Journal of Virological Methods 209 (2014) 25–29
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Journal of Virological Methods journal homepage: www.elsevier.com/locate/jviromet
Development of a two-step SYBR Green I based real time RT-PCR assay for detecting and quantifying peste des petits ruminants virus in clinical samples Tsegalem Abera ∗ , Ardhanary Thangavelu Department of Veterinary Microbiology, Madras Veterinary College, Chennai 600007, India
a b s t r a c t Article history: Received 29 June 2014 Received in revised form 6 August 2014 Accepted 12 August 2014 Available online 4 September 2014 Keywords: PPRV SYBR Green I Real time RT-PCR Matrix gene
A two-step SYBR Green I based real time RT-PCR targeting the matrix (M) gene of Peste des petits ruminants virus (PPRV) was developed. The specificity of the assay was assessed against viral nucleic acid extracted from a range of animal viruses of clinical and structural similarities to PPRV including canine distemper virus, measles virus, bluetongue virus and Newcastle disease virus. But none of the viruses and no template control showed an amplification signal. Sensitivity of the same assay was assessed based on plasmid DNA copy number and with respect to infectivity titre. The lower detection limit achieved was 2.88 plasmid DNA copies/l with corresponding Ct value of 35.93. Based on tissue culture infectivity titre the lower detection limits were 0.0001TCID50 /ml and 1TCID50 /ml for the SYBR green I based real time RT-PCR and conventional RT-PCR, respectively. The calculated coefficient of variations values for intra- and inter-assay variability were low, ranging from 0.21% to 1.83% and 0.44% to 1.97%, respectively. The performance of newly developed assay was evaluated on a total of 36 clinical samples suspected of PPR and compared with conventional RT-PCR. The SYBR Green I based real time RT-PCR assay detected PPRV in 32 (88.8%) of clinical samples compared to 19 (52.7%) by conventional RT-PCR. Thus, the two-step SYBR Green I based real time RT-PCR assay targeting the M gene of PPRV reported in this study was highly sensitive, specific and reproducible for detection and quantitation of PPRV nucleic acids. © 2014 Elsevier B.V. All rights reserved.
1. Introduction Peste des petits ruminants (PPR), also known as ‘goat plague’, is a viral disease of goats and sheep characterized by fever, sores in the mouth, diarrhoea, pneumonia, and sometimes death (OIE, 2010). PPR was first identified in West Africa in the 1940s, and can now be detected in a broad belt of sub-Saharan Africa, Arabia, the Middle East and Southern Asia (Dhar et al., 2002). The causative agent peste-des-petits ruminants virus (PPRV) is classified as a member of the genus Morbillivirus, family Paramyxoviridae under the order Mononegavirales. It has a negative sense single-stranded RNA genome encoding eight proteins (Bailey et al., 2005). Several conventional reverse transcription polymerase chain reactions (RT-PCRs) are available for detection of PPRV genomic material (Forsyth and Barrett, 1995; Couacy-Hymann et al., 2002;
∗ Corresponding author at: College of Veterinary Medicine, Jigjiga University, Jijiga, Ethiopia Po. Box 1020. Tel.: +251 913 387416; fax: +251 25 775 5947. E-mail address: [email protected] (T. Abera). http://dx.doi.org/10.1016/j.jviromet.2014.08.017 0166-0934/© 2014 Elsevier B.V. All rights reserved.
Balamurugan et al., 2006). However, these conventional RT-PCR assays are labour intensive, as they require gel analysis for the detection of PCR products with a consequent high risk of contamination (Bao et al., 2008). Real time RT-PCR has gained wider acceptance over conventional RT-PCR because it is more rapid, sensitive and reproducible. Few real time RT-PCR assays have been described for detection and quantitation of PPRV in clinical samples using TaqMan chemistry (Bao et al., 2008; Balamurugan et al., 2010; Kwiatek et al., 2010; Batten et al., 2011). In comparison with TaqMan-PCR, SYBR Green I based real-time RT-PCR assay has the advantages of being more cost-effective, easy to design, more precise and produce a more linear decay plot (Schmittgen et al., 2000). The use of M gene based two-step SYBR Green I real time RT-PCR for molecular diagnosis of PPR has not been reported so far. The availability of such technique will provide an alternative to the N gene based real-time RT-PCR diagnostic assays that already exist (especially in terms of its sensitivity); thereby helping in rapid clinical diagnosis of PPR. In this regard, the present study developed a two-step SYBR Green I based real time RT-PCR assay for the detection and quantitation of PPRV in clinical samples.
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T. Abera, A. Thangavelu / Journal of Virological Methods 209 (2014) 25–29
consisted of 70 ◦ C for 15 s, and followed by temperature increase to 95 ◦ C for 15 s at the rate of 1.25 ◦ C/s with continuous reading of fluorescence.
2. Materials and methods 2.1. Viruses, cells and clinical samples Viruses used in this study are listed in Table 1. Vero cells were used for virus propagation and titration. A total of 36 field samples from PPR suspected outbreaks from different areas of the Indian state of Tamil Nadu were analyzed. Samples received for analyses from different vet clinics were ocular, nasal, oral and rectal swabs, spleen, kidney, lung, lymph nodes and dung.
