Journal of Virological Methods 222 (2015) 34–40
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Development of a SYBR Green real-time PCR assay with melting curve analysis for simultaneous detection and differentiation of canine adenovirus type 1 and type 2 Andrea Balboni, Francesco Dondi, Santino Prosperi, Mara Battilani ∗ Department of Veterinary Medical Sciences, Alma Mater Studiorum-University of Bologna, Via Tolara di Sopra 50, 40064 Ozzano Emilia (BO), Italy
a b s t r a c t Article history: Received 4 March 2015 Received in revised form 11 May 2015 Accepted 21 May 2015 Available online 29 May 2015 Keywords: Canine adenovirus Dog Infectious canine hepatitis Infectious tracheobronchitis Melting curve analysis Real-time PCR
Canine adenovirus type 1 (CAdV-1) and canine adenovirus type 2 (CAdV-2) cause infectious canine hepatitis (ICH) and infectious tracheobronchitis (ITB) in dogs, respectively. Cases of ICH have been documented in recent years and recent surveys have demonstrated a wide percentage of asymptomatic CAdV-1 infection in the canine population. Since both CAdV types are detectable in the same biological matrices, and viral coinfection with CAdV-1 and CAdV-2 are reported with high frequency, it is urgent to have available a rapid, highly sensitive and specific assay for the diagnosis of CAdV infection and distinction between CAdV-1 and CAdV-2. In order to detect canine adenovirus in biological samples and to rapidly distinguish the two viral types, a SYBR Green real-time PCR assay was optimized to discriminate CAdV-1 and CAdV-2 via a melting curve analysis. The developed assay showed high sensitivity and reproducibility and was highly efficient and specific in discriminating the two CAdV types. This reliable and rapid technique may represent a simple, useful and economic option for simultaneous CAdV types detection, which would be feasible and attractive for all diagnostic laboratories, both for clinical purposes and for epidemiological investigations. © 2015 Elsevier B.V. All rights reserved.
1. Introduction Canine adenovirus type 1 (CAdV-1) is the aetiologic agent of infectious canine hepatitis (ICH), a severe systemic disease in dogs characterized by acute necrohaemorragic hepatitis (Greene, 2012). Canine adenovirus type 2 (CAdV-2) is one of the viral agents implicated in the aetiopathogenesis of infectious tracheobronchitis (ITB), also known as kennel cough, a mild self-limiting acute upper respiratory disease in dogs (Ford, 2012) which has a high prevalence in canine populations. The widespread use of a modified live CAdV-2 vaccine in countries like Italy has greatly reduced the incidence of
Abbreviations: CAdV, canine adenovirus; CAdV-1, canine adenovirus type 1; CAdV-2, canine adenovirus type 2; ICH, canine hepatitis; ITB, infectious tracheobronchitis; qPCR, quantitative real-time PCR; MCA, melting curve analysis; Tm , melting temperatures; LOD, limit of detection; SD, standard deviation; CV, coefficient of variation; R2 , coefficient of determination; E, reaction efficiency; S, slope; SNPs, single nucleotide polymorphisms; HRM, high-resolution melting curve. ∗ Corresponding author. Tel.: +39 051 2097081; fax: +39 051 2097039. E-mail address: [email protected] (M. Battilani). http://dx.doi.org/10.1016/j.jviromet.2015.05.009 0166-0934/© 2015 Elsevier B.V. All rights reserved.
ICH in the domestic canine population (Abdelmagid et al., 2004; Bass et al., 1980; Decaro et al., 2008). Infectious canine hepatitis is nowadays considered a neglected canine disease and veterinary practitioners rarely take into account CAdV-1 as causative agent of disease. Nevertheless, cases of ICH and of asymptomatic CAdV-1 infections have been documented in recent years in foxes and dogs (Balboni et al., 2013, 2014; Caudell et al., 2005; Decaro et al., 2007; Headley et al., 2013; Müller et al., 2010; Pratelli et al., 2001; Thompson et al., 2010), confirming that the CAdV-1 continues to circulate and to be pathogenic in dogs. Furthermore, CAdV types are detectable in the same biological matrices (Balboni et al., 2013, 2014; Decaro et al., 2004; Greene, 2012) and viral coinfection with CAdV-1 and CAdV-2 are detectable with high frequency (Balboni et al., 2013, 2014; Headley et al., 2013). For these reasons, the diagnosis of CAdV-1 infection and the distinction of this virus from CAdV-2 is important for both clinical and epidemiological investigations. Canine adenovirus types are distinguishable by genetic, antigenic and pathogenetic characteristics but there is immunological cross-reactivity among them (King et al., 2011). Serologic methods have limited application in the diagnostic protocols because they
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Table 1 Field CAdV strains used for assay development and validation. Virus CAdV-1 09-13 113-5 384 417 574 CAdV-2 60 S1 243 244 249 252 260 270 272 303
Year of sampling
Host
Biological matrix
GenBank accession number
References
2011 2011 1966 2013 2013
Fox Fox Dog Dog Dog
Faeces Liver FFPE-liver Liver RS
JX416838 JX416839 KP670422 KP670423 KP670424
Balboni et al. (2013) Balboni et al. (2013) – – –
2011 2011 2012 2012 2012 2012 2012 2012 2012 2012
Dog Dog Dog Dog Dog Dog Dog Dog Dog Dog
CS OPS urine RS RS RS RS RS RS RS
KF676978 KF676979 – – KF676981 – – – KF676982 –
– – Balboni et al. (2014) Balboni et al. (2014) Balboni et al. (2014) Balboni et al. (2014) Balboni et al. (2014) Balboni et al. (2014) Balboni et al. (2014) Balboni et al. (2014)
FFPE-liver: formalin-fixed paraffin-embedded liver. OPS: oro-pharyngeal swab. CS: cell supernatant. RS: rectal swab. References: viruses 384, 417, 574, 60 and S1 were identified in our lab and nucleotide sequences were directly submitted to GenBank database.
are mostly unable to distinguish the two viruses, and also for the widespread use of a modified live CAdV-2 vaccine. The molecular biology-based methods are more reliable but, to date, only one PCR assay able to distinguish the two CAdV types was developed (Hu et al., 2001; Chaturvedi et al., 2008). In order to reduce the time of execution and, at the same time, to increase the sensitivity of the molecular diagnosis, a SYBR Green real-time PCR (qPCR) assay was developed that is able to detect and to distinguish between CAdV-1 and CAdV-2 via a melting curve analysis (MCA). 2. Materials and methods 2.1. Experimental design and field CAdV strains used for assay development and validation
EF057101; M60937; S38238; JX416838; JX416839) and aligned using the ClustalW method implemented in BioEdit software version 7.2.5. A couple of degenerated primers overlapping E3 gene and flanking regions were obtained (Table 2). A BLAST search (http://blast.ncbi.nlm.nih.gov/Blast.cgi) was performed in order to verify the specificity of the selected primers. OligoAnalyzer (http://eu.idtdna.com/analyzer/applications/oligoanalyzer/ 3.1 ) and Oligo Calc (http://www.basic.northwestern.edu/biotools/ OligoCalc.html) web interfaces were used to exclude secondary structure and primer dimers. The designed primers amplify a product of 166 bp showing numerous nucleotide mutations between CAdV-1 and CAdV-2 (≥47 nucleotide substitutions, corresponding to ≥28.3% of the amplified DNA fragment) (Fig. 1), which implies a difference in Tm values between the two CAdV types. 2.4. Preparation of DNA standards
SYBR Green qPCR was developed and validated for the simultaneous detection and typing of CAdV-1 and CAdV-2 on the basis of melting temperature analysis of produced amplicons. Five CAdV-1 and 10 CAdV-2 strains reported in Table 1 were used to assess the analytical performance of the MCA-based SYBR Green qPCR and to validate the developed assay. Field viruses were previously identified in our laboratory by testing different biological matrices and using a PCR assay we were able to differentiate between the two adenovirus types (Chaturvedi et al., 2008; Hu et al., 2001). All the obtained PCR products were sequenced to confirm the CAdV typing. 2.2. DNA extraction Viral DNA extraction from organs, urine, rectal swabs, oropharyngeal swabs and cell supernatants was performed using the NucleoSpin Tissue Mini Kit (Macherey-Nagel, Düren, Germany) according to the manufacturer’s instructions. For formalin-fixed paraffin-embedded tissue samples, the deparaffinisation was performed by using the Solvent Plus reagent (Carlo Erba, Rodano, MI, Italy). The extracted DNA was eluted in 100 l of elution buffer and stored at −20 ◦ C.
