 2009). It has been shown experimentally that co-infection with PCV-2 and PPV causes more severe lesions and clinical disease in PMWS than infection with PCV-2 alone (Allan
∗ Corresponding authors at: College of Animal Science and Veterinary Medicine, Henan Agricultural University, Zhengzhou, Henan 450002, China. Tel.: +86 0 371 63554361; fax: +86 0 371 63558259. E-mail address: [email protected] (Z.-y. Wei). 1 The first and second author contributed equally to this work. 0166-0934/$ – see front matter © 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.jviromet.2012.06.024
et al., 1999; Kennedy et al., 2000; Krakowka et al., 2000; Yong et al., 2007). Designing a rapid and high throughput assay which is capable of detecting simultaneously the two agents will improve significantly the ability to identify the two diseases in swine herds rapidly. PCV-2 and PPV can be diagnosed by detecting the antibodies, viruses infected tissues and body fluids. However, there are several potential problems with these methods. Compared to the conventional detection methods, such as serology, immunohistochemistry and immunofluorescence for detecting antigen and the polymerase chain reaction (PCR) methods, real-time PCR technology is highly sensitive and specific, time-saving, easy to handle, and exhibits a decrease risk (Huang et al., 2004; Jiang et al., 2010; Ogawa et al., 2009). Individual real-time PCR assay has been developed for detection and identification of PCV-2 and PPV. Recently, multiplex real-time PCR, with melting curve analysis, has been described as a simple, reliable, and rapid method for detection and identification of certain bacteria, protozoa, and viruses (Balint et al., 2009; Lopez et al., 2009). The objectives of this study were to develop and validate a novel SYBR Green real-time PCR following by melting curve analysis of the amplicon, for the simultaneous detection of PCV-2 and PPV, and to investigate the effects of diagnostic specimen choice on its performance. The method established in current study could be used to detect PCV-2 and PPV in nasal swabs, tissues (lymph nodes, spleens, lungs and livers) and semen samples from naturally infected conventional pigs in different pig farms.
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2. Materials and methods
2.5. Duplex SYBR Green real-time PCR
2.1. Viruses and clinical specimens
The 171-bp PCV2-ORF2 specific DNA fragment and 313-bp PPVVP2 specific DNA fragment were amplified and inserted into the pGEM-T easy vector respectively to obtain the recombinant plasmids. According to the optimized reaction condition of Section 2.4, the duplex SYBR Green real-time PCR was amplified. Fluorescence was measured after extension at each cycle. All real-time reactions (standards, unknown samples, and controls) were performed in duplicate in neighboring wells on the sample plate. Results reported are an average of the duplicates.
The PPV standard strain 7909 was purchased from the China Institute of Veterinary Drug Control, Beijing, China. The PCV-2 HN strain was isolated from clinical specimens, and identified in the Key Laboratory for Animal-derived Food Safety of Henan Province, China. These viruses were propagated in PK-15 cells (purchased from China Institute of Veterinary Drug Control, China). Pseudorabies virus (PRV), Japanese encepphenia virus (JEV), Porcine reproductive and respiratory syndrome virus (PRRSV), Porcine circovirus type 1 (PCV-1) and Classical swine fever virus (CFSV) were provided by Key Laboratory for Animal-derived Food Safety of Henan Province, China. A total of 126 field samples were collected in different pig farms in several provinces of China in the period of May 2007 to July 2009, which included 46 tissue samples (lymph nodes, spleens, lungs and livers) and 8 nasal swab samples collected from diseased pigs and 72 semen samples obtained from clinically normal pigs. The sickness status was diagnosed based on clinical symptoms and pathological representations. 2.2. Template DNA preparations Viral genomic DNA was extracted either from 500 L lysates of PK-15 cells infected with each virus strain, or from 20 mg of frozen clinical specimens by using a commercial DNA extraction kit (QIAamp DNA Mini Kit, Qiagen, Hilden, Germany) according to the manufacturer’s direction. The isolated DNA was calculated and stored at −70 ◦ C. 2.3. Primers design and synthesize The VP2 nucleotide sequences of PPV strains/isolates and ORF2 nucleotide sequences of PCV-2 were retrieved from the GenBank and aligned using the software program DNAStar (DNASTAR, Inc., Madison). The primers were selected using the Primer Express (version 2.0) software and were based on a highly conserved sequence within the VP2 region of the PPV genome and the ORF2 region of the PCV-2. The primer pairs (forward primer: 5 -ATG GCG GGA GGA GTA GTT T-3 ; reverse primer: 5 -CCC TTT GAA TAC TAC AGC G3 ) used to amplify a fragment with a length of 171 bp for PCV-2 (GenBank Accession number AF027217). The primer pairs (forward primer: 5 -GGG CTT GGT TAG AAT CAC-3 ; reverse primer: 5 -TGG TGG TGA GGT TGC TGA T-3 ) used to amplify a fragment with a length of 313 bp for PPV (GenBank Accession number NC001718). The primers were designed and synthesized by Takara, China. 2.4. Optimization of the PPV/PCV-2 duplex SYBR Green real-time PCR assay A number of experiments were performed to optimize the PPV/PCV-2 duplex SYBR Green real-time PCR protocol, including reagent concentration and PCR cycling parameters. The primers as well as SYBR PreMix were titrated in chequerboard assays to determine the reagents concentrations that yielded the best results. Different annealing/elongation temperatures and times were evaluated. The following parameters were used in the duplex real-time PCR. Each reaction mix in a total volume of 25 L, including 2 L DNA, 12.5 L 2 × SYBR Green PreMix, 1 L of each primer. The reaction condition was: pre-denaturation at 95 ◦ C for 3 min, 40 cycles of denaturation at 95 ◦ C for 15 s, annealing at 55 ◦ C for 10 s, and extension at 72 ◦ C for 15 s.
