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A TaqMan-based real-time polymerase chain reaction for the detection of porcine parvovirus
Journal of Virological Methods 156 (2009) 84–88
Contents lists available at ScienceDirect
Journal of Virological Methods journal homepage: www.elsevier.com/locate/jviromet
A TaqMan-based real-time polymerase chain reaction for the detection of porcine parvovirus Hong-Ying Chen a,1 , Xiao-Kang Li b,1 , Bao-An Cui a,∗ , Zhan-Yong Wei a , Xin-Sheng Li a , Yan-Bin Wang a , Li Zhao a , Zhen-Ya Wang a a b
College of Animal Husbandry and Veterinary, Henan Agricultural University, Zhengzhou 450002, China College of Veterinary Medicine, South China Agricultural University, Guangzhou 510642, China
a b s t r a c t Article history: Received 6 February 2008 Received in revised form 7 October 2008 Accepted 21 October 2008 Available online 17 December 2008 Keywords: Porcine parvovirus Real-time PCR VP2 TaqMan Quantitation
A real-time polymerase chain reaction (PCR) using a TaqMan probe was developed to detect porcine parvovirus (PPV). Real-time PCR was optimized to quantify PPV using a detection system (Rotor Gene 2000 detector) and a dual-labeled fluorogenic probe. The gene-specific labeled fluorogenic probe for the VP2 gene of PPV was used to detect PPV. Quantitation of PPV was accomplished by a standard curve plotting cycle threshold values (Ct) against each dilution of standard plasmids. When the specificity of the assay using specific PPV primers was evaluated by testing the PPV standard strain and other viruses, no cross-reactions were detected with non-PPV reference viruses. The detection limit of real-time PCR for PPV was 2.08 log 10 genome copy equivalent (gce). In this study, a real-time PCR assay was performed on 80 clinical samples and compared with a conventional PCR assay. In 48 of 80 samples, PPV DNA was detected by the conventional PCR assay. All samples positive for PPV DNA by the conventional PCR assay were also positive by the real-time PCR assay, and 12 of 32 samples that tested negative for PPV DNA by the conventional method tested positive by the real-time PCR assay. Using the real-time PCR assay, the number of samples in which PPV was detected increased by 15%. Therefore, it is considered to be a useful tool for the detection of PPV. © 2008 Elsevier B.V. All rights reserved.
1. Introduction Porcine parvovirus (PPV) is an autonomous parvovirus belonging to the genus parvovirus, subfamily Parvovirinae, family Parvoviridae; it is the major causative virus in a reproductive failure syndrome in swine characterized by stillbirths, mummified fetuses, early embryonic death, and infertility. In addition, PPV has been implicated as the causative agent of diarrhea, skin disease and arthritis in swine. After PPV was isolated by Cartwright in 1967 from herd infertility, abortion, and stillbirth in pigs (Cartwright and Huck, 1967), PPV has been reported to occur worldwide with variable prevalence rates; it always causes recessive infection, which presents many challenges for diagnosis. Virus isolation is a fundamental diagnostic method, but PPV isolation in cell cultures is laborious and cannot be achieved for all PPV strains (Kim and Chae, 2004). Other diagnostic techniques, such as the hemagglutination (HA), enzyme-linked immunosorbent assay, immunofluorescence, in situ hybridization, conventional poly-
∗ Corresponding author. Tel.: +86 371 63558878; fax: +86 371 63558878. E-mail address: [email protected] (B.-A. Cui). 1 The first and second author contributed equally to this work. 0166-0934/$ – see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.jviromet.2008.10.029
merase chain reaction (PCR), and nested PCR have been developed. Comparing these techniques, nested PCR, and in situ hybridization were shown to be more sensitive than conventional PCR and the HA test (Kim and Chae, 2003; Soares et al., 1999). However, the nested PCR requires agarose gel analysis for the detection of amplification products and in situ hybridization may require several days to complete. Therefore, both assays are labor-intensive, and nested PCR has a very high risk of contamination. Recently, a real-time PCR has been used extensively for detection of amplicons that are amplified during the PCR cycling in real time (Olvera et al., 2004; Yoon et al., 2005; Ellis et al., 2007). Real-time PCR is an excellent diagnostic tool with high sensitivity, specificity, and a fast turnaround time. This system is called real-time PCR because the accumulated amplicons can be monitored directly during the DNA amplification process in real time. The development of fluorogenic PCR utilizing the 5 –3 nuclease activity of Taq DNA polymerase made it possible eliminate post-PCR processing such as visualization in agarose (Holland et al., 1991; Warrilow et al., 2002). In addition, the real-time PCR technique has been shown to provide good sensitivity and a linear relationship between the copy number and cycle threshold (Ct) values. The technique uses oligonucleotide probes labeled with fluorescent dyes, a reporter at the 5 end, and a quencher at the 3 end to monitor the accumulation of PCR products
H.-Y. Chen et al. / Journal of Virological Methods 156 (2009) 84–88
(Bustin, 2002). The quantitation of DNA is based on the determination of the threshold cycle when the amplified PCR product is first detected. The higher the initial DNA copy number input, the sooner the product of amplification is detected. Reports on virus detection based on real-time PCR technology have been described for various animal viruses (Schwaiger and Cassinotti, 2003; Yang et al., 2004; Yoon et al., 2005; Kang et al., 2007). Molecular beacon realtime PCR for the detection of PPV has been developed (McKillen et al., 2007); however, very few studies of a real-time PCR assay with a TaqMan probe for the detection of PPV exist. Therefore, a real-time PCR assay with a TaqMan probe was investigated in this study and used for laboratory detection and quantitation of PPV. In addition, the applicability of the real-time PCR was evaluated for the detection of PPV DNA in aborted pig fetuses. 