2.5. Conventional RT-PCR Conventional RT-PCR was performed on the cDNA preparations of the PPRV with a primer set of MF-Morb (5 -CTT GAT ACTC CCC AGA GATTC-3 ) and MRPPR3 (5 -TTC TCC CAT GAG CCG ACT ATGT3 ) as described by Balamurugan et al. (2006) which yielded 191 bp of PCR product. Amplification of target sequences was performed in a 2720 thermal cycler (Applied Biosystems).
2.2. Viral RNA extraction and cDNA synthesis 2.6. Generation of quantification standards Viral RNA was extracted from tissue culture supernatant using QIAamp® Viral RNA Mini Kit (Qiagen® , Germany), following manufacturer’s instructions. Trizol reagent (Invitrogen, CA, USA) was also used to isolate RNA from clinical samples. Synthesis of cDNA was carried out in 20 l reaction using RevertAidTM H minus first Strand cDNA Synthesis kit (Fermentas, USA) following manufacturer’s instructions. Briefly, 11 l of the purified RNA were added to a mixture containing 4 l RT buffer (5×), 1 l of random hexamer, 2 l 10 mM dNTP, 1 l RNase inhibitor and l l of Moloney murine leukaemia virus reverse transcriptase. The reaction was carried out at 25 ◦ C for 5 min, 42 ◦ C for 60 min and 70 ◦ C for 5 min and stored at −40 ◦ C until use. 2.3. Primer design and synthesis The forward and reverse primers were designed according to the sequences of matrix protein (M) gene (GenBank accession no. GQ452014.1) of PPRV Sungri-96 strain by using FastPCR software. The designed primers (Forward M3 F: 5 GGAGTGATTGAGGATAACGACC-3 (169–190) and Reverse M3 R: 5 -GCGTTAACAAGGACAGCGGAG-3 (350–370) were validated by OligoAnalyzer 1.2 and synthesized commercially (Sigma Aldrich, Bangalore, India). 2.4. Real time RT-PCR All reactions were performed using Realplex4 real time PCR machine (Eppendorf, Germany) using SYBR premix Ex Taq, (TaKaRa Bio Inc., Japan). The PCR was set up in a 10 l reaction volume containing 5 l of 2× SYBR Premix Ex Taq master mix, 1.5 l of cDNA, 1 l (10 M) of each primer and 2.5 l nuclease free water. The optimized cycling conditions were as follows: initial denaturation at 94 ◦ C for 5 min, followed by 40 cycles of denaturation at 94 ◦ C for 20 s, primer annealing at 56 ◦ C for 30 s, and extension at 72 ◦ C for 20 s. The fluorescence was measured at the end of each cycle. A melt curve analysis was performed following amplification to verify the specificity of the amplified products. Melting curve analysis
A fragment of 348 bp of M gene of PPRV containing the real time RT-PCR primers binding sites were amplified using the primer pair M4 F (forward primer): 5 CCAGGTAAGGGTCATCGATCC-3 (99–119) and M4 R (reverse primer): 5 -AAGCGGGACTAGGTTGACTGCA-3 (426–447) (In house designed). The RT-PCR product was cloned into T&A cloning vector according to the manufacturer’s instructions. Plasmid DNA was recovered from the transformed Escherichia coli BL-21 cells using AxyPrep Plasmid Miniprep Kit (Axygen Biosciences). The OD value of the plasmid DNA standard concentrations was measured at 260 nm/280 nm on Thermo Scientific NanoDropTM 1000 Spectrophotometer (NanoDrop Technologies, LLC, Wilmington, DE, USA). Plasmid copy number was calculated using the formula described by Adams (2006). The concentration of the obtained plasmid DNA was 95 ng/l which equates 2.88 × 1010 copies/l. 2.7. Specificity and sensitivity of the real time RT-PCR The specificity of the developed assay was assessed against viral nucleic acid extracted from a range of animal viruses of clinical and structural relevance to PPRV. The sensitivity of the assay was determined by running 10-fold serial dilutions of the plasmid standard in duplicates. Additionally, the sensitivity of the assay was evaluated by titrating PPRV Combatoire strain on Vero cells in a 96-well microtitre plate using standard cell culture procedure and the virus titre was calculated using Reed and Muench (1938) formula. PPRV having a titre of 105 TCID50 /ml was diluted 10-fold serially from 10−1 and 10−11 , and total RNA was extracted from each dilution, subsequently cDNA synthesis was performed. 2.8. Reproducibility The DNA standard ranging from 2.88 × 107 copies/l to 2.88 × 100 copies/l was tested repeatedly. Three separate dilution series were assayed in a single run to evaluate intra-assay
Table 1 Viruses used in the study. Virus
Strain
Source
Peste des petits ruminants virus (PPRV)
AR 87 vaccine strain Sunguri vaccine strain Coimbatore vaccine strain Coimbatore field isolate Recent field isolates Vaccine strain Vaccine strain Vaccine strain D58
