Multiplex PCR for rapid diagnosis and differentiation of pox and pox-like diseases in dromedary Camels

The optimization process was performed using plasmid DNA construct containing the targeted gene fragments of the three viruses. The performance of the three sets of primers was first tested in an individual PCR assay before combining them in a multiplex format. The optimum annealing temperature of the multiplex PCR was found to be 57.5 °C (Fig. 2). The developed multiplex PCR gave visible DNA bands when a constant concentration of the three plasmid constructs (10⁶ copy number) containing insert of the targeted genes were tested separately and when all mixed together (Fig. 3). The assay also performed well using Qiagen Hotstart PCR kit (Qiagen, Germany) and Multiplex PCR Master Mix (New England Biolabs, UK). The thermal cycling was performed in a Tpersonal Thermal Cycler (Biometra, Germany).

The developed multiplex PCR was confirmed to be specific in amplifying DNA fragments of the three plasmid constructs and in detecting all the known CPPV, CMLV and CdPV DNAs described in Table 1, which were diagnosed initially by monoplex PCR. The assay produced amplicons of expected sizes that were distinguishable in the agarose gel; 569, 478 and 226 bp PCR products from DNAs of known CPPV, CdPV and CMLV, respectively while no positive signal was obtained when using sheeppox virus (SPV), Lumpy skin disease virus (LSDV), Brucella melitensis (BM) or the no template control (NTC) (Fig. 4), suggesting that the assay was highly specific for detection of the target viruses. Furthermore, sequencing data confirmed the amplification of expected DNA sequences from the three viruses (data not shown).

The PCR sensitivity tests were performed using 10-fold serial dilution of the DNA plasmid constructs that were first diluted to contain 10⁹ copy numbers. DNA copy numbers ranging between 10 and 10⁸ were submitted to monoplex and multiplex PCR. The lower limit of detection (LOD) was found to be 10³ copy numbers of plasmid DNA for CPPV and CdPV and 10² for CMLV (Fig. 5). On the other hand, the LOD was found to be 19 pg for DNA extracted from purified CMLV-14 reference strain (data not shown). The sensitivity of the monoplex and multiplex PCR assays was similar for CMLV, but the sensitivity of both assays in detecting CPPV and CdPV was 10 fold lower than for CMLV (Fig. 5).

The multiplex PCR assay successfully amplified the expected DNA fragments from 86 clinical materials: CPPV (n = 42), CdPV (n = 27) and CMLV (n = 17) as shown in Table 2. Interestingly, the developed assay detected co-infection with CPPV and CdPV in one clinical specimen collected at Showak area of eastern Sudan in 2013 (Fig. 6). This result could only be detected by multiplex PCR as these specimens were previously diagnosed as CPPV positive by a monoplex PCR. The multiplex was able to detect CPPV, CdPV and CMLV DNAs in all analyzed clinical samples positive by monoplex PCR (Table 2).

Known viruses and DNAs used as positive controls for each pathogen in the present study are listed in Table 1. These include four reference CMLV strains: VD45 previously supplied by CIRAD-EMVT, France [56], Dubai camelpox vaccine (Ducapox), and DNAs extracted from purified CMLV-1 and CMLV-14 (kindly provided by Dr. Sophie Duraffour, Rega Institute, leuven, Belgium). Additionally, some previously published CMLVs isolated from outbreaks of the disease in Sudan [49], CPPV positive specimens collected from previous outbreaks [19] and skin specimens from an outbreak of papilloma infection in the Sudan [2], as well as the vaccine strain Sudan CMLV/115 [57] were also included.

Sheeppox virus (SPV) strain SGP0240, Lumpy skin disease virus (LSDV) strain isolated in the Sudan [58] and Brucella Melitensis (BM) strain REV 1 original seed (kindly provided by Dr. Iaam El Sanousi, Veterinary Research Institute, Sudan) were used as negative controls and in the specificity experiments.

To validate the specificity and sensitivity of the developed assay a total of eighty-six skin scabs and nodules which were collected from dromedary camels in Sudan and Saudi Arabia showing symptoms of pox and pox-like lesions between 1993 and 2014 (Table 2).

The multiplex PCR assay included two sets of published primers previously described in the literature, but had never been used simultaneously. The primer set PPP-1/PPP-4 [21] amplifies a 569 bp region of the conserved major viral glycoprotein (B2L) gene of PPV, which is a widely used in PPV diagnosis and genotyping. For the camelus dromedary papilloma virus (CdPV), we used the degenerate pan-papilloma virus specific primers described by Forslund et al. [54] that target the L1 ORF of PV (Table 3). For the CMLV, primer sets targeting genes encoding for the A-type inclusion body (ATI), the hemagglutinin (HA), the ankyrin repeat protein (C18L) and the DNA polymerase were initially tested in combination with the above-mentioned primer sets, but results were not satisfactory. Therefore, new pair of primers targeting the hemagglutinin (HA) of OPV was designed. Sequences of CMLV HA gene originating from different countries were obtained from the Genbank®. Regions of high homology in different CMLV strains were identified by sequence alignment using Clustal-O (http://​www.​ebi.​ac.​uk/​Tools/​msa/​clustalo/​). Primers were initially selected by using Primer Explorer V4 (http://​primerexplorer.​jp/​e/​) and then manually edited. Details on primer sets used in the present study are shown in Table 3.