2.6. Melt curve analysis A melt curve analysis was performed after each run, with the mixture being cooled of 45 s at 60 ◦ C, and then heated in one-degree increments at 95 ◦ C. Fluorescence was measured for 15 s at each increment and a graph plotting the second derivative of the melt displayed. The melting peaks were analyzed to distinguish PCV2 and/or PPV specific amplicons. Fluorescence normalization and data analysis were performed by thermal cycler program software (MxPro-Mx3005P v.3.00; Stratagene, La Jolla, CA).
2.7. Sensitivity, specificity and reproducibility of the real-time PCR A 1-mL aliquot of a culture solution of PCV-2 and PPV with a titer of 107 TCID50 /mL was subjected to a 10-fold serial dilution in PBS, with nine dilutions. The DNA of each dilution was extracted and subjected to duplex real-time PCR using the established amplification conditions in Section 2.5. The minimum concentration which can be detected will provide the sensitivity of the real-time PCR. To determine the analytical specificities of the PCR protocols, other five porcine (PRRSV, CSFV, PRV, PCV-1 and JEV) viral pathogens were tested. The intra-assay and inter-assay reproducibilities were evaluated using high, intermediate and low input of the standard PPV and PCV-2 plasmid preparations. The coefficients of variation for CT value obtained for each dilution were calculated. To determine the reproducibility of the real-time PCR, the standard plasmid was diluted to 1 × 107 , 1 × 106 , 1 × 105 , 1 × 104 , 1 × 103 , 1 × 102 , 1 × 101 , and 1 × 100 copies and tested by three different people at six different times per person. The variation among the three testers at each time was analyzed.
2.8. Data analyses After a SYBR Green PCR run, data acquisition and subsequent data analyses were done using the 5700 Sequence Detection System (SDS Version 1.3). In the system, the fluorescence of SYBR Green against the internal passive reference dye, ROX (Rn) is measured at the end of each cycle. A sample is considered positive when Rn exceeds the threshold value. The threshold value is set at the midpoint of Rn vs. cycle number plot. For all the amplifications described in this paper, the threshold value of Rn was taken as 0.25. The threshold cycle (C) is defined as the cycle at which a statistically significant increase in Rn is first detected. Target cDNA copy number and CT values are related inversely. A sample containing higher copies of the target cDNA will cross the threshold at an earlier cycle, compared to a sample with lower copies of the same target. The copy numbers of PCV-2 and PPV samples were determined by normalizing the CT values of the samples and then extrapolating the normalized CT values to the standard curve of the virus. For further statistical analyses, the CT values were exported into a Microsoft Excel Worksheet. Regression analyses of the CT
L.-l. Zheng et al. / Journal of Virological Methods 187 (2013) 15–19
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Fig. 2. The PCR amplification of PPV VP2. Fig. 1. The PCR amplification of PCV-2 ORF2.
values of the DNA dilution series were used to determine the amplification efficiency for PCV-2 and PPV. 2.9. Conventional PCR Viral DNA sequences were amplified by conventional PCR as described previously (Kim et al., 2001; Wu et al., 2008). Amplification of DNA for PCV-2-171 bp was achieved by 3 min at 95 ◦ C, followed by 40 cycles of 15 s at 95 ◦ C, 1 min at 55 ◦ C, and 1 min at 72 ◦ C, and a final extension of 5 min at 72 ◦ C. Amplification for PPV-313 bp was performed similarly, but with an annealing temperature of 57 ◦ C. The reactions were performed in triplicate in a DNA Engine (PTC-200) thermocycler, and amplicons were visualized by ethidium bromide staining on a 1.5% agarose gel with a DNA ladder. To ensure the quality of data, negative and positive controls were used in each PCR reaction. 2.10. Detection of filed samples A total of 126 field samples were tested in parallel by the duplex real-time PCR and conventional PCR. Ten positive and ten negative samples were selected at random, and subjected to virus isolation to validate the reliability of the duplex real-time PCR. 3. Results
curve analysis. Hence, PCV-2 and PPV amplicons can easily be distinguished by specific Tm values due to the different lengths and compositions of two amplicons. Melting curve analysis revealed that the PCV-2 melting curve displays a Tm of 82.3 ◦ C, whereas the PPV curve showed a Tm of 77.5 ◦ C (Fig. 3). 3.3. Sensitivity of the duplex real-time PCR The analytical sensitivity of SYBR Green PCR was determined by using a serial dilution of PCV-2 and PPV plasmid DNA as template for amplification. A linear relationship that between the input plasmid DNA and the CT values with regression coefficient (r2 ) which greater than 0.99 was obtained for both the viruses. The detection limits of the duplex real-time PCR was determined to be 175 copies/L for PPV and 156 copies/L for PCV-2, equivalent to 0.01 TCID50 /mL respectively, which are about 5–10 times more sensitive than those of the conventional PCRs (0.1 TCID50 /mL for PPV and PCV-2, respectively), and comparable to that of the PCR (0.03 and 0.05 TCID50 /mL for PPV and PCV-2, respectively) (Kim et al., 2001). 3.4. Specificity of the real-time PCR The assay was specific for the PPV and PCV-2. No specific amplification was detected with other porcine viruses such as PRV, JEV, PRRSV, PCV-1, and CFSV. In addition, the Tm of PPV and PCV-2 was recorded as 77.5 ◦ C and 82.3 ◦ C, respectively, and other viruses showed no specific melting peak (Fig. 4). The specific amplifications
3.1. The construction of standard templets of PPV and PCV-2 The 171 bp PCV-2-ORF2 specific DNA fragment and 313 bp PPVVP2 specific DNA fragment were amplified (Figs. 1 and 2) and inserted into the pGEM-T easy vector respectively to obtain the recombinant plasmids. The recombinant plasmids were used as the standard templets of PPV and PCV-2, which are required for the standard curve calculation in quantization assays. 3.2. Real-time PCR strategies optimization and melting curve analysis The procedure of real-time PCR was optimized according to the concentrations of primers and SYBR Green master mixture, the denature/extension temperature, and reaction time (data not shown). Duplex real-time PCR fragments are detected by melting
Fig. 3. Melting curve analysis for the duplex SYBR Green I based real-time PCR.