Dept. of Vet. Microbiology, Madras Veterinary College Dept. of Vet. Microbiology, Madras Veterinary College Dept. of Animal Biotechnology, Madras Veterinary College Dept. of Vet. Microbiology, Madras Veterinary College Dept. of Vet. Microbiology, Madras Veterinary College Nobivac Puppy DP, Intervet Serum institute of India LTD, Pune Dept. of Vet. Microbiology, Madras Veterinary College Dept. of Vet. Microbiology, Madras Veterinary College
Canine distemper virus (CDV) Measles virus (MV) Bluetongue virus (BTV) Newcastle disease virus (NDV)
T. Abera, A. Thangavelu / Journal of Virological Methods 209 (2014) 25–29
variations. Whereas, the inter-assay variations were measured by testing each dilution in three separate consecutive runs. The mean, standard deviation (SD) and coefficient of variation (CV) for both intra-assay and inter-assay variations were calculated separately for each standard DNA dilution based on their Ct values using Microsoft Excel software. 2.9. Spiking The performance of the newly developed assay was checked using clinical samples (2 nasal swabs from goats) that were spiked with known titre of PPR virus as described by Balamurugan et al. (2010). Infected cell culture fluid containing 105 TCID50 /ml viruses was diluted 1: 10 serially by using the two negative nasal swabs as diluent. The 103 and 104 TCID50 /ml spiked titre viruses were used for total RNA extraction. Control virus samples (103 and 104 TCID50/ ml titre virus suspensions) were also used for RNA extraction. 3. Results 3.1. Specificity The assay exhibited specificity as an amplification signal was observed only with PPRV nucleic acid. None of the viruses which have genetic or clinical similarities with PPRV (CDV, MV, BTV, and NDV) and no template control (NTC) showed an amplification signal. Vaccine and field isolates of PPRV (Arasur, Sunguri, Coimbatore and recent field isolates) were amplified in the real-time RT-PCR and all were positive with the melting point of 84.6 ± 0.15 (Fig. 1). 3.2. Detection limit A series of 10-fold dilutions of the plasmid DNA starting from 2.88 × 107 to 2.88 × 100 copies/l were prepared to construct standard curve. A linear regression relationship between the plasmid dilutions and the threshold cycle (Ct) values was observed with a coefficient of determination (R2 ) of 0.9947 and a slope of −3.0763. The lower detection limit achieved was 2.88 copies/l with corresponding Ct value of 35.93 (Fig. 2). The lower detection limit based on tissue culture infectivity titre was 0.0001 TCID50 /ml with a corresponding Ct value of 31.9 (data not shown).
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Table 2 Comparative evaluation of clinical samples suspected of PPRV using conventional and real time RT-PCR. Specimen type
Conventional RT-PCR result
RT-PCR PPRV (Ct ± S.D)a
Lung Ocular swab Oral swab Spleen PLN Spleen Mediastinal LN Nasal swab Nasal swab Dung Mesenteric LN Ocular swab Lung Nasal swab Nasal swab Nasal swab Nasal swab Nasal swab Ocular swab Oral swab Spleen Nasal swab Mandibular LN Ocular swab Rectal swab Kidney Nasal swab Ocular swab Mesenteric LN Oral swab Rectal swab Mesenteric LN Rectal swab Ocular swab Lung Spleen
+ + + + − + + − − − + + + + + + + + + − − + + + − + − − − − − − − − − −
13.90 19.05 19.72 20.13 20.23 20.69 20.52 20.77 20.84 21.02 22.05 22.18 22.46 23.55 24.38 24.58 25.02 25.91 26.21 27.61 26.11 28.19 28.75 28.33 29.92 29.72 30.73 24.40 31.90 31.84 32.31 36.31 – – – –
a
± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±
0.81 0.34 0.33 0.03 0.05 0.05 0.26 0.08 0.10 0.36 0.06 0.19 0.17 0.20 0.06 0.33 0.28 0.01 0.05 0.01 1.85 0.16 0.06 1.14 0.03 0.27 0.00 6.99 0.39 1.10 0.69 2.07
Results are displayed in ascending order of Ct values.
deviation (SD) and coefficient of variation (CV) values were, ranging from 0.05 to 0.56 and from 0.21 to 1.83%, respectively. The interassay CV and SD were in the range of 0.44 to 1.97% and 0.08 to 0.61, respectively. 3.4. Spiking
3.3. Reproducibility The intra-assay reproducibility was analyzed by using 10-fold dilutions of the plasmid DNA standard ranging from 2.88 × 107 to 2.88 × 100 copies/l in triplicates per run. The calculated standard
Spiking assay with 104 and 103 TCID50 /ml of cell culture infected PPRV was performed on two negative goat nasal swabs. The mean Ct values obtained for the spiked clinical samples and tissue culture virus were compared. Negative clinical samples spiked with known
Fig. 1. Specificity of M gene based real time RT-PCR: (a) amplification plot representing PPRV, CDV, MV, BTV, NDV and No template control. (b) Melting curve analysis.