Two pCR4 plasmids (Invitrogen, Carlsbad, CA, USA) containing one copy of the CAdV-1 and CAdV-2 target sequences, respectively, were produced for optimization of the real-time PCR assay and for construction of the assay standard curve. The CAdV-1 amplicon of 508 bp and the CAdV-2 amplicon of 1030 bp of the E3 gene and flanking regions obtained from dog 300-2012 (Balboni et al., 2014) were used as target sequences. The CAdV-1 and CAdV-2 DNA fragments were cloned into the pCR4 vector using the TOPO TA Cloning Kit (Invitrogen, Leek, the Netherlands), according to the manufacturer’s instructions. The resulting recombinant plasmid purification was carried out using the PureYield Plasmid Miniprep System (Promega, Madison, WI, USA) and the plasmids were linearized above the CAdV fragment sequence using restriction endonuclease Spe I (Fermentas, Burlington, Ontario, Canada) in order to avoid the presence of supercoiled plasmid DNA. The concentration and purity of the standard plasmids were assessed by measuring absorbance at 260 nm in a UV spectrophotometer (BioPhotometer, Eppendorf, Hamburg, Germany) and using the 260/280 nm ratio. The copy number of standard plasmids was calculated using the equation described by the US Environmental Protection Agency protocol (US Environmental Protection Agency, 2004).
2.3. Primer design 2.5. SYBR Green real-time PCR For appropriate primer design, sequences of 12 reference CAdV strains were retrieved from the GenBank database (http:// www.ncbi.nlm.nih.gov/genbank; accession numbers: AC 000020; S38212; JX416841; JK416842; AC 00003; GQ340423; U55001;
The real-time PCR was performed using the Rotor-Gene SYBR Green PCR Kit (QIAGEN, Hilden, Germany) and the Rotor-Gene 3000 system (Corbett Research, Mortlake, NSW, Australia). The
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Table 2 Primers used for the SYBR Green qPCR assay. Primer
Nucleotide sequence (5 –3 )
Nt. position in CAdV-1
Nt. position in CAdV-2
Fragment amplified (E3 and U-exon genes)
CAdV-qPCR-For3 CAdV-qPCR-Rev2
CTGASACTGCWATRMCTATATAYATTTCCA GACATAGARACRCAGGACCCAGA
25600–25626 25743–25765
26305–26334 26448–26470
166 bp
Nucleotide positions are referring to the following reference strains: RI261, GenBank Accession number AC 000003 (CAdV-1) and TorontoA26/61, GenBank Accession number AC 000020 (CAdV-2).
fluorescence signal was acquired on the FAM channel (multichannel machine, source 470 nm; detector 510 nm; gain set to 5) with a fluorescence reading taken at the end of each elongation step. Different annealing temperature (from 50 to 60 ◦ C) and primers concentration (from 1 to 1.4 M) were tested to optimize the assay performance. The optimized SYBR Green real-time PCR was performed in a final volume of 25 l containing 12.5 l of 2× Rotor-Gene SYBR Green Master Mix, 1 M of forward (CAdVqPCR-For3) and reverse (CAdV-qPCR-Rev2) primers, and 2.5 l of template DNA. Autoclaved nanopure water was added to arrive at a final volume of 25 l. Each run consisted of an initial incubation for activation of the hot-start DNA polymerase at 95 ◦ C for 5 min followed by 45 cycles of denaturation at 95 ◦ C for 10 s, annealing at 59 ◦ C for 20 s and polymerization at 60 ◦ C for 15 s. The melting experiments were performed after the last extension step; the temperature was increased by increments of 0.2 ◦ C from 60 to 95 ◦ C every 5 s. Melting curves were analyzed by the Rotor-Gene 6 software (Corbett Research, Mortlake, NSW, Australia) and the Tm was defined as the peak of the obtained curves. Specimens were considered positive if the fluorescence curve in the amplification plot showed an exponential increase, and a specific melting peak was observed.
2.6. Standard curve and limit of detection (LOD) The SYBR Green real-time PCR standard curve was generated for both CAdV-1 and CAdV-2 by serial 10-fold dilutions of the two recombinant plasmids with a known copy number (from 1 × 108 to 1 × 10−2 copies/l). These dilutions were tested in triplicate and used as quantification standards to construct the standard curve by plotting the plasmid copy number against the corresponding threshold cycle values. The threshold was determined using the Auto-Find Threshold function of the Rotor-Gene 3000, which scans the range of the threshold levels to obtain the best fit of the standard curve using the samples which have been defined as standards. A
melting curve analysis of the obtained amplification products was carried out. The LOD of the reaction was determined for both CAdV1 and CAdV-2 based on the highest dilution of respective plasmid possible to amplify with good reproducibility. In order to mimic realistic conditions, standard curves were also constructed using serial 10-fold dilutions of both CAdV-1 and CAdV-2 recombinant plasmids in negative extracts of representative biological matrices. A negative faecal extract was used for both CAdV-1 and CAdV-2 as representative sample for in vivo tests (Balboni et al., 2014). Instead, as representative samples for post mortem tests we used a negative liver extract for CAdV-1 and a negative lung extract for CAdV-2. 2.7. Diagnostic sensitivity and specificity The analytical sensitivity and efficiency of real-time PCR for the two CAdV types are reflected by the LOD, which was assessed as described above. The specificity of the assay was evaluated by testing, in triplicate, two field CAdV-1 (417/2013 and 574/2013) and two field CAdV-2 (60/2011 and S1/2011). To further determine the specificity of the amplification reaction, other DNA viruses affecting dogs and cats were tested: the canine parvovirus type 2 (Protoparvovirus, CPV-2b/461/2009, GenBank ID: KF373599), the canid herpesvirus 1 (Varicellovirus, 643/2014, a field virus identified in our lab), the canine oral papillomavirus (Lambdapapillomavirus, 146/2012, a field virus identified in our lab), the feline panleukopenia virus (Protoparvovirus, 1033/2009, Battilani et al., 2011) and the felid herpesvirus 1 (Varicellovirus, 10/2011, a field virus identified in our lab). To verify the specificity, a melting curve analysis and electrophoresis on 2% (w/v) agarose gel stained with ethidium bromide in 1× standard tris-acetate-EDTA (TAE) buffer were carried out for the amplification products. Furthermore, the genotype identification was confirmed by sequencing the products obtained to CAdV-1 and CAdV-2 recombinant plasmids.
Fig. 1. Multiple sequence alignment of primer binding sites. Alignment of the genomic tract comprising part of the E3 and U-exon genes amplified by the designed primers. The alignment include 4 CAdV-2 and 8 CAdV-1 reference strains. In grey: designed primers.
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Table 3 Intra- and inter-assay variability of the real-time PCR. Samples Intra-assay variability A1 A2 A3 A4 A5 A6 A7 A8 B1 B2 B3 B4 B5 B6 B7 B8 417/2013 574/2013 60/2011 S1/2011 Inter-assay variability C1 C2 C3 D1 D2 D3 417/2013 574/2013 60/2011 S1/2011
Replicate (and assay) numbers
Mean (SD)
log10 Mean (SD)
log10 CV (%)
3 (1) 3 (1) 3 (1) 3 (1) 3 (1) 3 (1) 3 (1) 3 (1) 3 (1) 3 (1) 3 (1) 3 (1) 3 (1) 3 (1) 3 (1) 3 (1) 3 (1) 3 (1) 3 (1) 3 (1)
8.36E+07 (1.52E+06) 9.98E+06 (4.65E+05) 1.08E+06 (3.09E+04) 1.23E+05 (2.87E+03) 1.01E+04 (5.74E+02) 1.03E+03 (8.56E+01) 9.75E+01 (3.71) 9.06E+00 (1.47) 8.45E+07 (1.28E+06) 9.75E+06 (4.63E+05) 1.11E+06 (2.16E+04) 1.04E+05 (6.38E+03) 1.08E+04 (3.86E+02) 1.15E+03 (1.17E+02) 1.21E+02 (1.36E+01) 7.32E+00 (1.62) 1.19E+07 (8.98E+05) 9.99E+02 (6.65E+01) 1.46E+08 (1.27E+07) 1.84E+05 (2.26E+04)
7.92 (0.01) 7 (0.02) 6.03 (0.01) 5.09 (0.01) 4 (0.02) 3.01 (0.04) 1.99 (0.02) 0.95 (0.07) 7.93 (0.01) 6.99 (0.02) 6.04 (0.01) 5.02 (0.03) 4.03 (0.01) 3.06 (0.04) 2.08 (0.05) 0.85 (0.1) 7.07 (0.03) 3 (0.03) 8.16 (0.04) 5.26 (0.05)
0.1 0.29 0.21 0.2 0.61 1.22 0.83 7.61 0.08 0.29 0.14 0.52 0.38 1.41 2.28 12.38 0.46 0.97 0.45 1.05
3 (3) 3 (3) 3 (3) 3 (3) 3 (3) 3 (3) 3 (3) 3 (3) 3 (3) 3 (3)
1.47E+08 (4.55E+07) 1.28E+05 (1.84E+04) 1.15E+02 (1.99E+01) 1.57E+08 (4.16E+07) 7.14E+04 (2.98E+04) 1.15E+02 (1.37E+01) 1.08E+07 (1.34E+06) 6.82E+02 (2.24E+02) 1.76E+08 (3.8E+07) 7.21E+04 (2.46E+04)
8.14 (0.16) 5.1 (0.06) 2.05 (0.07) 8.18 (0.11) 4.82 (0.18) 2.06 (0.05) 7.03 (0.06) 2.81 (0.13) 8.24 (0.09) 4.83 (0.18)
1.92 1.2 3.5 1.36 3.67 2.48 0.8 4.71 1.07 3.66
A1–A8: 10-fold of CAdV-1 recombinant plasmid dilutions (from 1 × 108 to 1 × 101 respectively). B1–B8: 10-fold of CAdV-2 recombinant plasmid dilutions (from 1 × 108 to 1 × 101 respectively). C1–C3: CAdV-1 recombinant plasmid dilutions with high, medium and low concentration respectively. D1–D3: CAdV-2 recombinant plasmid dilutions with high, medium and low concentration respectively. Two CAdV-1 field samples: 417/2013 and 574/2013. Two CAdV-2 field samples: 60/2011 and S1/2011. SD: standard deviation. CV: coefficients of variation.