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L.-l. Zheng et al. / Journal of Virological Methods 187 (2013) 15–19
Fig. 4. Melting curve analysis of the specificity of the duplex SYBR Green I based real-time PCR assay.
were also confirmed by the detection specific bands of 313 bp for PPV and 171 bp for PCV-2 by agarose gel electrophoresis. No bands of the expected sizes were observed from other viruses real-time PCR products (data not shown). 3.5. Reproducibility of the real-time PCR The standard plasmid was serially diluted and then subjected to the real-time PCR, results obtained by three people at six times were similar. The inter-assay variation coefficient (the variation among the three testers) was 1.34, 1.29, 1.40 for each of the six times. The coefficients of variation (CVs) of CT values for each input DNA copy number between different intra-assay and inter-assay runs were less than 2% for both PPV and PCV-2 detection channels. 3.6. Agreement between the duplex real-time PCR and conventional PCRs Among the 126 field samples tested, the duplex real-time PCR showed 70 samples (all from clinically diseased pigs) to contain PPV and 77 samples to contain PCV-2, while the conventional PCRs tested 60 samples positive for PPV and 68 positive for PCV-2 (Table 1). The agreement between the duplex real-time PCR and the conventional PCRs was 98.4% for PPV and PCV-2. 4. Discussion PCV-2 and PPV have caused great loss in porcine industry in the world, and co-infection of the two viruses has been demonstrated in a significant proportion of field cases of PMWS in pigs in many countries of the world. Rapid and accurate methods for detection are essential in identification of PCV-2 and PPV. However, to date, few publications have reported on the duplex PCR enabling differentiation between PPV and PCV-2. Previous studies have demonstrated detection of PCV2 and PPV using nuclease probes in the Key Laboratory for Animal-derived
Food Safety of Henan Province laboratory (Hong et al., 2009). However, use of probes can add significant cost to an assay comprising multiple gene targets and is highly influenced by the potential presence of mutations within the probe-binding site that can prevent annealing of the probe and subsequent detection. Alternatively, intercalating dyes such as SYBR Green can be used to detect the presence of generated PCR fragments and is considered to be a flexible approach that can be directly applied to any gene without the need to design and synthesize fluorescently labeled target-specific probes. Hence, the reasons why propose the assay as an alternative method to other tests, particularly for large scale PCR screening, are that the SYBR Green PCR method was not influenced by the different genetic backgrounds. Amplicons can be distinguished by melt curve analysis, whereby the Tm of PCR products is determined by the reduction in relative fluorescence as all dsDNA is denatured to their single-stranded form. The shape and peak location of the melt curve are functions of the GC/AT ratio, length, and sequence of the fragment (Ririe et al., 1997; Wehrle et al., 2009). Detection with SYBR Green is as sensitive as probe-based real-time PCR without the need for a specialized probe. The duplex SYBR Green real-time PCR is more sensitive compared to routine methods such as virus isolation which is time consuming and labor intensive (Kim et al., 2001) and nested PCR (nPCR) assays (Kim et al., 2001; Larochelle et al., 2000; McIntosh et al., 2006; Varga and James, 2005; Wilhelm et al., 2005). These assays can amplify a group of 3–5 genes simultaneously. This study describes the development of a SYBR Green-based duplex real-time PCR for the simultaneous detection of PPV and PCV-2 genomes in a single reaction vessel. The assay was proved to be sensitive, specific, and reproducible. No specific amplification was found for several clinically relevant porcine viruses other than PCV-2 and PPV within 40 cycles in the duplex real-time PCR demonstrating the high specificity of the assay. An inherent limitation in designing a multiplex real-time PCR assay is loss of sensitivity when combining a large number of primer sets in a single reaction. In this study, the reaction conditions were carefully optimized to obtain maximum sensitivity (data not shown). As a result, despite the presence of two primers set in the PCR reaction mixture, the multiplex PCR was able to detect all viruses tested at a high level of sensitivity. The real-time PCR was able to detect as low as 0.01 TCID50 /mL of PPV and PCV-2, which were at least 10 times more sensitive than the conventional PCRs. The result of preliminary clinical application indicated that PPV and PCV-2 co-infection is probably broad prevalent in the swine herds. In addition, 72 semen samples collected from clinically “normal” pigs in small scale herds. 37/72 were positive for PPV, 33/72 were positive for PCV-2 and 18/72 were positive for co-infection implying a potential problem which should not be ignored. Simultaneous detection and differentiation of the two viruses should be incorporated in the disease control of swine herds in China. In conclusion, the SYBR Green-based real-time PCR assay described in this study for the simultaneous detection of PPV and PCV-2 has been shown to be rapid, easy to handle, relatively low
Table 1 Agreements between the duplex real-time PCR and conventional PCRs. Samples from clinically normal pigs
Samples from diseased pigs
Total
PPV+
PPV+
PPV+
PCV2+
PCV2+
a
PCV2+ b
Duplex real-time PCR
0/37
15/37
70 /89
77 /89
70/126
92/126
Conventional PCR
0/37
13/37
60a /89
68b /89
60/126
81/126
98.4%
98.4%
Agreements +
Notes: , positive for PPV or PCV. a Thirteen out of the 70 PPV-positive samples were co-infections with PCV. b Thirteen out of the 68 PCV-positive samples were co-infections with PPV.