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titre of PPRV and tissue culture virus with same titre were equally well detected by the assay. 3.5. Assay performance on clinical samples The competence of the SYBR Green-based real time RT-PCR assay for the detection of PPR virus in clinical samples was evaluated by analysis of 36 samples from PPR suspected cases. Comparison of the newly developed real time RT-PCR assay was made with gel-based conventional RT-PCR assay. The results of both assays were similar for the evaluation of 23 samples. However, 13 samples tested negative by gel based RT-PCR were found to be positive by real time RT-PCR. None of the samples tested negative by real-time RT-PCR was found positive by the gel based PCR (Table 2). 4. Discussion In this study, RT-PCR conditions for efficient amplification and quantification of PPRV nucleic acid sequences of M gene in clinical samples using real time fluorescence measurement were defined. Overall, this newly developed SYBR Green I based real time RT-PCR assay had detection limit that was significantly lower than that of
conventional RT-PCR while maintaining a high level of specificity and reproducibility. The generated standard curve can be applicable for accurate quantification of PPRV. The assay maintained linearity over seven orders of magnitude. The assay showed high sensitivity with the ability of quantifying PPRV nucleic acid to as few as 2.88 copies/l. This can be useful in detecting low levels of PPRV and confirming early stage of PPRV infection where the virus titres are very low. Since the main drawbacks of using DNA binding dyes such as SYBR Green I is detecting any double stranded DNA generated during PCR. In this study, several approaches were followed to cross-check the specificity of the newly developed assays: (a) by confirming the exact expected size of the amplicon, which resulted in 201 bp product. (b) generation of consistent melt peak at 84.6 ± 0.15 (c) lack of cross reactivity with range of viruses which have genetic or structural similarities to PPRV (d) sequence analysis of PCR product, which showed high identity with sequences of PPRV isolates available in GenBank. Good reproducibility is an essential requirement for nucleic acid quantitation (Vaerman et al., 2004) and a mean CV value that is lower than 5% is acceptable (Amer and Almajhdi, 2011). In light of this, the assay reported here can generate reproducible results.
Fig. 2. The detection limit and standard curve of M gene based real time RT-PCR assay based on plasmid copy number. (a) Amplification plot for 10-fold serial dilutions ranging from 2.88 × 107 to 2.88 × 100 copies/l plasmid DNA. (b) Melting curve analysis (c) standard curve.
T. Abera, A. Thangavelu / Journal of Virological Methods 209 (2014) 25–29
The spiking assays showed that there was no measurable inhibition in the spiked negative nasal swabs when compared to pure tissue culture virus. This indicated that the PCR performance was the same in the spiked clinical samples and the tissue culture propagated virus. The assay was validated on field clinical samples. The results suggested that the SYBR Green I based real time RT-PCR assay has better diagnostic applicability than conventional RT-PCR. In summary, the assay developed in this study provides highly sensitive, specific, and reproducible results for detection and quantitation of PPRV nucleic acid in clinical samples. Acknowledgment This work was supported by National Agricultural Development Project in India (NADP). We are grateful to Dr. N. Daniel Joy Chandran for his unreserved help in facilitating the work. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.jviromet. 2014.08.017. References Adams, P.S., 2006. Data analysis and reporting. In: Dorak, M.T. (Ed.), Real Time PCR. Taylor & Francis Group, Newcastle, UK, pp. 40–62. Amer, H.M., Almajhdi, F.N., 2011. Development of a SYBR Green I based real-time RT-PCR assay for detection and quantification of bovine coronavirus. Mol. Cell. Probes 25, 101–107.