2.8. Intra- and inter-assay variability In order to determine the intra-assay variability of the technique for the standard plasmids and for samples, eight successive 10-fold dilutions (from 1 × 108 to 1 × 101 copies/l) of both CAdV1 and CAdV-2 recombinant plasmids, two CAdV-1 samples with different viral concentration (417/2013 and 574/2013), and two CAdV-2 samples with different viral concentrations (60/2011 and S1/2011) were tested in triplicate within the same run (Table 3). The inter-assay variability was evaluated by testing three dilutions with high, medium and low concentrations of both CAdV-1 and CAdV-2 recombinant plasmids, and the previous four CAdV samples in triplicate on three different days (Table 3). Mean, standard deviation (SD), and coefficient of variation (CV) were calculated. In particular, the CV was calculated as the percentage of the ratio of standard deviation and the mean values were obtained, in accordance with the US Environmental Protection Agency protocol (US Environmental Protection Agency, 2004). The repeatability of the melting temperature analysis was examined by comparing the results obtained from replicates of three dilutions with high, medium and low concentration of both CAdV-1 and CAdV-2 recombinant plasmids, and four CAdV samples (417/2013, 574/2013, 60/2011 and S1/2011) during a single qPCR reaction run (intra-assay) and comparing the mean values obtained from the same plasmids and samples on three different days (inter-assay) (Table 4). 2.9. Statistical analysis To evaluate whether MCA could be used to accurately type CAdV-1 and CAdV-2, Tm values of plasmids and samples
testing positive to the two viruses were compared using a twogroup Student’s t-test. 2.10. Detection and differentiation of CAdV type 1 and 2 in dog and fox samples using MCA-based SYBR Green qPCR After optimization of the SYBR Green qPCR assay, the 15 field CAdV samples listed in Table 1 were tested to validate the technique. For each run, duplicates of six 10-fold dilutions of the standard CAdV-2 plasmid, duplicates of the dog and fox DNA extracts, and a no template control were simultaneously subjected to analysis. The melting peak and Tm obtained from each sample were used to discriminate between CAdV-1 and CAdV-2. Samples were considered positive if the mean of the replicates was greater than the LOD. 3. Results 3.1. Development of MCA-based SYBR Green qPCR The linearity and efficiency of the SYBR Green real-time PCR were determined for CAdV-1 and CAdV-2 amplification by generating a standard curve for each of the two viruses in which serial 10-fold dilutions of recombinant plasmids were tested. The standard curve was generated by plotting the real-time PCR threshold cycle numbers (Ct ) of each dilution against the known copy numbers of recombinant plasmid. The resulting slope showed a linear relationship over 8 orders of magnitude ranging from 1 × 101 to 1 × 108 copies/l for both CAdV-1 and CAdV-2 standard plasmids. The slope was −3.36 with a coefficient of determination (R2 ) > 0.99 and a reaction efficiency (E) of 0.98 for CAdV-1 and −3.42 with a
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Table 4 Intra- and inter-assay variations of Tm values (◦ C) obtained from real-time PCR with melting curve analysis. Virus
Intra-assay variation Interval
CAdV-1 CAdV-2
76.04–76.56 80.96–81.3
Range 0.52 0.34
Inter-assay variation Mean 76.37 81.18
SD 0.16 0.08
R2 > 0.99 and an E of 0.96 for CAdV-2. The reaction efficiency was calculated from the slope (S) using E = 10(−1/s) − 1. The LOD determined on the CAdV-1 and CAdV-2 standard curves was found to be 0.049 fg (10 copies/l) and 0.055 fg (10 copies/l) respectively, thus showing a high sensitivity of the assay. Two distinct melting peaks were clearly visible for the CAdV1 and CAdV-2 standard plasmid dilutions (Fig. 2), indicating the formation of a single PCR product with no artefacts, such as non-specific amplification products or primer dimers (results not shown), for each of the two viruses, confirming the specificity of the reaction. The standard curves generated with 10-fold dilutions of CAdV-1 and CAdV-2 recombinant plasmids in negative extracts of representative biological matrices (faeces, liver and lung) showed linearity up to a LOD of 10 copies/l for both viruses and for all biological matrices. In tests performed on negative faecal extracts, the solitary specific melting peaks corresponding to CAdV-1 or CAdV2 amplification was showed without artefacts. In tests performed on negative liver or lung extracts, in addition to the specific peaks of CAdV amplification, some weak melting peaks at temperatures higher than 82.5 ◦ C were visible at plasmid concentrations below 103 copies/l; however, they did not affect the reading of specific melting peaks and these potential nonspecific products did not affect the amplification of target viral DNA, as demonstrated by the linearity of the standard curves. The developed assay was able to detect CAdV-1 (417/2013 and 574/2013) and CAdV-2 (60/2011 and S1/2011) samples. No positive results were obtained with any other DNA viruses tested (data not shown). Amplification products were also checked on
CV (%) 0.21 0.09
Interval
Range
Mean
SD
CV (%)
75.85–76.62 81.04–81.27
0.77 0.23
76.22 81.16
0.24 0.07
0.32 0.09
gel electrophoresis and a clear and well-defined specific band of approximately 166 bp was visualized for all replicates of the two recombinant plasmids and samples that tested positive in realtime PCR amplification. Furthermore, the nucleotide sequence of the products obtained from CAdV-1 and CAdV-2 recombinant plasmid amplification confirmed the genotype identification (data not shown). The intra-assay variability was determined, at first, on eight successive 10-fold dilutions (from 1 × 108 to 1 × 101 copies/l) of both CAdV-1 and CAdV-2 recombinant plasmids tested in triplicate and then, on two CAdV-1 and two CAdV-2 field samples with different viral concentrations tested in triplicate. The coefficient of variation (CV) that was obtained ranged from 0.08 to 2.28 for all the recombinant plasmids and samples with concentrations higher than or equal to 102 copies/l, while it increased for lower concentrations (Table 3). The determination of the inter-assay variability tested on three different dilutions of both CAdV-1 and CAdV-2 recombinant plasmids and two CAdV-1 and CAdV-2 field samples gave a CV ranging from 0.8 to 4.71 for all the viral concentrations tested (Table 3). The variability of plasmid dilutions and samples is therefore low for decreasing concentrations down to 102 copies/l and progressively higher for lower concentrations, as influenced by distribution statistics (Poisson’s law) which involves an increase in CV values for the quantification of low copy number. However, despite the higher variability, low viral concentrations were always detected in all repetitions using the SYBR Green assay. Furthermore, the standard deviation (SD) between the individual assays was below 0.25 log10 (Table 3), a level which is normally
Fig. 2. Discrimination of CAdV-1 and CAdV-2 by real-time PCR with melting curve analysis. In grey: signal obtained from the CAdV-1 standard plasmid. In black: signal obtained from the CAdV-2 standard plasmid. Derivative – dF/dT where F is the fluorescence and T is the time. deg: temperature (centigrade).