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cost, sensitive and specific. These features make it an excellent tool for laboratory detection of PPV and PCV-2 in tissue cultured samples as well as in field samples such as fetal tissues. The high degree of sensitivity and specificity observed with the tissue culturepropagated virus suggests that the assay would be a useful tool for field investigation of PPV and PCV-2 infection. Acknowledgement This work was supported by Innovation Scientists and Technicians troop Construction Projects of Zhengzhou City, Henan, China (no. 10XTD148) and Key Sci-tech Project of Zhengzhou City, Henan, China (0910SGYN12319-5). References Allan, G.M., Kennedy, S., McNeilly, F., Foster, J.C., Ellis, J.A., Krakowka, S.J., Meehan, B.M., Adair, B.M., 1999. Experimental reproduction of severe wasting disease by co-infection of pigs with porcine circovirus and porcine parvovirus. Journal of Comparative Pathology 121, 1–11. Balint, A., Tenk, M., Deim, Z., Rasmussen, T.B., Uttenthal, A., Csagola, A., Tuboly, T., Farsang, A., Fossum, C., Timmusk, S., Berg, M., Belak, S., 2009. Development of primer-probe energy transfer real-time PCR for the detection and quantification of porcine circovirus type 2. Acta Veterinaria Hungarica 57, 441–452. Choi, C., Chae, C., 2000. Distribution of porcine parvovirus in porcine circovirus 2infected pigs with postweaning multisystemic wasting syndrome as shown by in situ hybridization. Journal of Comparative Pathology 123, 302–305. Dunne, H.W., Gobble, J.L., Hokanson, J.F., Kradel, D.C., Bubash, G.R., 1965. Porcine reproductive failure associated with a newly identified ‘SMEDI’ group of picorna viruses. American Journal of Veterinary Research 26, 1284–1297. Ellis, J.A., Bratanich, A., Clark, E.G., Allan, G., Meehan, B., Haines, D.M., Harding, J., West, K.H., Krakowka, S., Konoby, C., Hassard, L., Martin, K., McNeilly, F., 2000. Coinfection by porcine circoviruses and porcine parvovirus in pigs with naturally acquired postweaning multisystemic wasting syndrome. Journal of Veterinary Diagnostic Investigation 12, 21–27. Hong, Y.C., Xiao, K.L., Bao, A.C., Zhan, Y.W., Xin, S.L., Yan, B.W., Li, Z., Zhen, Y.W., 2009. A TaqMan-based real-time polymerase chain reaction for the detection of porcine parvovirus. Journal of Virological Methods 156, 84–88. Huang, C., Hung, J.J., Wu, C.Y., Chien, M.S., 2004. Multiplex PCR for rapid detection of pseudorabies virus, porcine parvovirus and porcine circoviruses. Veterinary Microbiology 101, 209–214. Jiang, Y., Shang, H., Xu, H., Zhu, L., Chen, W., Zhao, L., Fang, L., 2010. Simultaneous detection of porcine circovirus type 2, classical swine fever virus, porcine parvovirus and porcine reproductive and respiratory syndrome virus in pigs by multiplex polymerase chain reaction. Journal of Veterinary Research 183, 172–175. Kennedy, S., Moffett, D., McNeilly, F., Meehan, B., Ellis, J., Krakowka, S., Allan, G.M., 2000. Reproduction of lesions of postweaning multisystemic wasting syndrome by infection of conventional pigs with porcine circovirus type 2 alone or in combination with porcine parvovirus. Journal of Comparative Pathology 122, 9–24. Kim, J., Chae, C., 2001. Differentiation of porcine circovirus 1 and 2 in formalin-fixed, paraffin-wax-embedded tissues from pigs with postweaning multisystemic wasting syndrome by in situ hybridisation. Research in Veterinary Science 70, 265–269.
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Contents lists available at SciVerse ScienceDirect
Journal of Virological Methods journal homepage: www.elsevier.com/locate/jviromet
Simultaneous detection of porcine parvovirus and porcine circovirus type 2 by duplex real-time PCR and amplicon melting curve analysis using SYBR Green Lan-lan Zheng a,1 , Ya-bin Wang b,1 , Ming-feng Li a , Hong-ying Chen b , Xian-po Guo b , Jing-wei Geng a , Zhen-ya Wang b , Zhan-yong Wei a,∗ , Bao-an Cui b,∗ a b
The College of Animal Science and Veterinary Medicine, Henan Agricultural University, Zhengzhou, Henan 450002, China Key Laboratory for Animal-derived Food Safety of Henan Province, Zhengzhou, Henan 450002, China
a b s t r a c t Article history: Received 10 November 2011 Received in revised form 20 June 2012 Accepted 27 June 2012 Available online 4 July 2012 Keywords: Porcine parvovirus (PPV) Porcine circovirus type 2 (PCV-2) Duplex real-time PCR SYBR-Green
The development of a SYBR Green-based duplex real-time PCR is described for simultaneous detection of porcine parvovirus (PPV) and porcine circovirus type 2 (PCV-2) genomes. Viral genomes were identified in the same sample by their distinctive melting temperature (Tm ) which is 77.5 ◦ C for PPV VP2 313 bp amplicon and 82.3 ◦ C for PCV-2 ORF2 171 bp amplicon, respectively. The detection limit of the method was 0.01 TCID50 /mL for PPV and PCV-2, about 10 times more sensitive than conventional PCR. In addition, PPV and PCV-2 viral load were measured in 126 field samples, confirming the sensitivity and specificity, and the result showed that 70/126 samples were positive for PPV and 92/126 samples were positive for PCV2 by the duplex real-time PCR. This method may be a useful alternative rapid and reliable method for the detection of PPV/PCV-2 co-infection. © 2012 Elsevier B.V. All rights reserved.