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Bailey, D., Banyard, A.C., Dash, P., Ozkul, A., Barrett, T., 2005. Full genome sequence of peste des petits ruminants virus, a member of the Morbillivirus genus. Virus Res. 110, 119–124. Balamurugan, V., Sen, A., Venkatesan, G., Yadav, V., Bhanot, V., Bhanuprakash, V., Singh, R.K., 2010. Application of semi-quantitative M gene-based hydrolysis probe (TaqMan) real-time RT-PCR assay for the detection of Peste des petites ruminants virus in the clinical samples for investigation into clinical prevalence of disease. Transbound. Emerg. Dis. 10, 1682–1865. Balamurugan, V., Sen, A., Saravanan, P., Singh, R.P., Singh, R.K., Rasool, T.J., Bandhyopadhyay, S.K., 2006. One-step multiplex RT-PCR assay for the detection of PPR virus in clinical samples. Vet. Res. Commun. 30, 566–666. Bao, J., Li, L., Wang, Z., Barrett, T., Suo, L., Zhao, W., Liu, Y., Liu, C., Li, J., 2008. Development of one-step real-time RT-PCR assay for detection and quantitation of pest des petits ruminants virus. J. Virol. Methods 148, 232–236. Batten, C.A., Banyard, A.C., King, D.P., Henstock, M.R., Edwards, L., Sander, A., Buczkowski, H., Oura, C., Barrett, T., 2011. A real time RT-PCR assay for the specific detection of peste des petits ruminants virus. J. Virol. Methods 171, 401–404. Couacy-Hymann, E., Roger, F., Hurard, C., Guillou, J.P., Libeau, G., Diallo, A., 2002. Rapid and sensitive detection of peste-des-petits-ruminants virus by a polymerase chain reaction assay. J. Virol. Methods 100, 17–25. Dhar, P., Sreenivasa, B.P., Barrett, T., Corteyn, M., Singh, R.P., Bandyopadhyay, S.K., 2002. Recent epidemiology of peste des petits ruminants virus (PPRV). Vet. Microbiol. 88, 153–159. Forsyth, M.A., Barrett, T., 1995. Evaluation of polymerase chain reaction for the detection and characterization of rinderpest and peste des petits ruminants viruses for epidemiological studies. Virus Res. 39, 151–163. Kwiatek, O., Keita, D., Gil, P., Fernandez-Pinero, J., Clavero, M.A., Albina, E., Libeau, G., 2010. Quantitative one-step real-time RT-PCR for the fast detection of the four genotypes of PPRV. J. Virol. Methods 165, 168–177. Reed, L.J., Muench, A.H., 1938. A simple method of estimating fifty percent end points. Am. J. Hyg. 27, 493–497. Schmittgen, T.D., Zakrajsek, B.A., Mills, A.G., Gorn, V., Singer, M.J., Reed, M.W., 2000. Quantitative reverse transcription-polymerase chain reaction to study mRNA decay: comparison of endpoint and real-time methods. Anal. Biochem. 285 (2), 194–200. Vaerman, J.L., Saussoy, P., Ingargiola, I., 2004. Evaluation of real time PCR data. J. Biol. Regul. Homeost. Agents 18, 212–214.
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Journal of Virological Methods journal homepage: www.elsevier.com/locate/jviromet
Development of a two-step SYBR Green I based real time RT-PCR assay for detecting and quantifying peste des petits ruminants virus in clinical samples Tsegalem Abera ∗ , Ardhanary Thangavelu Department of Veterinary Microbiology, Madras Veterinary College, Chennai 600007, India
a b s t r a c t Article history: Received 29 June 2014 Received in revised form 6 August 2014 Accepted 12 August 2014 Available online 4 September 2014 Keywords: PPRV SYBR Green I Real time RT-PCR Matrix gene
A two-step SYBR Green I based real time RT-PCR targeting the matrix (M) gene of Peste des petits ruminants virus (PPRV) was developed. The specificity of the assay was assessed against viral nucleic acid extracted from a range of animal viruses of clinical and structural similarities to PPRV including canine distemper virus, measles virus, bluetongue virus and Newcastle disease virus. But none of the viruses and no template control showed an amplification signal. Sensitivity of the same assay was assessed based on plasmid DNA copy number and with respect to infectivity titre. The lower detection limit achieved was 2.88 plasmid DNA copies/l with corresponding Ct value of 35.93. Based on tissue culture infectivity titre the lower detection limits were 0.0001TCID50 /ml and 1TCID50 /ml for the SYBR green I based real time RT-PCR and conventional RT-PCR, respectively. The calculated coefficient of variations values for intra- and inter-assay variability were low, ranging from 0.21% to 1.83% and 0.44% to 1.97%, respectively. The performance of newly developed assay was evaluated on a total of 36 clinical samples suspected of PPR and compared with conventional RT-PCR. The SYBR Green I based real time RT-PCR assay detected PPRV in 32 (88.8%) of clinical samples compared to 19 (52.7%) by conventional RT-PCR. Thus, the two-step SYBR Green I based real time RT-PCR assay targeting the M gene of PPRV reported in this study was highly sensitive, specific and reproducible for detection and quantitation of PPRV nucleic acids. © 2014 Elsevier B.V. All rights reserved.
1. Introduction Peste des petits ruminants (PPR), also known as ‘goat plague’, is a viral disease of goats and sheep characterized by fever, sores in the mouth, diarrhoea, pneumonia, and sometimes death (OIE, 2010). PPR was first identified in West Africa in the 1940s, and can now be detected in a broad belt of sub-Saharan Africa, Arabia, the Middle East and Southern Asia (Dhar et al., 2002). The causative agent peste-des-petits ruminants virus (PPRV) is classified as a member of the genus Morbillivirus, family Paramyxoviridae under the order Mononegavirales. It has a negative sense single-stranded RNA genome encoding eight proteins (Bailey et al., 2005). Several conventional reverse transcription polymerase chain reactions (RT-PCRs) are available for detection of PPRV genomic material (Forsyth and Barrett, 1995; Couacy-Hymann et al., 2002;
∗ Corresponding author at: College of Veterinary Medicine, Jigjiga University, Jijiga, Ethiopia Po. Box 1020. Tel.: +251 913 387416; fax: +251 25 775 5947. E-mail address: [email protected] (T. Abera). http://dx.doi.org/10.1016/j.jviromet.2014.08.017 0166-0934/© 2014 Elsevier B.V. All rights reserved.