2. Materials and methods 2.1. Viruses and sample preparation The PPV standard strain 7909 was purchased from Beijing Ordinary Microbiology Strain Store Center, Beijing, China and used as a standard virus for the detection of PPV by real-time PCR. The PPV 7909 strain was propagated in the PK-15 porcine kidney cell line. The porcine circovirus (PCV-2), porcine reproductive and respiratory syndrome virus (PRRSV), pseudorabies virus (PRV), Japanese encephalitis virus (JEV), canine parvovirus (CPV) and feline panleukopenia virus (FPV) were used as reference strains for the specificity test of the real-time PCR. In 2007, 80 fetuses that spontaneously aborted before 70 days of gestation were collected from several provinces in China and necropsied for determination of the reason for abortion. Tissue samples of the colon, duodenum, jejunum, heart, kidneys, liver, lung, spleen, thymus, and gonads were stored separately at −80 ◦ C. Viral DNA was extracted using a commercial test kit (QIAamp DNA Mini Kit, Qiagen, Hilden, Germany) according to the manufacturer’s instructions and stored at −20 ◦ C until needed. 2.2. Design of primers and a probe for real-time PCR The VP2 nucleotide sequences of PPV strains/isolates were retrieved from the GenBank and aligned using the software program DNAStar (DNASTAR, Inc., Madison). The accession numbers of the strains/isolates used for the alignment were the following: AY583318, U44978, AY390557, AY686602, AY459350, AY502115, AY597052, AY781130, AY786299, AY786300 (NJ-1), AY786301, AY786302, AY786303, AY788086, AY788087, AY788088, and AY788089. The primers and probe set 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. The primers were designed to detect the available PPV sequences in the relevant VP2 region (sense 5 -CCAAAAATGCAAACCCCAATA3 , antisense 5 -TCTGGCGGTGTTGGAGTTAAG-3 ) and amplify a fragment with a length of 194 bp. The TaqMan probe, FAMCTTGGAGCCGTGGAGCGAGCC-TAMRA, was used to detect any amplifications. This probe was labeled with a fluorescent reporter dye (FAM: 6-carboxyfluorescein) at the 5 end and a quencher dye (TAMRA: 6-carboxyteramethy-rhodamine) at the 3 end. 2.3. Construction of standard plasmids for real-time PCR To generate a PPV standard curve for the real-time reaction, a PCR product containing 751 bp covering the region of interest of VP2 and using the oligonucleotide primers PPV-1 (5 -ATACTTGGGGGAGGGCTT-3 ) and PPV-2 (5 TGTTCCTGGGTGTTGGTC-3 ) was cloned into the vector pGEM-T
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Easy vector according to the instructions of the manufacturer (Promega). The resulting plasmid, pPPV, was used to transform Escherichia coli DH5␣ cells and was purified using a QIAGEN plasmid purification kit (Qiagen Ltd., Hilden, Germany) according to the manufacturer’s instructions. The concentration of the plasmid preparation was determined by measuring the OD at 260 nm using a spectrophotometer (Thermo Electron, USA). Serial 10-fold dilutions of plasmid DNA in TE buffer (10 mM Tris–HCl, 1 mM EDTA) were used in the amplification reactions. The dilutions were stored at −20 ◦ C; stock plasmid was stored at −70 ◦ C. 2.4. Real-time PCR Real-time PCR assays using the TaqMan probe were carried out in a micro-reaction tube (Corbett Research, Australia). The reaction mixture for each tube of TaqMan reaction mix consisted of 2.5 l of 10× buffer, 0.5 l of 10 mM dNTP, 4.5 l of 25 mM MgCl2 , 0.4 l of 50 M fluorogenic FAM-labeled PPV probe, 0.5 l of 50 M forward primer, 0.5 l of 50 M reverse primer, 1 l of DNA solution, 14.1 l of distilled water, and 1 l of Ex Taq DNA (TaKaRa, Dalian, China) to produce a total volume of 25 l. The thermal profile for the realtime PCR was 94 ◦ C for 5 min, followed by 40 cycles of 94 ◦ C for 5 s and 60 ◦ C for 15 s. PCR amplification was performed by using the Rotor Gene 2000 real-time thermal cycler (Corbett Research, Australia). Positive and negative reference samples were tested along with the unknown samples in each run. 2.5. Conventional PCR Viral DNA sequences were amplified by conventional PCR as described previously (Kim and Chae, 2003) using the oligonucleotide primers PPV-3 (5 -GGGGAGGGCTTGGTTAGAAT-3 ) and PPV-4 (5 -TGGTTGGTGGTGAGGTTGCT-3 ). Five-l samples of the supernatant containing extracted DNA were used as the PCR templates. The amplification was performed in a 50-l reaction mixture containing 1× PCR buffer, 4 mM MgCl2 , 200 M of each dNTP, 1 M of each primer, and 1U of Taq DNA polymerase (TaKaRa, Dalian, China). The reaction was run in a PCT-200 Peltier thermal cycler (MJ Research, USA) under the following conditions: one cycle at 96 ◦ C 3 min, followed by 25 cycles of denaturation at 94 ◦ C for 30 s, primer annealing at 50 ◦ C for 30 s, and extension at 72 ◦ C for 30 s. PCR was ended with a final extension step at 72 ◦ C for 10 min. The reactions were performed in triplicate. To ensure the quality of data, negative and positive controls were used in each PCR reaction. DNA extraction and amplification were performed in different rooms. 3. Results 3.1. PPV DNA standard and optimization strategies for real-time PCR The recombinant plasmid pPPV has been constructed as the standard of PPV DNA, which is required for the standard curve calculation in quantitation assays. The constructed plasmid is 3766 bp. Conversion to genome equivalents was calculated as 1 OD260 equals 50 g/ml and one base pair equals 660 g/mol, resulting in a molecular weight for the plasmid (3766 bp) of 2.49 × 106 pg/pmol. One genome copy equivalent (gce) of PPV equals 4 attograms (ag) of this plasmid. The procedure was optimized with regard to the concentrations of primers and the denature/extension temperature. The concentration of primers, magnesium, and probe giving the highest fluorescence and the lowest threshold cycle were selected as
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H.-Y. Chen et al. / Journal of Virological Methods 156 (2009) 84–88
Fig. 1. Real-time PCR standard curve generated from plasmid DNA amplification plots. Standard curve was plotted in the sample plasmid on the x-axis and cycle threshold (Ct) on the y-axis. The x-axis represents pPPV in 10-fold dilutions and the y-axis the fluorescence data used for Ct determinations in dRn (baseline-corrected normalized fluorescence). The assays were linear over a 108 dilution range of pPPV with R2 values (square of the correlation coefficient) of 0.99 and reaction efficiencies of 93.4%.