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considered the minimum requirement for acceptable reproducibility of quantitative molecular assays. The melting temperature analysis showed a variation of Tm values between samples of 76.37 ◦ C ± 0.16 and 81.18 ◦ C ± 0.08 for CAdV-1 and CAdV-2, respectively. Percentage of variation coefficient (%CV) between samples was 0.21 for CAdV-1 and 0.09 for CAdV-2 (Table 4). Instead, the variations of Tm values between runs were 76.22 ◦ C ± 0.24 with %CV of 0.32 for CAdV-1 and 81.16 ◦ C ± 0.07 with %CV of 0.09 for CAdV-2 (Table 4). The two-group t-statistic for the comparison between the Tm value obtained after MCA for CAdV-1 and CAdV-2 showed statistical differences (p < 0.001). 3.2. Validation of MCA-based SYBR Green qPCR Fifteen samples, previously identified by conventional methods as CAdV-1 (n = 5) and CAdV-2 (n = 10), were correctly amplified and detected with SYBR Green qPCR. Based on MCA, the five CAdV-1 were correctly typed with a Tm value ranging between 76.04 and 76.55 ◦ C, and the 10 CAdV-2 were correctly typed with Tm value ranged between 81.01 and 81.25 ◦ C.
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(HRM) analysis, entails the use of a HRM-capable real-time PCR device (able to accurately control the temperature to acquire fluorescence data at high density during DNA melting, and to perform automated HRM curve comparison) and a so-called “saturating” DNA binding dye (able to increase the resolution and accuracy of melting analysis) (Erali et al., 2008; Tong and Giffard, 2012). The real-time PCR with HRM assay allow to detect variants in the DNA sequence and it was applied to bacterial, viral or parasite typing (Ruskova and Raclavsky, 2011). The SYBR Green real-time PCR assay developed in this study amplified a 166 bp fragment of CAdV genome containing almost 47 nucleotide substitutions between CAdV-1 and CAdV-2 that produces a difference in Tm greater than 4 ◦ C between the two viral types. This considerable difference in CAdV-1 and CAdV-2 melting temperatures does not require the use of the HRM analysis allowing the use of a conventional real-time PCR device. Therefore, this reliable and rapid technique, given its extreme sensitivity and reproducibility, may represent a simple, useful and economic option for CAdV detection and type discrimination, which would be feasible and attractive for all diagnostic laboratories, even those with limited instrumentation, both for clinical purposes and for epidemiological investigations. References
4. Discussion In this paper, a SYBR Green real-time PCR method was optimized and evaluated in terms of specificity, sensitivity, amplification efficiency, standard curve linearity, and overall performance for detection and discrimination of the two CAdV types through the melting curve analysis. Although the probe-based real-time PCR assay provides greater specificity than the SYBR Green qPCR, a real-time instrument able to detect different wavelengths would be required to detect CAdV-1 and CAdV-2 in the same reaction using two specific probes labelled with different fluorophores. Furthermore, a probe-based real-time PCR reaction is more expensive than a SYBR Green qPCR reaction because it requires the use of probes in addition to the primers and nucleotide mutations in the hybridization sites of the probes can affect the specificity of the reaction. Therefore, it was chosen to perform a melting curve analysis using SYBR Green dye because it requires a less complex instrumentation and it is simpler and cheaper to carry out for a diagnostic laboratory than to a probebased assay. MCA can be useful to detect nucleotide variability because PCR products with different lengths, nucleotide composition and guanine-cytosine (GC) content showed different melting temperatures (Tm ) (Herrmann et al., 2006). For this reason, MCAbased SYBR Green qPCR methods have already been successfully used to detect, quantify and genotype various human and veterinary pathogens (Hays et al., 2011; Liu et al., 2006; Martínez et al., 2008; Maurelli et al., 2009; Payungporn et al., 2008; Tanriverdi et al., 2002). The developed assay was capable of detecting low concentrations of the two viruses and showed satisfactory amplification efficiency and linearity both for CAdV-1 than for CAdV-2. The good reaction performance is also associated with extremely low intraand inter-assay variations. The results obtained from PCR (Chaturvedi et al., 2008; Hu et al., 2001), direct nucleotide sequencing, and melting curve analysis of the two CAdV types were in complete agreement. Thus, the results confirm that the MCA-based SYBR Green qPCR performed under the optimized conditions is reproducible and highly efficient and specific in discriminating between CAdV-1 and CAdV-2. In recent years the conventional melting analysis was improved to interrogate specific single nucleotide polymorphisms (SNPs) accurately and detect up to 0.2 ◦ C difference in melting temperature. This new technology, named high-resolution melting curve
Abdelmagid, O.Y., Larson, L., Payne, L., Tubbs, A., Wasmoen, T., Schultz, R., 2004. Evaluation of the efficacy and duration of immunity of a canine combination vaccine against virulent parvovirus, infectious canine hepatitis virus, and distemper virus experimental challenges. Vet. Ther. 5 (3), 173–186. Balboni, A., Mollace, C., Giunti, M., Dondi, F., Prosperi, S., Battilani, M., 2014. Investigation of the presence of canine adenovirus (CAdV) in owned dogs in Northern Italy. Res. Vet. Sci. 97 (3), 631–636. Balboni, A., Verin, R., Morandi, F., Poli, A., Prosperi, S., Battilani, M., 2013. Molecular epidemiology of canine adenovirus type 1 and type 2 in free-ranging red foxes (Vulpes vulpes) in Italy. Vet. Microbiol. 162 (2–4), 551–557. Bass, E.P., Gill, M.A., Beckenhauer, W.H., 1980. Evaluation of a canine adenovirus type 2 strain as a replacement for infectious canine hepatitis vaccine. J. Am. Vet. Med. Assoc. 177 (3), 234–242. Battilani, M., Balboni, A., Ustulin, M., Giunti, M., Scagliarini, A., Prosperi, S., 2011. Genetic complexity and multiple infections with more parvovirus species in naturally infected cats. Vet. Res. 42 (1), 43. Caudell, D., Confer, A.W., Fulton, R.W., Berry, A., Saliki, J.T., Fent, G.M., Ritchey, J.W., 2005. Diagnosis of infectious canine hepatitis virus (CAV-1) infection in puppies with encephalopathy. J. Vet. Diagn. Invest. 17 (1), 58–61. Chaturvedi, U., Tiwari, A.K., Ratta, B., Ravindra, P.V., Rajawat, Y.S., Palia, S.K., Rai, A., 2008. Detection of canine adenoviral infections in urine and faeces by the polymerase chain reaction. J. Virol. Methods 149, 260–263. Decaro, N., Camero, M., Greco, G., Zizzo, N., Tinelli, A., Campolo, M., Pratelli, A., Buonavoglia, C., 2004. Canine distemper and related diseases: report of a severe outbreak in a kennel. New Microbiol. 27 (2), 177–181. Decaro, N., Campolo, M., Elia, G., Buonavoglia, D., Colaianni, M.L., Lorusso, A., Mari, V., Buonavoglia, C., 2007. Infectious canine hepatitis: an “old” disease reemerging in Italy. Res. Vet. Sci. 83 (2), 269–273. Decaro, N., Martella, V., Buonavoglia, C., 2008. Canine adenoviruses and herpesvirus. Vet. Clin. North Am. Small Anim. Pract. 38 (4), 799–814. Erali, M., Voelkerding, K.V., Wittwer, C.T., 2008. High resolution melting applications for clinical laboratory medicine. Exp. Mol. Pathol. 85 (1), 50–58. Ford, R.B., 2012. Canine infectious respiratory disease. In: Greene, C.E. (Ed.), Infectious Diseases of the Dog and Cat. , Fourth ed. Saunders Elsevier, St. Louis, Missouri, pp. 55–65. Greene, C.E., 2012. Infectious canine hepatitis and canine acidophil cell hepatitis. In: Greene, C.E. (Ed.), Infectious Diseases of the Dog and Cat. , Fourth ed. Saunders Elsevier, St. Louis, Missouri, pp. 42–48. Hays, C., Duhamel, C., Cattoir, V., Bonhomme, J., 2011. Rapid and accurate identification of species belonging to the Candida parapsilosis complex by real-time PCR and melting curve analysis. J. Med. Microbiol. 60 (4), 477–480. Headley, S.A., Alfieri, A.A., Fritzen, J.T., Garcia, J.L., Weissenböck, H., da Silva, A.P., Bodnar, L., Okano, W., Alfieri, A.F., 2013. Concomitant canine distemper, infectious canine hepatitis, canine parvoviral enteritis, canine infectious tracheobronchitis, and toxoplasmosis in a puppy. J. Vet. Diagn. Invest. 25 (1), 129–135. Herrmann, M.G., Durtschi, J.D., Bromley, L.K., Wittwer, C.T., Voelkerding, K.V., 2006. Amplicon DNA melting analysis for mutation scanning and genotyping: crossplatform comparison of instruments and dyes. Clin. Chem. 52 (3), 494–503. Hu, R.L., Huang, G., Qiu, W., Zhong, Z.H., Xia, X.Z., Yin, Z., 2001. Detection and differentiation of CAV-1 and CAV-2 by polymerase chain reaction. Vet. Res. Commun. 25, 77–84. King, A.M.Q., Lefkowitz, E., Adams, M.J., Carstens, E.B., 2011. Virus Taxonomy: IXth Report of the International Committee on Taxonomy of Viruses. Elsevier Academic Press, London, pp. 125–141.