Balamurugan et al., 2006). However, these conventional RT-PCR assays are labour intensive, as they require gel analysis for the detection of PCR products with a consequent high risk of contamination (Bao et al., 2008). Real time RT-PCR has gained wider acceptance over conventional RT-PCR because it is more rapid, sensitive and reproducible. Few real time RT-PCR assays have been described for detection and quantitation of PPRV in clinical samples using TaqMan chemistry (Bao et al., 2008; Balamurugan et al., 2010; Kwiatek et al., 2010; Batten et al., 2011). In comparison with TaqMan-PCR, SYBR Green I based real-time RT-PCR assay has the advantages of being more cost-effective, easy to design, more precise and produce a more linear decay plot (Schmittgen et al., 2000). The use of M gene based two-step SYBR Green I real time RT-PCR for molecular diagnosis of PPR has not been reported so far. The availability of such technique will provide an alternative to the N gene based real-time RT-PCR diagnostic assays that already exist (especially in terms of its sensitivity); thereby helping in rapid clinical diagnosis of PPR. In this regard, the present study developed a two-step SYBR Green I based real time RT-PCR assay for the detection and quantitation of PPRV in clinical samples.
26
T. Abera, A. Thangavelu / Journal of Virological Methods 209 (2014) 25–29
consisted of 70 ◦ C for 15 s, and followed by temperature increase to 95 ◦ C for 15 s at the rate of 1.25 ◦ C/s with continuous reading of fluorescence.
2. Materials and methods 2.1. Viruses, cells and clinical samples Viruses used in this study are listed in Table 1. Vero cells were used for virus propagation and titration. A total of 36 field samples from PPR suspected outbreaks from different areas of the Indian state of Tamil Nadu were analyzed. Samples received for analyses from different vet clinics were ocular, nasal, oral and rectal swabs, spleen, kidney, lung, lymph nodes and dung.
2.5. Conventional RT-PCR Conventional RT-PCR was performed on the cDNA preparations of the PPRV with a primer set of MF-Morb (5 -CTT GAT ACTC CCC AGA GATTC-3 ) and MRPPR3 (5 -TTC TCC CAT GAG CCG ACT ATGT3 ) as described by Balamurugan et al. (2006) which yielded 191 bp of PCR product. Amplification of target sequences was performed in a 2720 thermal cycler (Applied Biosystems).
2.2. Viral RNA extraction and cDNA synthesis 2.6. Generation of quantification standards Viral RNA was extracted from tissue culture supernatant using QIAamp® Viral RNA Mini Kit (Qiagen® , Germany), following manufacturer’s instructions. Trizol reagent (Invitrogen, CA, USA) was also used to isolate RNA from clinical samples. Synthesis of cDNA was carried out in 20 l reaction using RevertAidTM H minus first Strand cDNA Synthesis kit (Fermentas, USA) following manufacturer’s instructions. Briefly, 11 l of the purified RNA were added to a mixture containing 4 l RT buffer (5×), 1 l of random hexamer, 2 l 10 mM dNTP, 1 l RNase inhibitor and l l of Moloney murine leukaemia virus reverse transcriptase. The reaction was carried out at 25 ◦ C for 5 min, 42 ◦ C for 60 min and 70 ◦ C for 5 min and stored at −40 ◦ C until use. 2.3. Primer design and synthesis The forward and reverse primers were designed according to the sequences of matrix protein (M) gene (GenBank accession no. GQ452014.1) of PPRV Sungri-96 strain by using FastPCR software. The designed primers (Forward M3 F: 5 GGAGTGATTGAGGATAACGACC-3 (169–190) and Reverse M3 R: 5 -GCGTTAACAAGGACAGCGGAG-3 (350–370) were validated by OligoAnalyzer 1.2 and synthesized commercially (Sigma Aldrich, Bangalore, India). 2.4. Real time RT-PCR All reactions were performed using Realplex4 real time PCR machine (Eppendorf, Germany) using SYBR premix Ex Taq, (TaKaRa Bio Inc., Japan). The PCR was set up in a 10 l reaction volume containing 5 l of 2× SYBR Premix Ex Taq master mix, 1.5 l of cDNA, 1 l (10 M) of each primer and 2.5 l nuclease free water. The optimized cycling conditions were as follows: initial denaturation at 94 ◦ C for 5 min, followed by 40 cycles of denaturation at 94 ◦ C for 20 s, primer annealing at 56 ◦ C for 30 s, and extension at 72 ◦ C for 20 s. The fluorescence was measured at the end of each cycle. A melt curve analysis was performed following amplification to verify the specificity of the amplified products. Melting curve analysis