1. Introduction Porcine circovirus (PCV) is a small, nonenveloped, singlestranded DNA virus with a circular genome and belongs to the Circoviridae family. In the late 1990s, PCV-2 was associated with the post-weaning multisystemic wasting syndrome (PMWS) which is characterized by progressive weight loss, respiratory signs, and jaundice. Depletion of lymphocytes in the lymphoid follicles and their replacement by macrophages are the hallmark lesions observed in this syndrome (Allan et al., 1999; Meehan et al., 1998). Porcine parvovirus (PPV) is a small non-enveloped single-stranded DNA virus of Parvoviridae, and is the major causative virus of the syndrome of reproductive failure in swine, which includes stillbirths and infertility (Dunne et al., 1965; Mengeling and Cutlip, 1976). Co-infection with PCV-2 and PPV has been demonstrated in a significant proportion of field cases of PMWS in pigs in many countries (Choi and Chae, 2000; Ellis et al., 2000; Kim and Chae, 2001; Kim et al., 2002; Yue et al., 2009). It has been shown experimentally that co-infection with PCV-2 and PPV causes more severe lesions and clinical disease in PMWS than infection with PCV-2 alone (Allan
∗ Corresponding authors at: College of Animal Science and Veterinary Medicine, Henan Agricultural University, Zhengzhou, Henan 450002, China. Tel.: +86 0 371 63554361; fax: +86 0 371 63558259. E-mail address: [email protected] (Z.-y. Wei). 1 The first and second author contributed equally to this work. 0166-0934/$ – see front matter © 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.jviromet.2012.06.024
et al., 1999; Kennedy et al., 2000; Krakowka et al., 2000; Yong et al., 2007). Designing a rapid and high throughput assay which is capable of detecting simultaneously the two agents will improve significantly the ability to identify the two diseases in swine herds rapidly. PCV-2 and PPV can be diagnosed by detecting the antibodies, viruses infected tissues and body fluids. However, there are several potential problems with these methods. Compared to the conventional detection methods, such as serology, immunohistochemistry and immunofluorescence for detecting antigen and the polymerase chain reaction (PCR) methods, real-time PCR technology is highly sensitive and specific, time-saving, easy to handle, and exhibits a decrease risk (Huang et al., 2004; Jiang et al., 2010; Ogawa et al., 2009). Individual real-time PCR assay has been developed for detection and identification of PCV-2 and PPV. Recently, multiplex real-time PCR, with melting curve analysis, has been described as a simple, reliable, and rapid method for detection and identification of certain bacteria, protozoa, and viruses (Balint et al., 2009; Lopez et al., 2009). The objectives of this study were to develop and validate a novel SYBR Green real-time PCR following by melting curve analysis of the amplicon, for the simultaneous detection of PCV-2 and PPV, and to investigate the effects of diagnostic specimen choice on its performance. The method established in current study could be used to detect PCV-2 and PPV in nasal swabs, tissues (lymph nodes, spleens, lungs and livers) and semen samples from naturally infected conventional pigs in different pig farms.
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2. Materials and methods
2.5. Duplex SYBR Green real-time PCR
2.1. Viruses and clinical specimens
The 171-bp PCV2-ORF2 specific DNA fragment and 313-bp PPVVP2 specific DNA fragment were amplified and inserted into the pGEM-T easy vector respectively to obtain the recombinant plasmids. According to the optimized reaction condition of Section 2.4, the duplex SYBR Green real-time PCR was amplified. Fluorescence was measured after extension at each cycle. All real-time reactions (standards, unknown samples, and controls) were performed in duplicate in neighboring wells on the sample plate. Results reported are an average of the duplicates.