Fig. 2. Specificity of real-time PCR. No cross-reactions were detected with PCV-2, PRRSV, PRV, JEV, CPV, FPV, and PK15 cells.
follows: 1 M forward and reverse primers, 4.5 mM MgCl2 , and 0.8 M probe. 3.2. Standard curve for quantitation of PPV Real-time PCR amplifications were performed with serial dilutions (101 to 109 gce) of standard plasmids to assess the quantitation assay. Samples were tested three times. The result showed that a detectable fluorescence signal above the threshold occurred at 16.38 cycles from the amplification profile with number of cycles versus normalized fluorescence values (data not shown). The Rotor Gene detection system software generated a standard curve by plotting the Ct values against each standard dilution of standard plasmids. Fig. 1 shows the real-time PCR standard curve generated from plasmid DNA amplification plots. A linear standard curve was obtained from 1 × 104 to 1 × 108 gce per reaction mixture, which resulted in Ct values ranging from 16.38 to 29.84 cycles. The assays were linear over a 108 dilution range of template DNA with R2 values (square of the correlation coefficient) of 0.99 and reaction efficiencies of 93.4%. 3.3. Specificity of the assay When several reference viruses were screened by the TaqMan probe under the optimal conditions of the assay, the 7909 strain was positive according to real-time PCR. As shown in Fig. 2, no crossreactions were detected with PCV-2, PRRSV, PRV, JEV, CPV, FPV, and PK 5 cells.
Fig. 3. Sensitivity of real-time PCR. When the extracted DNA was diluted serially 10-fold with PBS to 10−7 , fluorescence was detected, no reaction was detected at 10−8 .
tivity titer equivalent. DNA concentrations extrapolated from the standard curve were compared with the titers determined in the parallel TCID50 experiment (data not shown). One TCID50 equaled 1.836 ± 0.31 log10 gce. Therefore, the detection limit of real-time PCR was 2.08 log10 gce. 3.5. Reproducibility of the real-time PCR for PPV When the standard PPV plasmid DNA was used for the evaluation of the coefficients of variation (CVs) of the real-time PCR, the intra- and inter-assay CVs for Ct values ranged between 0.35 and 0.81%, and 0.30 and 1.32%, respectively (Table 1).
3.4. Sensitivity of the assay In order to determine the detection limit and efficiency of the assay, the titer of a PPV suspension was determined by the standard cell-based PPV-specific 50% tissue culture infectious dose (TCID50 ) assay and was 106.8 TCID50 /ml. The DNA of PPV was extracted and used as a template. The extracted DNA was diluted serially 10-fold with phosphate-buffered saline solution (PBS) up to 10−8 . When the extracted DNA was diluted to 10−7 , fluorescence was detected; no reaction was detected at 10−8 (Fig. 3). Sensitivity of the PCR assay for the detection of PPV was expressed as the infec-
3.6. Comparison of results obtained by real-time PCR and conventional PCR Twenty-five of eighty samples were positive by HA for PPV. All samples positive by this test were also positive in real-time and conventional PCR assays. The compared results of the real-time and conventional PCR assays are shown in Table 2. All samples positive by the conventional PCR for PPV DNA were also positive by the realtime PCR assay; whereas, 12 of the samples negative for PPV DNA by the conventional PCR assays were positive in the real-time PCR
Table 1 Variance analysis of Ct values quantified by real-time PCR in serially diluted standard plasmid solutions. Concentration of standard plasmid (copies/ml)
n
Intra-assay variability Ct
107 105 103
10 10 10
Inter-assay variability CV (%)
Mean
S.D.
12.71 20.12 27.72
0.08 0.07 0.22
0.63 0.35 0.81
Ct
CV (%)
Mean
S.D.
12.90 19.97 27.48
0.17 0.06 0.16
1.32 0.30 0.58
H.-Y. Chen et al. / Journal of Virological Methods 156 (2009) 84–88 Table 2 Comparison of real-time PCR and conventional PCR. Conventional PCR assay result (n)
Real-time PCR assay result (n) Positive
Negative
Total
Positive Negative Total
48 12 60
0 20 20
48 32 80
assays. The copy number of PPV DNA in tissue samples that was positive by both methods ranged from 1 × 105 to 1 × 1012 gce per 500 ng DNA. The copy number of the PPV DNA of 12 liver samples, which were positive by the real-time PCR assay but negative by the conventional PCR assay, was 1 × 103 to 1 × 105 gce per 500 ng DNA. 4. Discussion In this study, real-time PCR for laboratory detection of PPV was investigated in different samples. Specificity, sensitivity and quantitative range of real-time PCR were also evaluated. The established real-time PCR assay for PPV DNA quantitation was linear over a 108 dilution range of template concentrations with a R2 value (the square of the correlation coefficient) of 0.99 and a reaction efficiency of 93.4% (Fig. 1). The detection limit for the real-time PCR was shown to be 2.08 log10 gce. Porcine pathogens such as PRV, PCV-2, PRRSV, and other parvoviruses like FPV and CPV showed no Ct, and were therefore not detected by this assay. When compared to the previous reports for the detection of PPV (Molitor et al., 1991; Soares et al., 1999), the real-time PCR method has several advantages over conventional PCR. Firstly, real-time PCR yields a more rapid and sensitive test result than conventional PCR (Warrilow et al., 2002; Schwaiger and Cassinotti, 2003). Secondly, real-time PCR is less likely to produce a false positive due to contamination during the sample preparation process. Thirdly, real-time PCR can save time, because a conventional PCR assay requires post-PCR analysis such as gel electrophoresis, but the amplification of a specific PCR product in real-time PCR is measured in real time during PCR cycling. For precise estimation of viral loads in samples, a competitive quantitative PCR was applied that can detect and quantify target DNA simultaneously (Liu et al., 2000); however, it requires an exogenous competitor as a control, which must be constructed and characterized, and it requires post-PCR electrophoresis. Realtime