A fragment of 348 bp of M gene of PPRV containing the real time RT-PCR primers binding sites were amplified using the primer pair M4 F (forward primer): 5 CCAGGTAAGGGTCATCGATCC-3 (99–119) and M4 R (reverse primer): 5 -AAGCGGGACTAGGTTGACTGCA-3 (426–447) (In house designed). The RT-PCR product was cloned into T&A cloning vector according to the manufacturer’s instructions. Plasmid DNA was recovered from the transformed Escherichia coli BL-21 cells using AxyPrep Plasmid Miniprep Kit (Axygen Biosciences). The OD value of the plasmid DNA standard concentrations was measured at 260 nm/280 nm on Thermo Scientific NanoDropTM 1000 Spectrophotometer (NanoDrop Technologies, LLC, Wilmington, DE, USA). Plasmid copy number was calculated using the formula described by Adams (2006). The concentration of the obtained plasmid DNA was 95 ng/l which equates 2.88 × 1010 copies/l. 2.7. Specificity and sensitivity of the real time RT-PCR The specificity of the developed assay was assessed against viral nucleic acid extracted from a range of animal viruses of clinical and structural relevance to PPRV. The sensitivity of the assay was determined by running 10-fold serial dilutions of the plasmid standard in duplicates. Additionally, the sensitivity of the assay was evaluated by titrating PPRV Combatoire strain on Vero cells in a 96-well microtitre plate using standard cell culture procedure and the virus titre was calculated using Reed and Muench (1938) formula. PPRV having a titre of 105 TCID50 /ml was diluted 10-fold serially from 10−1 and 10−11 , and total RNA was extracted from each dilution, subsequently cDNA synthesis was performed. 2.8. Reproducibility The DNA standard ranging from 2.88 × 107 copies/l to 2.88 × 100 copies/l was tested repeatedly. Three separate dilution series were assayed in a single run to evaluate intra-assay
Table 1 Viruses used in the study. Virus
Strain
Source
Peste des petits ruminants virus (PPRV)
AR 87 vaccine strain Sunguri vaccine strain Coimbatore vaccine strain Coimbatore field isolate Recent field isolates Vaccine strain Vaccine strain Vaccine strain D58
Dept. of Vet. Microbiology, Madras Veterinary College Dept. of Vet. Microbiology, Madras Veterinary College Dept. of Animal Biotechnology, Madras Veterinary College Dept. of Vet. Microbiology, Madras Veterinary College Dept. of Vet. Microbiology, Madras Veterinary College Nobivac Puppy DP, Intervet Serum institute of India LTD, Pune Dept. of Vet. Microbiology, Madras Veterinary College Dept. of Vet. Microbiology, Madras Veterinary College
Canine distemper virus (CDV) Measles virus (MV) Bluetongue virus (BTV) Newcastle disease virus (NDV)
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variations. Whereas, the inter-assay variations were measured by testing each dilution in three separate consecutive runs. The mean, standard deviation (SD) and coefficient of variation (CV) for both intra-assay and inter-assay variations were calculated separately for each standard DNA dilution based on their Ct values using Microsoft Excel software. 2.9. Spiking The performance of the newly developed assay was checked using clinical samples (2 nasal swabs from goats) that were spiked with known titre of PPR virus as described by Balamurugan et al. (2010). Infected cell culture fluid containing 105 TCID50 /ml viruses was diluted 1: 10 serially by using the two negative nasal swabs as diluent. The 103 and 104 TCID50 /ml spiked titre viruses were used for total RNA extraction. Control virus samples (103 and 104 TCID50/ ml titre virus suspensions) were also used for RNA extraction. 3. Results 3.1. Specificity The assay exhibited specificity as an amplification signal was observed only with PPRV nucleic acid. None of the viruses which have genetic or clinical similarities with PPRV (CDV, MV, BTV, and NDV) and no template control (NTC) showed an amplification signal. Vaccine and field isolates of PPRV (Arasur, Sunguri, Coimbatore and recent field isolates) were amplified in the real-time RT-PCR and all were positive with the melting point of 84.6 ± 0.15 (Fig. 1). 3.2. Detection limit A series of 10-fold dilutions of the plasmid DNA starting from 2.88 × 107 to 2.88 × 100 copies/l were prepared to construct standard curve. A linear regression relationship between the plasmid dilutions and the threshold cycle (Ct) values was observed with a coefficient of determination (R2 ) of 0.9947 and a slope of −3.0763. The lower detection limit achieved was 2.88 copies/l with corresponding Ct value of 35.93 (Fig. 2). The lower detection limit based on tissue culture infectivity titre was 0.0001 TCID50 /ml with a corresponding Ct value of 31.9 (data not shown).
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Table 2 Comparative evaluation of clinical samples suspected of PPRV using conventional and real time RT-PCR. Specimen type
Conventional RT-PCR result
RT-PCR PPRV (Ct ± S.D)a
Lung Ocular swab Oral swab Spleen PLN Spleen Mediastinal LN Nasal swab Nasal swab Dung Mesenteric LN Ocular swab Lung Nasal swab Nasal swab Nasal swab Nasal swab Nasal swab Ocular swab Oral swab Spleen Nasal swab Mandibular LN Ocular swab Rectal swab Kidney Nasal swab Ocular swab Mesenteric LN Oral swab Rectal swab Mesenteric LN Rectal swab Ocular swab Lung Spleen
+ + + + − + + − − − + + + + + + + + + − − + + + − + − − − − − − − − − −
13.90 19.05 19.72 20.13 20.23 20.69 20.52 20.77 20.84 21.02 22.05 22.18 22.46 23.55 24.38 24.58 25.02 25.91 26.21 27.61 26.11 28.19 28.75 28.33 29.92 29.72 30.73 24.40 31.90 31.84 32.31 36.31 – – – –
a
± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±
0.81 0.34 0.33 0.03 0.05 0.05 0.26 0.08 0.10 0.36 0.06 0.19 0.17 0.20 0.06 0.33 0.28 0.01 0.05 0.01 1.85 0.16 0.06 1.14 0.03 0.27 0.00 6.99 0.39 1.10 0.69 2.07
Results are displayed in ascending order of Ct values.