The PPV standard strain 7909 was purchased from the China Institute of Veterinary Drug Control, Beijing, China. The PCV-2 HN strain was isolated from clinical specimens, and identified in the Key Laboratory for Animal-derived Food Safety of Henan Province, China. These viruses were propagated in PK-15 cells (purchased from China Institute of Veterinary Drug Control, China). Pseudorabies virus (PRV), Japanese encepphenia virus (JEV), Porcine reproductive and respiratory syndrome virus (PRRSV), Porcine circovirus type 1 (PCV-1) and Classical swine fever virus (CFSV) were provided by Key Laboratory for Animal-derived Food Safety of Henan Province, China. A total of 126 field samples were collected in different pig farms in several provinces of China in the period of May 2007 to July 2009, which included 46 tissue samples (lymph nodes, spleens, lungs and livers) and 8 nasal swab samples collected from diseased pigs and 72 semen samples obtained from clinically normal pigs. The sickness status was diagnosed based on clinical symptoms and pathological representations. 2.2. Template DNA preparations Viral genomic DNA was extracted either from 500 L lysates of PK-15 cells infected with each virus strain, or from 20 mg of frozen clinical specimens by using a commercial DNA extraction kit (QIAamp DNA Mini Kit, Qiagen, Hilden, Germany) according to the manufacturer’s direction. The isolated DNA was calculated and stored at −70 ◦ C. 2.3. Primers design and synthesize The VP2 nucleotide sequences of PPV strains/isolates and ORF2 nucleotide sequences of PCV-2 were retrieved from the GenBank and aligned using the software program DNAStar (DNASTAR, Inc., Madison). The primers were selected using the Primer Express (version 2.0) software and were based on a highly conserved sequence within the VP2 region of the PPV genome and the ORF2 region of the PCV-2. The primer pairs (forward primer: 5 -ATG GCG GGA GGA GTA GTT T-3 ; reverse primer: 5 -CCC TTT GAA TAC TAC AGC G3 ) used to amplify a fragment with a length of 171 bp for PCV-2 (GenBank Accession number AF027217). The primer pairs (forward primer: 5 -GGG CTT GGT TAG AAT CAC-3 ; reverse primer: 5 -TGG TGG TGA GGT TGC TGA T-3 ) used to amplify a fragment with a length of 313 bp for PPV (GenBank Accession number NC001718). The primers were designed and synthesized by Takara, China. 2.4. Optimization of the PPV/PCV-2 duplex SYBR Green real-time PCR assay A number of experiments were performed to optimize the PPV/PCV-2 duplex SYBR Green real-time PCR protocol, including reagent concentration and PCR cycling parameters. The primers as well as SYBR PreMix were titrated in chequerboard assays to determine the reagents concentrations that yielded the best results. Different annealing/elongation temperatures and times were evaluated. The following parameters were used in the duplex real-time PCR. Each reaction mix in a total volume of 25 L, including 2 L DNA, 12.5 L 2 × SYBR Green PreMix, 1 L of each primer. The reaction condition was: pre-denaturation at 95 ◦ C for 3 min, 40 cycles of denaturation at 95 ◦ C for 15 s, annealing at 55 ◦ C for 10 s, and extension at 72 ◦ C for 15 s.
2.6. Melt curve analysis A melt curve analysis was performed after each run, with the mixture being cooled of 45 s at 60 ◦ C, and then heated in one-degree increments at 95 ◦ C. Fluorescence was measured for 15 s at each increment and a graph plotting the second derivative of the melt displayed. The melting peaks were analyzed to distinguish PCV2 and/or PPV specific amplicons. Fluorescence normalization and data analysis were performed by thermal cycler program software (MxPro-Mx3005P v.3.00; Stratagene, La Jolla, CA).
2.7. Sensitivity, specificity and reproducibility of the real-time PCR A 1-mL aliquot of a culture solution of PCV-2 and PPV with a titer of 107 TCID50 /mL was subjected to a 10-fold serial dilution in PBS, with nine dilutions. The DNA of each dilution was extracted and subjected to duplex real-time PCR using the established amplification conditions in Section 2.5. The minimum concentration which can be detected will provide the sensitivity of the real-time PCR. To determine the analytical specificities of the PCR protocols, other five porcine (PRRSV, CSFV, PRV, PCV-1 and JEV) viral pathogens were tested. The intra-assay and inter-assay reproducibilities were evaluated using high, intermediate and low input of the standard PPV and PCV-2 plasmid preparations. The coefficients of variation for CT value obtained for each dilution were calculated. To determine the reproducibility of the real-time PCR, the standard plasmid was diluted to 1 × 107 , 1 × 106 , 1 × 105 , 1 × 104 , 1 × 103 , 1 × 102 , 1 × 101 , and 1 × 100 copies and tested by three different people at six different times per person. The variation among the three testers at each time was analyzed.
2.8. Data analyses After a SYBR Green PCR run, data acquisition and subsequent data analyses were done using the 5700 Sequence Detection System (SDS Version 1.3). In the system, the fluorescence of SYBR Green against the internal passive reference dye, ROX (Rn) is measured at the end of each cycle. A sample is considered positive when Rn exceeds the threshold value. The threshold value is set at the midpoint of Rn vs. cycle number plot. For all the amplifications described in this paper, the threshold value of Rn was taken as 0.25. The threshold cycle (C) is defined as the cycle at which a statistically significant increase in Rn is first detected. Target cDNA copy number and CT values are related inversely. A sample containing higher copies of the target cDNA will cross the threshold at an earlier cycle, compared to a sample with lower copies of the same target. The copy numbers of PCV-2 and PPV samples were determined by normalizing the CT values of the samples and then extrapolating the normalized CT values to the standard curve of the virus. For further statistical analyses, the CT values were exported into a Microsoft Excel Worksheet. Regression analyses of the CT
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Fig. 2. The PCR amplification of PPV VP2. Fig. 1. The PCR amplification of PCV-2 ORF2.