PCR is performed in a closed tube and requires no post-PCR electrophoresis. Therefore, contamination with amplicons can be avoided. This assay is more reproducible and convenient than competitive quantitative PCR tests. The SYBR Green I-based real-time PCR is used to detect PPV (Wilhelm et al., 2006); the limitation of this method relates to false positives because SYBR Green I dye can bind to any double-stranded DNA, even non-specific amplicons. However, TaqMan probes and molecular beacons provide the further specificity needed for diagnostic PCR. The molecular beacon method utilizes a reporter probe that is wrapped around into a hairpin shape. It also has a quencher dye that must be in close contact to the reporter to work. An important difference of the molecular beacon method compared to the TaqMan method is that the probe remains intact throughout the PCR product, and is rebound to the target at every cycle. It has been suggested that the TaqMan probe and molecular beacon possess a similar sensitivity for the detection of antibiotic resistance-conferring single nucleotide polymorphisms in mixed Mycobacterium 199 tuberculosis (Yesilkaya et al., 2006). The assay was able to detect PPV in all samples shown to be PPV positive by the conventional PCR and the HA test. The comparison of real-time with conventional PCR proved to be useful in
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assessing the sensitivity of the real-time PCR assay with the TaqMan probe. The limitation of the comparison between conventional and real-time PCR is the absence of a gold standard. There were 12 samples that were negative by the conventional PCR but positive by real-time PCR (Table 2). The new assay increased the number of samples in which PPV was detected by 15% over the conventional PCR. Another approach to increase the sensitivity of the PCR assay is to perform nested PCR (Prikhod’ko et al., 2003), which has been reported to be more sensitive than the conventional PCR. However, a major disadvantage of nested PCR is the high risk of carryover and cross-contamination. Since the LightCycler PCR system uses sealed capillaries, the risk of contamination is much lower. The conventional method of TCID50 determination is laborious, expensive, time consuming, and requires susceptible cells. Compared with the conventional culture method, the quantitation of PPV investigated in this study was rapid and reproducible. Because the primer and probe set is designed based on a highly conserved region within the VP2 region of the PPV genome, the assay allows rapid quantitation of PPV. Although quantitation is not required for a diagnostic test, real-time PCR could be useful for several applications such as virus titration within a short period of time. In conclusion, the TaqMan real-time PCR assay described in this study for the detection and quantitation of PPV has been shown to be rapid, easy to handle, sensitive, and specific. These features make it an excellent tool for laboratory detection of PPV 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 culture-propagated virus suggests that the assay would be a useful tool for field investigation of PPV infection. Acknowledgement This work was supported by a grant from the Outstanding Person Innovation Fund of Henan, China (No. 0621002100). References Bustin, S.A., 2002. Quantitation of mRNA using real-time reverse transcription PCR (RT-PCR): trends and problems. J. Mol. Endocrinol. 29, 23–39. Cartwright, S.F., Huck, R.A., 1967. Viruses isolated in association with herd infertility abortions and stillbirths in pigs. Vet. Rec. 81, 196–197. Ellis, J.S., Smith, J.W., Braham, S., Lock, M., Barlow, K., Zambon, M.C., 2007. Design and validation of an H5 TaqMan real-time one-step reverse transcription-PCR and confirmatory assays for diagnosis and verification of influenza A virus H5 infections in humans. J. Clin. Microbiol. 45 (5), 1535–1543. Holland, P.M., Abramson, R.D., Watson, R., Gelfand, D.H., 1991. Detection of specific polymerase chain reaction product by utilizing the 5–3 exonuclease activity of Thermus aquaticus DNA polymerase. Proc. Natl. Acad. Sci. U.S.A. 88, 7276–7280. Kang, S.E., Nam, Y.S., Hong, K.W., 2007. Rapid detection of Enterobacter sakazakii using TaqMan real-time PCR assay. J. Microbiol. Biotechnol. 17 (3), 516–519. Kim, J., Chae, C., 2003. Multiplex nested PCR compared with in situ hybridization for the differentiation of porcine circoviruses and porcine parvovirus from pigs with postweaning multisystemic wasting syndrome. Can. J. Vet. Res. 67, 133–137. Kim, J., Chae, C., 2004. A comparison of virus isolation, polymerase chain reaction, immunohistochemistry, and in situ hybridization for the detection of porcine circovirus 2 and porcine parvovirus in experimentally and naturally coinfected pigs. J. Vet. Diagn. Invest. 16 (1), 45–50. Liu, Q., Wang, L., Willson, P., Babiuk, L.A., 2000. Quantitative, competitive PCR analysis of porcine circovirus DNA in serum from pigs with postweaning multisystemic wasting syndrome. J. Clin. Microbiol. 38 (9), 3474–3477. McKillen, J., Hjertner, B., Millar, A., McNeilly, F., Belák, S., Adair, B., Allan, G., 2007. Molecular beacon real-time PCR detection of swine viruses. J. Virol. Methods 140 (1–2), 155–165. Molitor, T.W., Oraveerakul, K., Zhang, Q.Q., Choi, C.S., Ludemann, L.R., 1991. Polymerase chain reaction (PCR) amplification for the detection of porcine parvovirus. J. Virol. Methods 32 (2–3), 201–211. Olvera, A., Sibila, M., Calsamiglia, M., Segalés, J., Domingo, M., 2004. Comparison of porcine circovirus type 2 load in serum quantified by a real time PCR in postweaning multisystemic wasting syndrome and porcine dermatitis and nephropathy syndrome naturally affected pigs. J. Virol. Methods 117 (1), 75–80. Prikhod’ko, G.G., Reyes, H., Vasilyeva, I., Busby, T.F., 2003. Establishment of a porcine parvovirus (PPV) DNA standard and evaluation of a new LightCycler nested-PCR assay for detection of PPV. J. Virol. Methods 111, 13–19.