deviation (SD) and coefficient of variation (CV) values were, ranging from 0.05 to 0.56 and from 0.21 to 1.83%, respectively. The interassay CV and SD were in the range of 0.44 to 1.97% and 0.08 to 0.61, respectively. 3.4. Spiking
3.3. Reproducibility The intra-assay reproducibility was analyzed by using 10-fold dilutions of the plasmid DNA standard ranging from 2.88 × 107 to 2.88 × 100 copies/l in triplicates per run. The calculated standard
Spiking assay with 104 and 103 TCID50 /ml of cell culture infected PPRV was performed on two negative goat nasal swabs. The mean Ct values obtained for the spiked clinical samples and tissue culture virus were compared. Negative clinical samples spiked with known
Fig. 1. Specificity of M gene based real time RT-PCR: (a) amplification plot representing PPRV, CDV, MV, BTV, NDV and No template control. (b) Melting curve analysis.
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titre of PPRV and tissue culture virus with same titre were equally well detected by the assay. 3.5. Assay performance on clinical samples The competence of the SYBR Green-based real time RT-PCR assay for the detection of PPR virus in clinical samples was evaluated by analysis of 36 samples from PPR suspected cases. Comparison of the newly developed real time RT-PCR assay was made with gel-based conventional RT-PCR assay. The results of both assays were similar for the evaluation of 23 samples. However, 13 samples tested negative by gel based RT-PCR were found to be positive by real time RT-PCR. None of the samples tested negative by real-time RT-PCR was found positive by the gel based PCR (Table 2). 4. Discussion In this study, RT-PCR conditions for efficient amplification and quantification of PPRV nucleic acid sequences of M gene in clinical samples using real time fluorescence measurement were defined. Overall, this newly developed SYBR Green I based real time RT-PCR assay had detection limit that was significantly lower than that of
conventional RT-PCR while maintaining a high level of specificity and reproducibility. The generated standard curve can be applicable for accurate quantification of PPRV. The assay maintained linearity over seven orders of magnitude. The assay showed high sensitivity with the ability of quantifying PPRV nucleic acid to as few as 2.88 copies/l. This can be useful in detecting low levels of PPRV and confirming early stage of PPRV infection where the virus titres are very low. Since the main drawbacks of using DNA binding dyes such as SYBR Green I is detecting any double stranded DNA generated during PCR. In this study, several approaches were followed to cross-check the specificity of the newly developed assays: (a) by confirming the exact expected size of the amplicon, which resulted in 201 bp product. (b) generation of consistent melt peak at 84.6 ± 0.15 (c) lack of cross reactivity with range of viruses which have genetic or structural similarities to PPRV (d) sequence analysis of PCR product, which showed high identity with sequences of PPRV isolates available in GenBank. Good reproducibility is an essential requirement for nucleic acid quantitation (Vaerman et al., 2004) and a mean CV value that is lower than 5% is acceptable (Amer and Almajhdi, 2011). In light of this, the assay reported here can generate reproducible results.
Fig. 2. The detection limit and standard curve of M gene based real time RT-PCR assay based on plasmid copy number. (a) Amplification plot for 10-fold serial dilutions ranging from 2.88 × 107 to 2.88 × 100 copies/l plasmid DNA. (b) Melting curve analysis (c) standard curve.
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The spiking assays showed that there was no measurable inhibition in the spiked negative nasal swabs when compared to pure tissue culture virus. This indicated that the PCR performance was the same in the spiked clinical samples and the tissue culture propagated virus. The assay was validated on field clinical samples. The results suggested that the SYBR Green I based real time RT-PCR assay has better diagnostic applicability than conventional RT-PCR. In summary, the assay developed in this study provides highly sensitive, specific, and reproducible results for detection and quantitation of PPRV nucleic acid in clinical samples. Acknowledgment This work was supported by National Agricultural Development Project in India (NADP). We are grateful to Dr. N. Daniel Joy Chandran for his unreserved help in facilitating the work. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.jviromet. 2014.08.017. References Adams, P.S., 2006. Data analysis and reporting. In: Dorak, M.T. (Ed.), Real Time PCR. Taylor & Francis Group, Newcastle, UK, pp. 40–62. Amer, H.M., Almajhdi, F.N., 2011. Development of a SYBR Green I based real-time RT-PCR assay for detection and quantification of bovine coronavirus. Mol. Cell. Probes 25, 101–107.
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