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Liu, J., Yan, M., Wang, Z., Wang, L., Zhou, Y., Xiao, B., 2006. Molecular diagnosis of alpha-thalassemia by combining real-time PCR with SYBR Green1 and dissociation curve analysis. Transl. Res. 148 (1), 6–12. Martínez, E., Riera, P., Sitjà, M., Fang, Y., Oliveira, S., Maldonado, J., 2008. Simultaneous detection and genotyping of porcine reproductive and respiratory syndrome virus (PRRSV) by real-time RT-PCR and amplicon melting curve analysis using SYBR Green. Res. Vet. Sci. 85 (1), 184–193. Maurelli, M.P., Rinaldi, L., Capuano, F., Perugini, A.G., Cringoli, G., 2009. Development of a real-time PCR for the differentiation of the G1 and G2/G3 genotypes of Echinococcus granulosus. Parasitol. Res. 105 (1), 255–259. Müller, C., Sieber-Ruckstuhl, N., Decaro, N., Keller, S., Quante, S., Tschuor, F., Wenger, M., Reusch, C., 2010. Infectious canine hepatitis in 4 dogs in Switzerland (Article in German). Schweiz. Arch. Tierheilkd. 152 (2), 63–88. Payungporn, S., Chieochansin, T., Thongmee, C., Panjaworayan, N., Samransamruajkit, R., Theamboolers, A., Poovorawan, Y., 2008. Detection and discrimination of WU/KI polyomaviruses by real-time PCR with melting curve analysis. J. Virol. Methods 153 (1), 70–73. Pratelli, A., Martella, V., Elia, G., Tempesta, M., Guarda, F., Capucchio, M.T., Carmichael, L.E., Buonavoglia, C., 2001. Severe enteric disease in an animal
shelter associated with dual infections by canine adenovirus type 1 and canine coronavirus. J. Vet. Med. B: Infect. Dis. Vet. Public Health 48 (5), 385–392. Ruskova, L., Raclavsky, V., 2011. The potential of high resolution melting analysis (hrma) to streamline, facilitate and enrich routine diagnostics in medical microbiology. Biomed. Pap. Med. Fac. Univ. Palacky Olomouc Czech Repub. 155 (3), 239–252. Tanriverdi, S., Tanyeli, A., Bas¸lamis¸li, F., Köksal, F., Kilinc¸, Y., Feng, X., Batzer, G., Tzipori, S., Widmer, G., 2002. Detection and genotyping of oocysts of Cryptosporidium parvum by real-time PCR and melting curve analysis. J. Clin. Microbiol. 40 (9), 3237–3244. Thompson, H., O’Keeffe, A.M., Lewis, J.C.M., Stocker, L.R., Laurenson, M.K., Philbey, A.W., 2010. Infectious canine hepatitis in red foxes (Vulpes vulpes) in the United Kingdom. Vet. Rec. 166, 111–114. Tong, S.Y., Giffard, P.M., 2012. Microbiological applications of high-resolution melting analysis. J. Clin. Microbiol. 50 (11), 3418–3421. US Environmental Protection Agency, 2004. Quality assurance/Quality Control Guidance for Laboratories Performing PCR Analysis on Environmental Samples. US Environmental Protection Agency, Washington, DC http://www.epa.gov/ nerlcwww/qa qc pcr10 04.pdf
Contents lists available at ScienceDirect
Journal of Virological Methods journal homepage: www.elsevier.com/locate/jviromet
Development of a SYBR Green real-time PCR assay with melting curve analysis for simultaneous detection and differentiation of canine adenovirus type 1 and type 2 Andrea Balboni, Francesco Dondi, Santino Prosperi, Mara Battilani ∗ Department of Veterinary Medical Sciences, Alma Mater Studiorum-University of Bologna, Via Tolara di Sopra 50, 40064 Ozzano Emilia (BO), Italy
a b s t r a c t Article history: Received 4 March 2015 Received in revised form 11 May 2015 Accepted 21 May 2015 Available online 29 May 2015 Keywords: Canine adenovirus Dog Infectious canine hepatitis Infectious tracheobronchitis Melting curve analysis Real-time PCR
Canine adenovirus type 1 (CAdV-1) and canine adenovirus type 2 (CAdV-2) cause infectious canine hepatitis (ICH) and infectious tracheobronchitis (ITB) in dogs, respectively. Cases of ICH have been documented in recent years and recent surveys have demonstrated a wide percentage of asymptomatic CAdV-1 infection in the canine population. Since both CAdV types are detectable in the same biological matrices, and viral coinfection with CAdV-1 and CAdV-2 are reported with high frequency, it is urgent to have available a rapid, highly sensitive and specific assay for the diagnosis of CAdV infection and distinction between CAdV-1 and CAdV-2. In order to detect canine adenovirus in biological samples and to rapidly distinguish the two viral types, a SYBR Green real-time PCR assay was optimized to discriminate CAdV-1 and CAdV-2 via a melting curve analysis. The developed assay showed high sensitivity and reproducibility and was highly efficient and specific in discriminating the two CAdV types. This reliable and rapid technique may represent a simple, useful and economic option for simultaneous CAdV types detection, which would be feasible and attractive for all diagnostic laboratories, both for clinical purposes and for epidemiological investigations. © 2015 Elsevier B.V. All rights reserved.
1. Introduction Canine adenovirus type 1 (CAdV-1) is the aetiologic agent of infectious canine hepatitis (ICH), a severe systemic disease in dogs characterized by acute necrohaemorragic hepatitis (Greene, 2012). Canine adenovirus type 2 (CAdV-2) is one of the viral agents implicated in the aetiopathogenesis of infectious tracheobronchitis (ITB), also known as kennel cough, a mild self-limiting acute upper respiratory disease in dogs (Ford, 2012) which has a high prevalence in canine populations. The widespread use of a modified live CAdV-2 vaccine in countries like Italy has greatly reduced the incidence of
Abbreviations: CAdV, canine adenovirus; CAdV-1, canine adenovirus type 1; CAdV-2, canine adenovirus type 2; ICH, canine hepatitis; ITB, infectious tracheobronchitis; qPCR, quantitative real-time PCR; MCA, melting curve analysis; Tm , melting temperatures; LOD, limit of detection; SD, standard deviation; CV, coefficient of variation; R2 , coefficient of determination; E, reaction efficiency; S, slope; SNPs, single nucleotide polymorphisms; HRM, high-resolution melting curve. ∗ Corresponding author. Tel.: +39 051 2097081; fax: +39 051 2097039. E-mail address: [email protected] (M. Battilani). http://dx.doi.org/10.1016/j.jviromet.2015.05.009 0166-0934/© 2015 Elsevier B.V. All rights reserved.