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Schwaiger, M., Cassinotti, P., 2003. Development of a quantitative real-time RT-PCR assay with internal control for the laboratory detection of tick borne encephalitis virus (TBEV) RNA. J. Clin. Virol. 27, 136–145. Soares, R.M., Durignon, E.L., Bersano, J.G., Richtzenhain, L.J., 1999. Detection of porcine parvovirus DNA by the polymerase chain reaction assay using primers to the highly conserved nonstructural protein gene, NS-1. J. Virol. Methods 78, 191–198. Warrilow, D., Northill, J.A., Pyke, A., Smith, G.A., 2002. Single rapid TaqMan fluorogenic probe based PCR assay that detects all four dengue serotypes. J. Med. Virol. 66, 524–528. Wilhelm, S., Zimmermann, P., Selbitz, H.J., Truyen, U., 2006. Real-time PCR protocol for the detection of porcine parvovirus in field samples. J. Virol. Methods 134 (1–2), 257–260.
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Contents lists available at ScienceDirect
Journal of Virological Methods journal homepage: www.elsevier.com/locate/jviromet
A TaqMan-based real-time polymerase chain reaction for the detection of porcine parvovirus Hong-Ying Chen a,1 , Xiao-Kang Li b,1 , Bao-An Cui a,∗ , Zhan-Yong Wei a , Xin-Sheng Li a , Yan-Bin Wang a , Li Zhao a , Zhen-Ya Wang a a b
College of Animal Husbandry and Veterinary, Henan Agricultural University, Zhengzhou 450002, China College of Veterinary Medicine, South China Agricultural University, Guangzhou 510642, China
a b s t r a c t Article history: Received 6 February 2008 Received in revised form 7 October 2008 Accepted 21 October 2008 Available online 17 December 2008 Keywords: Porcine parvovirus Real-time PCR VP2 TaqMan Quantitation
A real-time polymerase chain reaction (PCR) using a TaqMan probe was developed to detect porcine parvovirus (PPV). Real-time PCR was optimized to quantify PPV using a detection system (Rotor Gene 2000 detector) and a dual-labeled fluorogenic probe. The gene-specific labeled fluorogenic probe for the VP2 gene of PPV was used to detect PPV. Quantitation of PPV was accomplished by a standard curve plotting cycle threshold values (Ct) against each dilution of standard plasmids. When the specificity of the assay using specific PPV primers was evaluated by testing the PPV standard strain and other viruses, no cross-reactions were detected with non-PPV reference viruses. The detection limit of real-time PCR for PPV was 2.08 log 10 genome copy equivalent (gce). In this study, a real-time PCR assay was performed on 80 clinical samples and compared with a conventional PCR assay. In 48 of 80 samples, PPV DNA was detected by the conventional PCR assay. All samples positive for PPV DNA by the conventional PCR assay were also positive by the real-time PCR assay, and 12 of 32 samples that tested negative for PPV DNA by the conventional method tested positive by the real-time PCR assay. Using the real-time PCR assay, the number of samples in which PPV was detected increased by 15%. Therefore, it is considered to be a useful tool for the detection of PPV. © 2008 Elsevier B.V. All rights reserved.
1. Introduction Porcine parvovirus (PPV) is an autonomous parvovirus belonging to the genus parvovirus, subfamily Parvovirinae, family Parvoviridae; it is the major causative virus in a reproductive failure syndrome in swine characterized by stillbirths, mummified fetuses, early embryonic death, and infertility. In addition, PPV has been implicated as the causative agent of diarrhea, skin disease and arthritis in swine. After PPV was isolated by Cartwright in 1967 from herd infertility, abortion, and stillbirth in pigs (Cartwright and Huck, 1967), PPV has been reported to occur worldwide with variable prevalence rates; it always causes recessive infection, which presents many challenges for diagnosis. Virus isolation is a fundamental diagnostic method, but PPV isolation in cell cultures is laborious and cannot be achieved for all PPV strains (Kim and Chae, 2004). Other diagnostic techniques, such as the hemagglutination (HA), enzyme-linked immunosorbent assay, immunofluorescence, in situ hybridization, conventional poly-
∗ Corresponding author. Tel.: +86 371 63558878; fax: +86 371 63558878. E-mail address: [email protected] (B.-A. Cui). 1 The first and second author contributed equally to this work. 0166-0934/$ – see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.jviromet.2008.10.029
merase chain reaction (PCR), and nested PCR have been developed. Comparing these techniques, nested PCR, and in situ hybridization were shown to be more sensitive than conventional PCR and the HA test (Kim and Chae, 2003; Soares et al., 1999). However, the nested PCR requires agarose gel analysis for the detection of amplification products and in situ hybridization may require several days to complete. Therefore, both assays are labor-intensive, and nested PCR has a very high risk of contamination. Recently, a real-time PCR has been used extensively for detection of amplicons that are amplified during the PCR cycling in real time (Olvera et al., 2004; Yoon et al., 2005; Ellis et al., 2007). Real-time PCR is an excellent diagnostic tool with high sensitivity, specificity, and a fast turnaround time. This system is called real-time PCR because the accumulated amplicons can be monitored directly during the DNA amplification process in real time. The development of fluorogenic PCR utilizing the 5 –3 nuclease activity of Taq DNA polymerase made it possible eliminate post-PCR processing such as visualization in agarose (Holland et al., 1991; Warrilow et al., 2002). In addition, the real-time PCR technique has been shown to provide good sensitivity and a linear relationship between the copy number and cycle threshold (Ct) values. The technique uses oligonucleotide probes labeled with fluorescent dyes, a reporter at the 5 end, and a quencher at the 3 end to monitor the accumulation of PCR products
H.-Y. Chen et al. / Journal of Virological Methods 156 (2009) 84–88