values of the DNA dilution series were used to determine the amplification efficiency for PCV-2 and PPV. 2.9. Conventional PCR Viral DNA sequences were amplified by conventional PCR as described previously (Kim et al., 2001; Wu et al., 2008). Amplification of DNA for PCV-2-171 bp was achieved by 3 min at 95 ◦ C, followed by 40 cycles of 15 s at 95 ◦ C, 1 min at 55 ◦ C, and 1 min at 72 ◦ C, and a final extension of 5 min at 72 ◦ C. Amplification for PPV-313 bp was performed similarly, but with an annealing temperature of 57 ◦ C. The reactions were performed in triplicate in a DNA Engine (PTC-200) thermocycler, and amplicons were visualized by ethidium bromide staining on a 1.5% agarose gel with a DNA ladder. To ensure the quality of data, negative and positive controls were used in each PCR reaction. 2.10. Detection of filed samples A total of 126 field samples were tested in parallel by the duplex real-time PCR and conventional PCR. Ten positive and ten negative samples were selected at random, and subjected to virus isolation to validate the reliability of the duplex real-time PCR. 3. Results
curve analysis. Hence, PCV-2 and PPV amplicons can easily be distinguished by specific Tm values due to the different lengths and compositions of two amplicons. Melting curve analysis revealed that the PCV-2 melting curve displays a Tm of 82.3 ◦ C, whereas the PPV curve showed a Tm of 77.5 ◦ C (Fig. 3). 3.3. Sensitivity of the duplex real-time PCR The analytical sensitivity of SYBR Green PCR was determined by using a serial dilution of PCV-2 and PPV plasmid DNA as template for amplification. A linear relationship that between the input plasmid DNA and the CT values with regression coefficient (r2 ) which greater than 0.99 was obtained for both the viruses. The detection limits of the duplex real-time PCR was determined to be 175 copies/L for PPV and 156 copies/L for PCV-2, equivalent to 0.01 TCID50 /mL respectively, which are about 5–10 times more sensitive than those of the conventional PCRs (0.1 TCID50 /mL for PPV and PCV-2, respectively), and comparable to that of the PCR (0.03 and 0.05 TCID50 /mL for PPV and PCV-2, respectively) (Kim et al., 2001). 3.4. Specificity of the real-time PCR The assay was specific for the PPV and PCV-2. No specific amplification was detected with other porcine viruses such as PRV, JEV, PRRSV, PCV-1, and CFSV. In addition, the Tm of PPV and PCV-2 was recorded as 77.5 ◦ C and 82.3 ◦ C, respectively, and other viruses showed no specific melting peak (Fig. 4). The specific amplifications
3.1. The construction of standard templets of PPV and PCV-2 The 171 bp PCV-2-ORF2 specific DNA fragment and 313 bp PPVVP2 specific DNA fragment were amplified (Figs. 1 and 2) and inserted into the pGEM-T easy vector respectively to obtain the recombinant plasmids. The recombinant plasmids were used as the standard templets of PPV and PCV-2, which are required for the standard curve calculation in quantization assays. 3.2. Real-time PCR strategies optimization and melting curve analysis The procedure of real-time PCR was optimized according to the concentrations of primers and SYBR Green master mixture, the denature/extension temperature, and reaction time (data not shown). Duplex real-time PCR fragments are detected by melting
Fig. 3. Melting curve analysis for the duplex SYBR Green I based real-time PCR.
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Fig. 4. Melting curve analysis of the specificity of the duplex SYBR Green I based real-time PCR assay.
were also confirmed by the detection specific bands of 313 bp for PPV and 171 bp for PCV-2 by agarose gel electrophoresis. No bands of the expected sizes were observed from other viruses real-time PCR products (data not shown). 3.5. Reproducibility of the real-time PCR The standard plasmid was serially diluted and then subjected to the real-time PCR, results obtained by three people at six times were similar. The inter-assay variation coefficient (the variation among the three testers) was 1.34, 1.29, 1.40 for each of the six times. The coefficients of variation (CVs) of CT values for each input DNA copy number between different intra-assay and inter-assay runs were less than 2% for both PPV and PCV-2 detection channels. 3.6. Agreement between the duplex real-time PCR and conventional PCRs Among the 126 field samples tested, the duplex real-time PCR showed 70 samples (all from clinically diseased pigs) to contain PPV and 77 samples to contain PCV-2, while the conventional PCRs tested 60 samples positive for PPV and 68 positive for PCV-2 (Table 1). The agreement between the duplex real-time PCR and the conventional PCRs was 98.4% for PPV and PCV-2. 4. Discussion PCV-2 and PPV have caused great loss in porcine industry in the world, and co-infection of the two viruses has been demonstrated in a significant proportion of field cases of PMWS in pigs in many countries of the world. Rapid and accurate methods for detection are essential in identification of PCV-2 and PPV. However, to date, few publications have reported on the duplex PCR enabling differentiation between PPV and PCV-2. Previous studies have demonstrated detection of PCV2 and PPV using nuclease probes in the Key Laboratory for Animal-derived
Food Safety of Henan Province laboratory (Hong et al., 2009). However, use of probes can add significant cost to an assay comprising multiple gene targets and is highly influenced by the potential presence of mutations within the probe-binding site that can prevent annealing of the probe and subsequent detection. Alternatively, intercalating dyes such as SYBR Green can be used to detect the presence of generated PCR fragments and is considered to be a flexible approach that can be directly applied to any gene without the need to design and synthesize fluorescently labeled target-specific probes. Hence, the reasons why propose the assay as an alternative method to other tests, particularly for large scale PCR screening, are that the SYBR Green PCR method was not influenced by the different genetic backgrounds. Amplicons can be distinguished by melt curve analysis, whereby the Tm of PCR products is determined by the reduction in relative fluorescence as all dsDNA is denatured to their single-stranded form. The shape and peak location of the melt curve