ICH in the domestic canine population (Abdelmagid et al., 2004; Bass et al., 1980; Decaro et al., 2008). Infectious canine hepatitis is nowadays considered a neglected canine disease and veterinary practitioners rarely take into account CAdV-1 as causative agent of disease. Nevertheless, cases of ICH and of asymptomatic CAdV-1 infections have been documented in recent years in foxes and dogs (Balboni et al., 2013, 2014; Caudell et al., 2005; Decaro et al., 2007; Headley et al., 2013; Müller et al., 2010; Pratelli et al., 2001; Thompson et al., 2010), confirming that the CAdV-1 continues to circulate and to be pathogenic in dogs. Furthermore, CAdV types are detectable in the same biological matrices (Balboni et al., 2013, 2014; Decaro et al., 2004; Greene, 2012) and viral coinfection with CAdV-1 and CAdV-2 are detectable with high frequency (Balboni et al., 2013, 2014; Headley et al., 2013). For these reasons, the diagnosis of CAdV-1 infection and the distinction of this virus from CAdV-2 is important for both clinical and epidemiological investigations. Canine adenovirus types are distinguishable by genetic, antigenic and pathogenetic characteristics but there is immunological cross-reactivity among them (King et al., 2011). Serologic methods have limited application in the diagnostic protocols because they
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Table 1 Field CAdV strains used for assay development and validation. Virus CAdV-1 09-13 113-5 384 417 574 CAdV-2 60 S1 243 244 249 252 260 270 272 303
Year of sampling
Host
Biological matrix
GenBank accession number
References
2011 2011 1966 2013 2013
Fox Fox Dog Dog Dog
Faeces Liver FFPE-liver Liver RS
JX416838 JX416839 KP670422 KP670423 KP670424
Balboni et al. (2013) Balboni et al. (2013) – – –
2011 2011 2012 2012 2012 2012 2012 2012 2012 2012
Dog Dog Dog Dog Dog Dog Dog Dog Dog Dog
CS OPS urine RS RS RS RS RS RS RS
KF676978 KF676979 – – KF676981 – – – KF676982 –
– – Balboni et al. (2014) Balboni et al. (2014) Balboni et al. (2014) Balboni et al. (2014) Balboni et al. (2014) Balboni et al. (2014) Balboni et al. (2014) Balboni et al. (2014)
FFPE-liver: formalin-fixed paraffin-embedded liver. OPS: oro-pharyngeal swab. CS: cell supernatant. RS: rectal swab. References: viruses 384, 417, 574, 60 and S1 were identified in our lab and nucleotide sequences were directly submitted to GenBank database.
are mostly unable to distinguish the two viruses, and also for the widespread use of a modified live CAdV-2 vaccine. The molecular biology-based methods are more reliable but, to date, only one PCR assay able to distinguish the two CAdV types was developed (Hu et al., 2001; Chaturvedi et al., 2008). In order to reduce the time of execution and, at the same time, to increase the sensitivity of the molecular diagnosis, a SYBR Green real-time PCR (qPCR) assay was developed that is able to detect and to distinguish between CAdV-1 and CAdV-2 via a melting curve analysis (MCA). 2. Materials and methods 2.1. Experimental design and field CAdV strains used for assay development and validation
EF057101; M60937; S38238; JX416838; JX416839) and aligned using the ClustalW method implemented in BioEdit software version 7.2.5. A couple of degenerated primers overlapping E3 gene and flanking regions were obtained (Table 2). A BLAST search (http://blast.ncbi.nlm.nih.gov/Blast.cgi) was performed in order to verify the specificity of the selected primers. OligoAnalyzer (http://eu.idtdna.com/analyzer/applications/oligoanalyzer/ 3.1 ) and Oligo Calc (http://www.basic.northwestern.edu/biotools/ OligoCalc.html) web interfaces were used to exclude secondary structure and primer dimers. The designed primers amplify a product of 166 bp showing numerous nucleotide mutations between CAdV-1 and CAdV-2 (≥47 nucleotide substitutions, corresponding to ≥28.3% of the amplified DNA fragment) (Fig. 1), which implies a difference in Tm values between the two CAdV types. 2.4. Preparation of DNA standards
SYBR Green qPCR was developed and validated for the simultaneous detection and typing of CAdV-1 and CAdV-2 on the basis of melting temperature analysis of produced amplicons. Five CAdV-1 and 10 CAdV-2 strains reported in Table 1 were used to assess the analytical performance of the MCA-based SYBR Green qPCR and to validate the developed assay. Field viruses were previously identified in our laboratory by testing different biological matrices and using a PCR assay we were able to differentiate between the two adenovirus types (Chaturvedi et al., 2008; Hu et al., 2001). All the obtained PCR products were sequenced to confirm the CAdV typing. 2.2. DNA extraction Viral DNA extraction from organs, urine, rectal swabs, oropharyngeal swabs and cell supernatants was performed using the NucleoSpin Tissue Mini Kit (Macherey-Nagel, Düren, Germany) according to the manufacturer’s instructions. For formalin-fixed paraffin-embedded tissue samples, the deparaffinisation was performed by using the Solvent Plus reagent (Carlo Erba, Rodano, MI, Italy). The extracted DNA was eluted in 100 l of elution buffer and stored at −20 ◦ C.
Two pCR4 plasmids (Invitrogen, Carlsbad, CA, USA) containing one copy of the CAdV-1 and CAdV-2 target sequences, respectively, were produced for optimization of the real-time PCR assay and for construction of the assay standard curve. The CAdV-1 amplicon of 508 bp and the CAdV-2 amplicon of 1030 bp of the E3 gene and flanking regions obtained from dog 300-2012 (Balboni et al., 2014) were used as target sequences. The CAdV-1 and CAdV-2 DNA fragments were cloned into the pCR4 vector using the TOPO TA Cloning Kit (Invitrogen, Leek, the Netherlands), according to the manufacturer’s instructions. The resulting recombinant plasmid purification was carried out using the PureYield Plasmid Miniprep System (Promega, Madison, WI, USA) and the plasmids were linearized above the CAdV fragment sequence using restriction endonuclease Spe I (Fermentas, Burlington, Ontario, Canada) in order to avoid the presence of supercoiled plasmid DNA. The concentration and purity of the standard plasmids were assessed by measuring absorbance at 260 nm in a UV spectrophotometer (BioPhotometer, Eppendorf, Hamburg, Germany) and using the 260/280 nm ratio. The copy number of standard plasmids was calculated using the equation described by the US Environmental Protection Agency protocol (US Environmental Protection Agency, 2004).
2.3. Primer design 2.5. SYBR Green real-time PCR For appropriate primer design, sequences of 12 reference CAdV strains were retrieved from the GenBank database (http:// www.ncbi.nlm.nih.gov/genbank; accession numbers: AC 000020; S38212; JX416841; JK416842; AC 00003; GQ340423; U55001;
The real-time PCR was performed using the Rotor-Gene SYBR Green PCR Kit (QIAGEN, Hilden, Germany) and the Rotor-Gene 3000 system (Corbett Research, Mortlake, NSW, Australia). The
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Table 2 Primers used for the SYBR Green qPCR assay. Primer
Nucleotide sequence (5 –3 )
Nt. position in CAdV-1
Nt. position in CAdV-2
Fragment amplified (E3 and U-exon genes)
CAdV-qPCR-For3 CAdV-qPCR-Rev2
CTGASACTGCWATRMCTATATAYATTTCCA GACATAGARACRCAGGACCCAGA
25600–25626 25743–25765
26305–26334 26448–26470
166 bp
Nucleotide positions are referring to the following reference strains: RI261, GenBank Accession number AC 000003 (CAdV-1) and TorontoA26/61, GenBank Accession number AC 000020 (CAdV-2).
fluorescence signal was acquired on the FAM channel (multichannel machine, source 470 nm; detector 510 nm; gain set to 5) with a fluorescence reading taken at the end of each elongation step. Different annealing temperature (from 50 to 60 ◦ C) and primers concentration (from 1 to 1.4 M) were tested to optimize the assay performance. The optimized SYBR Green real-time PCR was performed in a final volume of 25 l containing 12.5 l of 2× Rotor-Gene SYBR Green Master Mix, 1 M of forward (CAdVqPCR-For3) and reverse (CAdV-qPCR-Rev2) primers, and 2.5 l of template DNA. Autoclaved nanopure water was added to arrive at a final volume of 25 l. Each run consisted of an initial incubation for activation of the hot-start DNA polymerase at 95 ◦ C for 5 min followed by 45 cycles of denaturation at 95 ◦ C for 10 s, annealing at 59 ◦ C for 20 s and polymerization at 60 ◦ C for 15 s. The melting experiments were performed after the last extension step; the temperature was increased by increments of 0.2 ◦ C from 60 to 95 ◦ C every 5 s. Melting curves were analyzed by the Rotor-Gene 6 software (Corbett Research, Mortlake, NSW, Australia) and the Tm was defined as the peak of the obtained curves. Specimens were considered positive if the fluorescence curve in the amplification plot showed an exponential increase, and a specific melting peak was observed.