(Bustin, 2002). The quantitation of DNA is based on the determination of the threshold cycle when the amplified PCR product is first detected. The higher the initial DNA copy number input, the sooner the product of amplification is detected. Reports on virus detection based on real-time PCR technology have been described for various animal viruses (Schwaiger and Cassinotti, 2003; Yang et al., 2004; Yoon et al., 2005; Kang et al., 2007). Molecular beacon realtime PCR for the detection of PPV has been developed (McKillen et al., 2007); however, very few studies of a real-time PCR assay with a TaqMan probe for the detection of PPV exist. Therefore, a real-time PCR assay with a TaqMan probe was investigated in this study and used for laboratory detection and quantitation of PPV. In addition, the applicability of the real-time PCR was evaluated for the detection of PPV DNA in aborted pig fetuses. 2. Materials and methods 2.1. Viruses and sample preparation The PPV standard strain 7909 was purchased from Beijing Ordinary Microbiology Strain Store Center, Beijing, China and used as a standard virus for the detection of PPV by real-time PCR. The PPV 7909 strain was propagated in the PK-15 porcine kidney cell line. The porcine circovirus (PCV-2), porcine reproductive and respiratory syndrome virus (PRRSV), pseudorabies virus (PRV), Japanese encephalitis virus (JEV), canine parvovirus (CPV) and feline panleukopenia virus (FPV) were used as reference strains for the specificity test of the real-time PCR. In 2007, 80 fetuses that spontaneously aborted before 70 days of gestation were collected from several provinces in China and necropsied for determination of the reason for abortion. Tissue samples of the colon, duodenum, jejunum, heart, kidneys, liver, lung, spleen, thymus, and gonads were stored separately at −80 ◦ C. Viral DNA was extracted using a commercial test kit (QIAamp DNA Mini Kit, Qiagen, Hilden, Germany) according to the manufacturer’s instructions and stored at −20 ◦ C until needed. 2.2. Design of primers and a probe for real-time PCR The VP2 nucleotide sequences of PPV strains/isolates were retrieved from the GenBank and aligned using the software program DNAStar (DNASTAR, Inc., Madison). The accession numbers of the strains/isolates used for the alignment were the following: AY583318, U44978, AY390557, AY686602, AY459350, AY502115, AY597052, AY781130, AY786299, AY786300 (NJ-1), AY786301, AY786302, AY786303, AY788086, AY788087, AY788088, and AY788089. The primers and probe set 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. The primers were designed to detect the available PPV sequences in the relevant VP2 region (sense 5 -CCAAAAATGCAAACCCCAATA3 , antisense 5 -TCTGGCGGTGTTGGAGTTAAG-3 ) and amplify a fragment with a length of 194 bp. The TaqMan probe, FAMCTTGGAGCCGTGGAGCGAGCC-TAMRA, was used to detect any amplifications. This probe was labeled with a fluorescent reporter dye (FAM: 6-carboxyfluorescein) at the 5 end and a quencher dye (TAMRA: 6-carboxyteramethy-rhodamine) at the 3 end. 2.3. Construction of standard plasmids for real-time PCR To generate a PPV standard curve for the real-time reaction, a PCR product containing 751 bp covering the region of interest of VP2 and using the oligonucleotide primers PPV-1 (5 -ATACTTGGGGGAGGGCTT-3 ) and PPV-2 (5 TGTTCCTGGGTGTTGGTC-3 ) was cloned into the vector pGEM-T
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Easy vector according to the instructions of the manufacturer (Promega). The resulting plasmid, pPPV, was used to transform Escherichia coli DH5␣ cells and was purified using a QIAGEN plasmid purification kit (Qiagen Ltd., Hilden, Germany) according to the manufacturer’s instructions. The concentration of the plasmid preparation was determined by measuring the OD at 260 nm using a spectrophotometer (Thermo Electron, USA). Serial 10-fold dilutions of plasmid DNA in TE buffer (10 mM Tris–HCl, 1 mM EDTA) were used in the amplification reactions. The dilutions were stored at −20 ◦ C; stock plasmid was stored at −70 ◦ C. 2.4. Real-time PCR Real-time PCR assays using the TaqMan probe were carried out in a micro-reaction tube (Corbett Research, Australia). The reaction mixture for each tube of TaqMan reaction mix consisted of 2.5 l of 10× buffer, 0.5 l of 10 mM dNTP, 4.5 l of 25 mM MgCl2 , 0.4 l of 50 M fluorogenic FAM-labeled PPV probe, 0.5 l of 50 M forward primer, 0.5 l of 50 M reverse primer, 1 l of DNA solution, 14.1 l of distilled water, and 1 l of Ex Taq DNA (TaKaRa, Dalian, China) to produce a total volume of 25 l. The thermal profile for the realtime PCR was 94 ◦ C for 5 min, followed by 40 cycles of 94 ◦ C for 5 s and 60 ◦ C for 15 s. PCR amplification was performed by using the Rotor Gene 2000 real-time thermal cycler (Corbett Research, Australia). Positive and negative reference samples were tested along with the unknown samples in each run. 2.5. Conventional PCR Viral DNA sequences were amplified by conventional PCR as described previously (Kim and Chae, 2003) using the oligonucleotide primers PPV-3 (5 -GGGGAGGGCTTGGTTAGAAT-3 ) and PPV-4 (5 -TGGTTGGTGGTGAGGTTGCT-3 ). Five-l samples of the supernatant containing extracted DNA were used as the PCR templates. The amplification was performed in a 50-l reaction mixture containing 1× PCR buffer, 4 mM MgCl2 , 200 M of each dNTP, 1 M of each primer, and 1U of Taq DNA polymerase (TaKaRa, Dalian, China). The reaction was run in a PCT-200 Peltier thermal cycler (MJ Research, USA) under the following conditions: one cycle at 96 ◦ C 3 min, followed by 25 cycles of denaturation at 94 ◦ C for 30 s, primer annealing at 50 ◦ C for 30 s, and extension at 72 ◦ C for 30 s. PCR was ended with a final extension step at 72 ◦ C for 10 min. The reactions were performed in triplicate. To ensure the quality of data, negative and positive controls were used in each PCR reaction. DNA extraction and amplification were performed in different rooms. 3. Results 3.1. PPV DNA standard and optimization strategies for real-time PCR The recombinant plasmid pPPV has been constructed as the standard of PPV DNA, which is required for the standard curve calculation in quantitation assays. The constructed plasmid is 3766 bp. Conversion to genome equivalents was calculated as 1 OD260 equals 50 g/ml and one base pair equals 660 g/mol, resulting in a molecular weight for the plasmid (3766 bp) of 2.49 × 106 pg/pmol. One genome copy equivalent (gce) of PPV equals 4 attograms (ag) of this plasmid. The procedure was optimized with regard to the concentrations of primers and the denature/extension temperature. The concentration of primers, magnesium, and probe giving the highest fluorescence and the lowest threshold cycle were selected as
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Fig. 1. Real-time PCR standard curve generated from plasmid DNA amplification plots. Standard curve was plotted in the sample plasmid on the x-axis and cycle threshold (Ct) on the y-axis. The x-axis represents pPPV in 10-fold dilutions and the y-axis the fluorescence data used for Ct determinations in dRn (baseline-corrected normalized fluorescence). The assays were linear over a 108 dilution range of pPPV with R2 values (square of the correlation coefficient) of 0.99 and reaction efficiencies of 93.4%.