PCR amplifications of the B2L gene of PPV and L1 ORF of PV were performed with primers shown in Table 1 in a total reaction volume of 25 μl as described by Inoshima et al. [21] and Forslund et al. [54], respectively. For the amplification of the CMLV HA gene PCR reaction was performed in a final volume of 25 μl that contained 1× PCR buffer (Vivantis Technologies, Malaysia), 10 mM dNTPs mix, 0.4 μM of each primer (HA-F and HA-R), one unit Taq DNA polymerase (Vivantis Technologies, Malaysia) and one μl DNA template. The PCR amplification was carried out in a Tpersonal Thermocycler (BIOMETRA, Germany) under the following conditions: initial denaturation at 94 °C for three min followed by 35 cycles each included denaturation step at 94 °C for 30 s, annealing step at 56 °C for 30 s and extension step at 72 °C for 30 s. A final extension step at 72 °C for seven min was included. PCR products were resolved by electrophoresis in 1.5 % agarose in TAE buffer (40 mM Tris–acetate pH 8.0,1 mM EDTA) and the gel, stained with ethidium bromide and photographed using ultraviolet gel documentation system (BIOMETRA, Germany).

As quantitative calibration standards, three plasmids containing targeted DNA fragments were prepared. PCR assays were carried out to amplify selected genes using monoplex protocols described above from reference strains CCE 41 (CPPV), CdPV1 (CdPV) and CP/Mg/92/1 (CMLV) (Table 1). The PCR products were separated by agarose gel electrophoresis, purified with a PCR purification kit (Vivantis Technologies, Malaysia) and the PCR product was cloned into pBASE/TA vector. The ligation product was transformed into JM109 E.coli strain. Positive clones were identified and the plasmid purified using a commercial kit and sent for sequencing in an automated ABI 3730 DNA sequencer (Applied Biosystems, USA) using the BigDye® Terminator v3.1 cycle sequencing kit chemistry.

A number of experiments were carried, several chemical and thermal conditions were evaluated, and the assay optimized by adjusting primers and MgCl₂ concentrations as well as the thermal cycling temperatures and duration. The best conditions were established based on amplicon yield and specificity. Monoplex PCR experiments were carried out before combining them in multiplex assays. Ordinary and hot-start Taq DNA polymerases were tested. PCR was tested with conventional PCR approach in addition to a hot-start amplification that employed heating all PCR components excluding the Taq DNA polymerase at 94 °C and then addition of the enzyme. The multiplex PCR was performed in 50 μl volume containing 1 μL (10⁶ copy number) of plasmid DNA, 0.8 μM of PPV primers (PPV-1 and PPV-4) and papilloma virus (PV) (FAP 59 and FAP64), 0.4 μM of OPV primers (HA-F and HA-R), 1× PCR buffer (Vivantis Technologies, Malaysia) including 160 mM (NH₄)₂ SO₄, 500 mM Tris–HCl (pH 9.2),17.5 mM MgCl₂ and 0.1 % Triton™ ×-100) and 10 mM dNTPs mix. A manual hot-start procedure was followed in which the above PCR components were heated up to 95 °C for two min and one unit of Chrome Max Taq DNA polymerase (Vivantis Technologies, Malaysia) was added at the same temperature followed by incubation at 95 °C for nine min, 30 cycles of denaturation (94 °C, one min), annealing (57.5 °C, one min) and extension (72 °C, one min) and a final extension at 72 °C for 10 min. The cycling program for the multiplex PCR has eight steps with a total running time of two h, 34 min and 18 s. PCR products were resolved by electrophoresis in 1.5 % agarose in tris-acetate EDTA (TAE) buffer (40 mM Tris–acetate pH 8.0,1 mM EDTA) and the gel was stained with ethidium bromide and photographed. For gel detection, eight μl of PCR product was stained with ethidium bromide and resolved on a 1.5 % agarose gel using TAE buffer.

To estimate the specificity, the developed multiplex assay was tested with all the three plasmid constructs (10⁶ copy number), known CPPV (8 ng), CdPV (20 ng) and CMLV (10 ng), SPV (20 ng), LSDV (20 ng) and BM (15 ng) DNAs in 1 μl templates volume. Furthermore, the expected PCR products obtained from the experiments were sequenced to evaluate the specificity of the assay.

The sensitivity of multiplex PCR and the corresponding monoplex PCR was performed using serial 10-fold dilution of the plasmid constructs. Plasmid constructs were first reconstituted in molecular grade-H₂O, quantified (NanoDrop-1000, Thermo Fisher Waltham, USA), 10-fold serially diluted to contain concentrations of 10-10⁸ copy numbers /μl and then submitted to monoplex and multiplex PCR. Furthermore, 10-fold serial dilution was carried out for DNA extracted from purified cell culture-strain of CMLV (CML-14) to evaluate the sensitivity of the monoplex and multiplex PCR.

To validate the developed multiplex PCR assay successfully amplified the expected DNA fragments from 86 DNA samples extracted from homogenized clinical skin specimens as shown in Table 2. A volume of 3 μl DNA was as template in a total reaction volume of 50 μl. All collected CCE, camelpox and camel papillomatosis clinical samples were previously tested using PPV-specific PCR [21], OPV-specific PCR [59] and Pan-Papilloma PCR [54] and were confirmed as CPPV, CdPV and CMLV infections, respectively.