2.6. Standard curve and limit of detection (LOD) The SYBR Green real-time PCR standard curve was generated for both CAdV-1 and CAdV-2 by serial 10-fold dilutions of the two recombinant plasmids with a known copy number (from 1 × 108 to 1 × 10−2 copies/l). These dilutions were tested in triplicate and used as quantification standards to construct the standard curve by plotting the plasmid copy number against the corresponding threshold cycle values. The threshold was determined using the Auto-Find Threshold function of the Rotor-Gene 3000, which scans the range of the threshold levels to obtain the best fit of the standard curve using the samples which have been defined as standards. A
melting curve analysis of the obtained amplification products was carried out. The LOD of the reaction was determined for both CAdV1 and CAdV-2 based on the highest dilution of respective plasmid possible to amplify with good reproducibility. In order to mimic realistic conditions, standard curves were also constructed using serial 10-fold dilutions of both CAdV-1 and CAdV-2 recombinant plasmids in negative extracts of representative biological matrices. A negative faecal extract was used for both CAdV-1 and CAdV-2 as representative sample for in vivo tests (Balboni et al., 2014). Instead, as representative samples for post mortem tests we used a negative liver extract for CAdV-1 and a negative lung extract for CAdV-2. 2.7. Diagnostic sensitivity and specificity The analytical sensitivity and efficiency of real-time PCR for the two CAdV types are reflected by the LOD, which was assessed as described above. The specificity of the assay was evaluated by testing, in triplicate, two field CAdV-1 (417/2013 and 574/2013) and two field CAdV-2 (60/2011 and S1/2011). To further determine the specificity of the amplification reaction, other DNA viruses affecting dogs and cats were tested: the canine parvovirus type 2 (Protoparvovirus, CPV-2b/461/2009, GenBank ID: KF373599), the canid herpesvirus 1 (Varicellovirus, 643/2014, a field virus identified in our lab), the canine oral papillomavirus (Lambdapapillomavirus, 146/2012, a field virus identified in our lab), the feline panleukopenia virus (Protoparvovirus, 1033/2009, Battilani et al., 2011) and the felid herpesvirus 1 (Varicellovirus, 10/2011, a field virus identified in our lab). To verify the specificity, a melting curve analysis and electrophoresis on 2% (w/v) agarose gel stained with ethidium bromide in 1× standard tris-acetate-EDTA (TAE) buffer were carried out for the amplification products. Furthermore, the genotype identification was confirmed by sequencing the products obtained to CAdV-1 and CAdV-2 recombinant plasmids.
Fig. 1. Multiple sequence alignment of primer binding sites. Alignment of the genomic tract comprising part of the E3 and U-exon genes amplified by the designed primers. The alignment include 4 CAdV-2 and 8 CAdV-1 reference strains. In grey: designed primers.
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Table 3 Intra- and inter-assay variability of the real-time PCR. Samples Intra-assay variability A1 A2 A3 A4 A5 A6 A7 A8 B1 B2 B3 B4 B5 B6 B7 B8 417/2013 574/2013 60/2011 S1/2011 Inter-assay variability C1 C2 C3 D1 D2 D3 417/2013 574/2013 60/2011 S1/2011
Replicate (and assay) numbers
Mean (SD)
log10 Mean (SD)
log10 CV (%)
3 (1) 3 (1) 3 (1) 3 (1) 3 (1) 3 (1) 3 (1) 3 (1) 3 (1) 3 (1) 3 (1) 3 (1) 3 (1) 3 (1) 3 (1) 3 (1) 3 (1) 3 (1) 3 (1) 3 (1)
8.36E+07 (1.52E+06) 9.98E+06 (4.65E+05) 1.08E+06 (3.09E+04) 1.23E+05 (2.87E+03) 1.01E+04 (5.74E+02) 1.03E+03 (8.56E+01) 9.75E+01 (3.71) 9.06E+00 (1.47) 8.45E+07 (1.28E+06) 9.75E+06 (4.63E+05) 1.11E+06 (2.16E+04) 1.04E+05 (6.38E+03) 1.08E+04 (3.86E+02) 1.15E+03 (1.17E+02) 1.21E+02 (1.36E+01) 7.32E+00 (1.62) 1.19E+07 (8.98E+05) 9.99E+02 (6.65E+01) 1.46E+08 (1.27E+07) 1.84E+05 (2.26E+04)
7.92 (0.01) 7 (0.02) 6.03 (0.01) 5.09 (0.01) 4 (0.02) 3.01 (0.04) 1.99 (0.02) 0.95 (0.07) 7.93 (0.01) 6.99 (0.02) 6.04 (0.01) 5.02 (0.03) 4.03 (0.01) 3.06 (0.04) 2.08 (0.05) 0.85 (0.1) 7.07 (0.03) 3 (0.03) 8.16 (0.04) 5.26 (0.05)
0.1 0.29 0.21 0.2 0.61 1.22 0.83 7.61 0.08 0.29 0.14 0.52 0.38 1.41 2.28 12.38 0.46 0.97 0.45 1.05
3 (3) 3 (3) 3 (3) 3 (3) 3 (3) 3 (3) 3 (3) 3 (3) 3 (3) 3 (3)
1.47E+08 (4.55E+07) 1.28E+05 (1.84E+04) 1.15E+02 (1.99E+01) 1.57E+08 (4.16E+07) 7.14E+04 (2.98E+04) 1.15E+02 (1.37E+01) 1.08E+07 (1.34E+06) 6.82E+02 (2.24E+02) 1.76E+08 (3.8E+07) 7.21E+04 (2.46E+04)
8.14 (0.16) 5.1 (0.06) 2.05 (0.07) 8.18 (0.11) 4.82 (0.18) 2.06 (0.05) 7.03 (0.06) 2.81 (0.13) 8.24 (0.09) 4.83 (0.18)
1.92 1.2 3.5 1.36 3.67 2.48 0.8 4.71 1.07 3.66
A1–A8: 10-fold of CAdV-1 recombinant plasmid dilutions (from 1 × 108 to 1 × 101 respectively). B1–B8: 10-fold of CAdV-2 recombinant plasmid dilutions (from 1 × 108 to 1 × 101 respectively). C1–C3: CAdV-1 recombinant plasmid dilutions with high, medium and low concentration respectively. D1–D3: CAdV-2 recombinant plasmid dilutions with high, medium and low concentration respectively. Two CAdV-1 field samples: 417/2013 and 574/2013. Two CAdV-2 field samples: 60/2011 and S1/2011. SD: standard deviation. CV: coefficients of variation.
2.8. Intra- and inter-assay variability In order to determine the intra-assay variability of the technique for the standard plasmids and for samples, eight successive 10-fold dilutions (from 1 × 108 to 1 × 101 copies/l) of both CAdV1 and CAdV-2 recombinant plasmids, two CAdV-1 samples with different viral concentration (417/2013 and 574/2013), and two CAdV-2 samples with different viral concentrations (60/2011 and S1/2011) were tested in triplicate within the same run (Table 3). The inter-assay variability was evaluated by testing three dilutions with high, medium and low concentrations of both CAdV-1 and CAdV-2 recombinant plasmids, and the previous four CAdV samples in triplicate on three different days (Table 3). Mean, standard deviation (SD), and coefficient of variation (CV) were calculated. In particular, the CV was calculated as the percentage of the ratio of standard deviation and the mean values were obtained, in accordance with the US Environmental Protection Agency protocol (US Environmental Protection Agency, 2004). The repeatability of the melting temperature analysis was examined by comparing the results obtained from replicates of three dilutions with high, medium and low concentration of both CAdV-1 and CAdV-2 recombinant plasmids, and four CAdV samples (417/2013, 574/2013, 60/2011 and S1/2011) during a single qPCR reaction run (intra-assay) and comparing the mean values obtained from the same plasmids and samples on three different days (inter-assay) (Table 4). 2.9. Statistical analysis To evaluate whether MCA could be used to accurately type CAdV-1 and CAdV-2, Tm values of plasmids and samples
testing positive to the two viruses were compared using a twogroup Student’s t-test. 2.10. Detection and differentiation of CAdV type 1 and 2 in dog and fox samples using MCA-based SYBR Green qPCR After optimization of the SYBR Green qPCR assay, the 15 field CAdV samples listed in Table 1 were tested to validate the technique. For each run, duplicates of six 10-fold dilutions of the standard CAdV-2 plasmid, duplicates of the dog and fox DNA extracts, and a no template control were simultaneously subjected to analysis. The melting peak and Tm obtained from each sample were used to discriminate between CAdV-1 and CAdV-2. Samples were considered positive if the mean of the replicates was greater than the LOD. 3. Results 3.1. Development of MCA-based SYBR Green qPCR The linearity and efficiency of the SYBR Green real-time PCR were determined for CAdV-1 and CAdV-2 amplification by generating a standard curve for each of the two viruses in which serial 10-fold dilutions of recombinant plasmids were tested. The standard curve was generated by plotting the real-time PCR threshold cycle numbers (Ct ) of each dilution against the known copy numbers of recombinant plasmid. The resulting slope showed a linear relationship over 8 orders of magnitude ranging from 1 × 101 to 1 × 108 copies/l for both CAdV-1 and CAdV-2 standard plasmids. The slope was −3.36 with a coefficient of determination (R2 ) > 0.99 and a reaction efficiency (E) of 0.98 for CAdV-1 and −3.42 with a
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Table 4 Intra- and inter-assay variations of Tm values (◦ C) obtained from real-time PCR with melting curve analysis. Virus
Intra-assay variation Interval
CAdV-1 CAdV-2
76.04–76.56 80.96–81.3
Range 0.52 0.34
Inter-assay variation Mean 76.37 81.18
SD 0.16 0.08