Fig. 2. Specificity of real-time PCR. No cross-reactions were detected with PCV-2, PRRSV, PRV, JEV, CPV, FPV, and PK15 cells.
follows: 1 M forward and reverse primers, 4.5 mM MgCl2 , and 0.8 M probe. 3.2. Standard curve for quantitation of PPV Real-time PCR amplifications were performed with serial dilutions (101 to 109 gce) of standard plasmids to assess the quantitation assay. Samples were tested three times. The result showed that a detectable fluorescence signal above the threshold occurred at 16.38 cycles from the amplification profile with number of cycles versus normalized fluorescence values (data not shown). The Rotor Gene detection system software generated a standard curve by plotting the Ct values against each standard dilution of standard plasmids. Fig. 1 shows the real-time PCR standard curve generated from plasmid DNA amplification plots. A linear standard curve was obtained from 1 × 104 to 1 × 108 gce per reaction mixture, which resulted in Ct values ranging from 16.38 to 29.84 cycles. The assays were linear over a 108 dilution range of template DNA with R2 values (square of the correlation coefficient) of 0.99 and reaction efficiencies of 93.4%. 3.3. Specificity of the assay When several reference viruses were screened by the TaqMan probe under the optimal conditions of the assay, the 7909 strain was positive according to real-time PCR. As shown in Fig. 2, no crossreactions were detected with PCV-2, PRRSV, PRV, JEV, CPV, FPV, and PK 5 cells.
Fig. 3. Sensitivity of real-time PCR. When the extracted DNA was diluted serially 10-fold with PBS to 10−7 , fluorescence was detected, no reaction was detected at 10−8 .
tivity titer equivalent. DNA concentrations extrapolated from the standard curve were compared with the titers determined in the parallel TCID50 experiment (data not shown). One TCID50 equaled 1.836 ± 0.31 log10 gce. Therefore, the detection limit of real-time PCR was 2.08 log10 gce. 3.5. Reproducibility of the real-time PCR for PPV When the standard PPV plasmid DNA was used for the evaluation of the coefficients of variation (CVs) of the real-time PCR, the intra- and inter-assay CVs for Ct values ranged between 0.35 and 0.81%, and 0.30 and 1.32%, respectively (Table 1).
3.4. Sensitivity of the assay In order to determine the detection limit and efficiency of the assay, the titer of a PPV suspension was determined by the standard cell-based PPV-specific 50% tissue culture infectious dose (TCID50 ) assay and was 106.8 TCID50 /ml. The DNA of PPV was extracted and used as a template. The extracted DNA was diluted serially 10-fold with phosphate-buffered saline solution (PBS) up to 10−8 . When the extracted DNA was diluted to 10−7 , fluorescence was detected; no reaction was detected at 10−8 (Fig. 3). Sensitivity of the PCR assay for the detection of PPV was expressed as the infec-
3.6. Comparison of results obtained by real-time PCR and conventional PCR Twenty-five of eighty samples were positive by HA for PPV. All samples positive by this test were also positive in real-time and conventional PCR assays. The compared results of the real-time and conventional PCR assays are shown in Table 2. All samples positive by the conventional PCR for PPV DNA were also positive by the realtime PCR assay; whereas, 12 of the samples negative for PPV DNA by the conventional PCR assays were positive in the real-time PCR
Table 1 Variance analysis of Ct values quantified by real-time PCR in serially diluted standard plasmid solutions. Concentration of standard plasmid (copies/ml)
n
Intra-assay variability Ct
107 105 103
10 10 10
Inter-assay variability CV (%)
Mean
S.D.
12.71 20.12 27.72
0.08 0.07 0.22
0.63 0.35 0.81
Ct
CV (%)
Mean
S.D.
12.90 19.97 27.48
0.17 0.06 0.16
1.32 0.30 0.58
H.-Y. Chen et al. / Journal of Virological Methods 156 (2009) 84–88 Table 2 Comparison of real-time PCR and conventional PCR. Conventional PCR assay result (n)
Real-time PCR assay result (n) Positive
Negative
Total
Positive Negative Total
48 12 60
0 20 20
48 32 80
assays. The copy number of PPV DNA in tissue samples that was positive by both methods ranged from 1 × 105 to 1 × 1012 gce per 500 ng DNA. The copy number of the PPV DNA of 12 liver samples, which were positive by the real-time PCR assay but negative by the conventional PCR assay, was 1 × 103 to 1 × 105 gce per 500 ng DNA. 4. Discussion In this study, real-time PCR for laboratory detection of PPV was investigated in different samples. Specificity, sensitivity and quantitative range of real-time PCR were also evaluated. The established real-time PCR assay for PPV DNA quantitation was linear over a 108 dilution range of template concentrations with a R2 value (the square of the correlation coefficient) of 0.99 and a reaction efficiency of 93.4% (Fig. 1). The detection limit for the real-time PCR was shown to be 2.08 log10 gce. Porcine pathogens such as PRV, PCV-2, PRRSV, and other parvoviruses like FPV and CPV showed no Ct, and were therefore not detected by this assay. When compared to the previous reports for the detection of PPV (Molitor et al., 1991; Soares et al., 1999), the real-time PCR method has several advantages over conventional PCR. Firstly, real-time PCR yields a more rapid and sensitive test result than conventional PCR (Warrilow et al., 2002; Schwaiger and Cassinotti, 2003). Secondly, real-time PCR is less likely to produce a false positive due to contamination during the sample preparation process. Thirdly, real-time PCR can save time, because a conventional PCR assay requires post-PCR analysis such as gel electrophoresis, but the amplification of a specific PCR product in real-time PCR is measured in real time during PCR cycling. For precise estimation of viral loads in samples, a competitive quantitative PCR was applied that can detect and quantify target DNA simultaneously (Liu et al., 2000); however, it requires an exogenous competitor as a control, which must be constructed and characterized, and it requires post-PCR electrophoresis. Realtime PCR is performed in a closed