are functions of the GC/AT ratio, length, and sequence of the fragment (Ririe et al., 1997; Wehrle et al., 2009). Detection with SYBR Green is as sensitive as probe-based real-time PCR without the need for a specialized probe. The duplex SYBR Green real-time PCR is more sensitive compared to routine methods such as virus isolation which is time consuming and labor intensive (Kim et al., 2001) and nested PCR (nPCR) assays (Kim et al., 2001; Larochelle et al., 2000; McIntosh et al., 2006; Varga and James, 2005; Wilhelm et al., 2005). These assays can amplify a group of 3–5 genes simultaneously. This study describes the development of a SYBR Green-based duplex real-time PCR for the simultaneous detection of PPV and PCV-2 genomes in a single reaction vessel. The assay was proved to be sensitive, specific, and reproducible. No specific amplification was found for several clinically relevant porcine viruses other than PCV-2 and PPV within 40 cycles in the duplex real-time PCR demonstrating the high specificity of the assay. An inherent limitation in designing a multiplex real-time PCR assay is loss of sensitivity when combining a large number of primer sets in a single reaction. In this study, the reaction conditions were carefully optimized to obtain maximum sensitivity (data not shown). As a result, despite the presence of two primers set in the PCR reaction mixture, the multiplex PCR was able to detect all viruses tested at a high level of sensitivity. The real-time PCR was able to detect as low as 0.01 TCID50 /mL of PPV and PCV-2, which were at least 10 times more sensitive than the conventional PCRs. The result of preliminary clinical application indicated that PPV and PCV-2 co-infection is probably broad prevalent in the swine herds. In addition, 72 semen samples collected from clinically “normal” pigs in small scale herds. 37/72 were positive for PPV, 33/72 were positive for PCV-2 and 18/72 were positive for co-infection implying a potential problem which should not be ignored. Simultaneous detection and differentiation of the two viruses should be incorporated in the disease control of swine herds in China. In conclusion, the SYBR Green-based real-time PCR assay described in this study for the simultaneous detection of PPV and PCV-2 has been shown to be rapid, easy to handle, relatively low
Table 1 Agreements between the duplex real-time PCR and conventional PCRs. Samples from clinically normal pigs
Samples from diseased pigs
Total
PPV+
PPV+
PPV+
PCV2+
PCV2+
a
PCV2+ b
Duplex real-time PCR
0/37
15/37
70 /89
77 /89
70/126
92/126
Conventional PCR
0/37
13/37
60a /89
68b /89
60/126
81/126
98.4%
98.4%
Agreements +
Notes: , positive for PPV or PCV. a Thirteen out of the 70 PPV-positive samples were co-infections with PCV. b Thirteen out of the 68 PCV-positive samples were co-infections with PPV.
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cost, sensitive and specific. These features make it an excellent tool for laboratory detection of PPV and PCV-2 in tissue cultured samples as well as in field samples such as fetal tissues. The high degree of sensitivity and specificity observed with the tissue culturepropagated virus suggests that the assay would be a useful tool for field investigation of PPV and PCV-2 infection. Acknowledgement This work was supported by Innovation Scientists and Technicians troop Construction Projects of Zhengzhou City, Henan, China (no. 10XTD148) and Key Sci-tech Project of Zhengzhou City, Henan, China (0910SGYN12319-5). References Allan, G.M., Kennedy, S., McNeilly, F., Foster, J.C., Ellis, J.A., Krakowka, S.J., Meehan, B.M., Adair, B.M., 1999. Experimental reproduction of severe wasting disease by co-infection of pigs with porcine circovirus and porcine parvovirus. Journal of Comparative Pathology 121, 1–11. Balint, A., Tenk, M., Deim, Z., Rasmussen, T.B., Uttenthal, A., Csagola, A., Tuboly, T., Farsang, A., Fossum, C., Timmusk, S., Berg, M., Belak, S., 2009. Development of primer-probe energy transfer real-time PCR for the detection and quantification of porcine circovirus type 2. Acta Veterinaria Hungarica 57, 441–452. Choi, C., Chae, C., 2000. Distribution of porcine parvovirus in porcine circovirus 2infected pigs with postweaning multisystemic wasting syndrome as shown by in situ hybridization. Journal of Comparative Pathology 123, 302–305. Dunne, H.W., Gobble, J.L., Hokanson, J.F., Kradel, D.C., Bubash, G.R., 1965. Porcine reproductive failure associated with a newly identified ‘SMEDI’ group of picorna viruses. American Journal of Veterinary Research 26, 1284–1297. Ellis, J.A., Bratanich, A., Clark, E.G., Allan, G., Meehan, B., Haines, D.M., Harding, J., West, K.H., Krakowka, S., Konoby, C., Hassard, L., Martin, K., McNeilly, F., 2000. Coinfection by porcine circoviruses and porcine parvovirus in pigs with naturally acquired postweaning multisystemic wasting syndrome. Journal of Veterinary Diagnostic Investigation 12, 21–27. Hong, Y.C., Xiao, K.L., Bao, A.C., Zhan, Y.W., Xin, S.L., Yan, B.W., Li, Z., Zhen, Y.W., 2009. A TaqMan-based real-time polymerase chain reaction for the detection of porcine parvovirus. Journal of Virological Methods 156, 84–88. Huang, C., Hung, J.J., Wu, C.Y., Chien, M.S., 2004. Multiplex PCR for rapid detection of pseudorabies virus, porcine parvovirus and porcine circoviruses. Veterinary Microbiology 101, 209–214. Jiang, Y., Shang, H., Xu, H., Zhu, L., Chen, W., Zhao, L., Fang, L., 2010. Simultaneous detection of porcine circovirus type 2, classical swine fever virus, porcine parvovirus and porcine reproductive and respiratory syndrome virus in pigs by multiplex polymerase chain reaction. Journal of Veterinary Research 183, 172–175. Kennedy, S., Moffett, D., McNeilly, F., Meehan, B., Ellis, J., Krakowka, S., Allan, G.M., 2000. Reproduction of lesions of postweaning multisystemic wasting syndrome by infection of conventional pigs with porcine circovirus type 2 alone or in combination with porcine parvovirus. Journal of Comparative Pathology 122, 9–24. Kim, J., Chae, C., 2001. Differentiation of porcine circovirus 1 and 2 in formalin-fixed, paraffin-wax-embedded tissues from pigs with postweaning multisystemic wasting syndrome by in situ hybridisation. Research in Veterinary Science 70, 265–269.
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