R2 > 0.99 and an E of 0.96 for CAdV-2. The reaction efficiency was calculated from the slope (S) using E = 10(−1/s) − 1. The LOD determined on the CAdV-1 and CAdV-2 standard curves was found to be 0.049 fg (10 copies/l) and 0.055 fg (10 copies/l) respectively, thus showing a high sensitivity of the assay. Two distinct melting peaks were clearly visible for the CAdV1 and CAdV-2 standard plasmid dilutions (Fig. 2), indicating the formation of a single PCR product with no artefacts, such as non-specific amplification products or primer dimers (results not shown), for each of the two viruses, confirming the specificity of the reaction. The standard curves generated with 10-fold dilutions of CAdV-1 and CAdV-2 recombinant plasmids in negative extracts of representative biological matrices (faeces, liver and lung) showed linearity up to a LOD of 10 copies/l for both viruses and for all biological matrices. In tests performed on negative faecal extracts, the solitary specific melting peaks corresponding to CAdV-1 or CAdV2 amplification was showed without artefacts. In tests performed on negative liver or lung extracts, in addition to the specific peaks of CAdV amplification, some weak melting peaks at temperatures higher than 82.5 ◦ C were visible at plasmid concentrations below 103 copies/l; however, they did not affect the reading of specific melting peaks and these potential nonspecific products did not affect the amplification of target viral DNA, as demonstrated by the linearity of the standard curves. The developed assay was able to detect CAdV-1 (417/2013 and 574/2013) and CAdV-2 (60/2011 and S1/2011) samples. No positive results were obtained with any other DNA viruses tested (data not shown). Amplification products were also checked on
CV (%) 0.21 0.09
Interval
Range
Mean
SD
CV (%)
75.85–76.62 81.04–81.27
0.77 0.23
76.22 81.16
0.24 0.07
0.32 0.09
gel electrophoresis and a clear and well-defined specific band of approximately 166 bp was visualized for all replicates of the two recombinant plasmids and samples that tested positive in realtime PCR amplification. Furthermore, the nucleotide sequence of the products obtained from CAdV-1 and CAdV-2 recombinant plasmid amplification confirmed the genotype identification (data not shown). The intra-assay variability was determined, at first, on eight successive 10-fold dilutions (from 1 × 108 to 1 × 101 copies/l) of both CAdV-1 and CAdV-2 recombinant plasmids tested in triplicate and then, on two CAdV-1 and two CAdV-2 field samples with different viral concentrations tested in triplicate. The coefficient of variation (CV) that was obtained ranged from 0.08 to 2.28 for all the recombinant plasmids and samples with concentrations higher than or equal to 102 copies/l, while it increased for lower concentrations (Table 3). The determination of the inter-assay variability tested on three different dilutions of both CAdV-1 and CAdV-2 recombinant plasmids and two CAdV-1 and CAdV-2 field samples gave a CV ranging from 0.8 to 4.71 for all the viral concentrations tested (Table 3). The variability of plasmid dilutions and samples is therefore low for decreasing concentrations down to 102 copies/l and progressively higher for lower concentrations, as influenced by distribution statistics (Poisson’s law) which involves an increase in CV values for the quantification of low copy number. However, despite the higher variability, low viral concentrations were always detected in all repetitions using the SYBR Green assay. Furthermore, the standard deviation (SD) between the individual assays was below 0.25 log10 (Table 3), a level which is normally
Fig. 2. Discrimination of CAdV-1 and CAdV-2 by real-time PCR with melting curve analysis. In grey: signal obtained from the CAdV-1 standard plasmid. In black: signal obtained from the CAdV-2 standard plasmid. Derivative – dF/dT where F is the fluorescence and T is the time. deg: temperature (centigrade).
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considered the minimum requirement for acceptable reproducibility of quantitative molecular assays. The melting temperature analysis showed a variation of Tm values between samples of 76.37 ◦ C ± 0.16 and 81.18 ◦ C ± 0.08 for CAdV-1 and CAdV-2, respectively. Percentage of variation coefficient (%CV) between samples was 0.21 for CAdV-1 and 0.09 for CAdV-2 (Table 4). Instead, the variations of Tm values between runs were 76.22 ◦ C ± 0.24 with %CV of 0.32 for CAdV-1 and 81.16 ◦ C ± 0.07 with %CV of 0.09 for CAdV-2 (Table 4). The two-group t-statistic for the comparison between the Tm value obtained after MCA for CAdV-1 and CAdV-2 showed statistical differences (p < 0.001). 3.2. Validation of MCA-based SYBR Green qPCR Fifteen samples, previously identified by conventional methods as CAdV-1 (n = 5) and CAdV-2 (n = 10), were correctly amplified and detected with SYBR Green qPCR. Based on MCA, the five CAdV-1 were correctly typed with a Tm value ranging between 76.04 and 76.55 ◦ C, and the 10 CAdV-2 were correctly typed with Tm value ranged between 81.01 and 81.25 ◦ C.
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(HRM) analysis, entails the use of a HRM-capable real-time PCR device (able to accurately control the temperature to acquire fluorescence data at high density during DNA melting, and to perform automated HRM curve comparison) and a so-called “saturating” DNA binding dye (able to increase the resolution and accuracy of melting analysis) (Erali et al., 2008; Tong and Giffard, 2012). The real-time PCR with HRM assay allow to detect variants in the DNA sequence and it was applied to bacterial, viral or parasite typing (Ruskova and Raclavsky, 2011). The SYBR Green real-time PCR assay developed in this study amplified a 166 bp fragment of CAdV genome containing almost 47 nucleotide substitutions between CAdV-1 and CAdV-2 that produces a difference in Tm greater than 4 ◦ C between the two viral types. This considerable difference in CAdV-1 and CAdV-2 melting temperatures does not require the use of the HRM analysis allowing the use of a conventional real-time PCR device. Therefore, this reliable and rapid technique, given its extreme sensitivity and reproducibility, may represent a simple, useful and economic option for CAdV detection and type discrimination, which would be feasible and attractive for all diagnostic laboratories, even those with limited instrumentation, both for clinical purposes and for epidemiological investigations. References
4. Discussion In this paper, a SYBR Green real-time PCR method was optimized and evaluated in terms of specificity, sensitivity, amplification efficiency, standard curve linearity, and overall performance for detection and discrimination of the two CAdV types through the melting curve analysis. Although the probe-based real-time PCR assay provides greater specificity than the SYBR Green qPCR, a real-time instrument able to detect different wavelengths would be required to detect CAdV-1 and CAdV-2 in the same reaction using two specific probes labelled with different fluorophores. Furthermore, a probe-based real-time PCR reaction is more expensive than a SYBR Green qPCR reaction because it requires the use of probes in addition to the primers and nucleotide mutations in the hybridization sites of the probes can affect the specificity of the reaction. Therefore, it was chosen to perform a melting curve analysis using SYBR Green dye because it requires a less complex instrumentation and it is simpler and cheaper to carry out for a diagnostic laboratory than to a probebased assay. MCA can be useful to detect nucleotide variability because PCR products with different lengths, nucleotide composition and guanine-cytosine (GC) content showed different melting temperatures (Tm ) (Herrmann et al., 2006). For this reason, MCAbased SYBR Green qPCR methods have already been successfully used to detect, quantify and genotype various human and veterinary pathogens (Hays et al., 2011; Liu et al., 2006; Martínez et al., 2008; Maurelli et al., 2009; Payungporn et al., 2008; Tanriverdi et al., 2002). The developed assay was capable of detecting low concentrations of the two viruses and showed satisfactory amplification efficiency and linearity both for CAdV-1 than for CAdV-2. The good reaction performance is also associated with extremely low intraand inter-assay variations. The results obtained from PCR (Chaturvedi et al., 2008; Hu et al., 2001), direct nucleotide sequencing, and melting curve analysis of the two CAdV types were in complete agreement. Thus, the results confirm that the MCA-based SYBR Green qPCR performed under the optimized conditions is reproducible and highly efficient and specific in discriminating between CAdV-1 and CAdV-2. In recent years the conventional melting analysis was improved to interrogate specific single nucleotide polymorphisms (SNPs) accurately and detect up to 0.2 ◦ C difference in melting temperature. This new technology, named high-resolution melting curve
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