tube and requires no post-PCR electrophoresis. Therefore, contamination with amplicons can be avoided. This assay is more reproducible and convenient than competitive quantitative PCR tests. The SYBR Green I-based real-time PCR is used to detect PPV (Wilhelm et al., 2006); the limitation of this method relates to false positives because SYBR Green I dye can bind to any double-stranded DNA, even non-specific amplicons. However, TaqMan probes and molecular beacons provide the further specificity needed for diagnostic PCR. The molecular beacon method utilizes a reporter probe that is wrapped around into a hairpin shape. It also has a quencher dye that must be in close contact to the reporter to work. An important difference of the molecular beacon method compared to the TaqMan method is that the probe remains intact throughout the PCR product, and is rebound to the target at every cycle. It has been suggested that the TaqMan probe and molecular beacon possess a similar sensitivity for the detection of antibiotic resistance-conferring single nucleotide polymorphisms in mixed Mycobacterium 199 tuberculosis (Yesilkaya et al., 2006). The assay was able to detect PPV in all samples shown to be PPV positive by the conventional PCR and the HA test. The comparison of real-time with conventional PCR proved to be useful in
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assessing the sensitivity of the real-time PCR assay with the TaqMan probe. The limitation of the comparison between conventional and real-time PCR is the absence of a gold standard. There were 12 samples that were negative by the conventional PCR but positive by real-time PCR (Table 2). The new assay increased the number of samples in which PPV was detected by 15% over the conventional PCR. Another approach to increase the sensitivity of the PCR assay is to perform nested PCR (Prikhod’ko et al., 2003), which has been reported to be more sensitive than the conventional PCR. However, a major disadvantage of nested PCR is the high risk of carryover and cross-contamination. Since the LightCycler PCR system uses sealed capillaries, the risk of contamination is much lower. The conventional method of TCID50 determination is laborious, expensive, time consuming, and requires susceptible cells. Compared with the conventional culture method, the quantitation of PPV investigated in this study was rapid and reproducible. Because the primer and probe set is designed based on a highly conserved region within the VP2 region of the PPV genome, the assay allows rapid quantitation of PPV. Although quantitation is not required for a diagnostic test, real-time PCR could be useful for several applications such as virus titration within a short period of time. In conclusion, the TaqMan real-time PCR assay described in this study for the detection and quantitation of PPV has been shown to be rapid, easy to handle, sensitive, and specific. These features make it an excellent tool for laboratory detection of PPV 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 culture-propagated virus suggests that the assay would be a useful tool for field investigation of PPV infection. Acknowledgement This work was supported by a grant from the Outstanding Person Innovation Fund of Henan, China (No. 0621002100). References Bustin, S.A., 2002. Quantitation of mRNA using real-time reverse transcription PCR (RT-PCR): trends and problems. J. Mol. Endocrinol. 29, 23–39. Cartwright, S.F., Huck, R.A., 1967. Viruses isolated in association with herd infertility abortions and stillbirths in pigs. Vet. Rec. 81, 196–197. Ellis, J.S., Smith, J.W., Braham, S., Lock, M., Barlow, K., Zambon, M.C., 2007. Design and validation of an H5 TaqMan real-time one-step reverse transcription-PCR and confirmatory assays for diagnosis and verification of influenza A virus H5 infections in humans. J. Clin. Microbiol. 45 (5), 1535–1543. Holland, P.M., Abramson, R.D., Watson, R., Gelfand, D.H., 1991. Detection of specific polymerase chain reaction product by utilizing the 5–3 exonuclease activity of Thermus aquaticus DNA polymerase. Proc. Natl. Acad. Sci. U.S.A. 88, 7276–7280. Kang, S.E., Nam, Y.S., Hong, K.W., 2007. Rapid detection of Enterobacter sakazakii using TaqMan real-time PCR assay. J. Microbiol. Biotechnol. 17 (3), 516–519. Kim, J., Chae, C., 2003. Multiplex nested PCR compared with in situ hybridization for the differentiation of porcine circoviruses and porcine parvovirus from pigs with postweaning multisystemic wasting syndrome. Can. J. Vet. Res. 67, 133–137. Kim, J., Chae, C., 2004. A comparison of virus isolation, polymerase chain reaction, immunohistochemistry, and in situ hybridization for the detection of porcine circovirus 2 and porcine parvovirus in experimentally and naturally coinfected pigs. J. Vet. Diagn. Invest. 16 (1), 45–50. Liu, Q., Wang, L., Willson, P., Babiuk, L.A., 2000. Quantitative, competitive PCR analysis of porcine circovirus DNA in serum from pigs with postweaning multisystemic wasting syndrome. J. Clin. Microbiol. 38 (9), 3474–3477. McKillen, J., Hjertner, B., Millar, A., McNeilly, F., Belák, S., Adair, B., Allan, G., 2007. Molecular beacon real-time PCR detection of swine viruses. J. Virol. Methods 140 (1–2), 155–165. Molitor, T.W., Oraveerakul, K., Zhang, Q.Q., Choi, C.S., Ludemann, L.R., 1991. Polymerase chain reaction (PCR) amplification for the detection of porcine parvovirus. J. Virol. Methods 32 (2–3), 201–211. Olvera, A., Sibila, M., Calsamiglia, M., Segalés, J., Domingo, M., 2004. Comparison of porcine circovirus type 2 load in serum quantified by a real time PCR in postweaning multisystemic wasting syndrome and porcine dermatitis and nephropathy syndrome naturally affected pigs. J. Virol. Methods 117 (1), 75–80. Prikhod’ko, G.G., Reyes, H., Vasilyeva, I., Busby, T.F., 2003. Establishment of a porcine parvovirus (PPV) DNA standard and evaluation of a new LightCycler nested-PCR assay for detection of PPV. J. Virol. Methods 111, 13–19.
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