2.1 Collection of symptomatic leaf samples and DNA extraction
Leaf samples of chilli (Capsicum annum L.), potato (Solanum tuberosum) and sow thistle (Sonchus arvensis) showing typical begomoviral symptoms (curling and/or mosaic; Fig. 1) were collected from adjoining areas of Jalandhar region of Punjab in Northern India. These plants were selected because the previous reports suggested that there are chances of amplification of all four targets in these samples (Kumar et al., 2011; Jeevalatha et al., 2017; Mubin et al., 2010). Samples were collected in Falcon tubes, put in liquid nitrogen and stored at -80ºC for further use. Total DNA was extracted from the samples using DNeasy® Plant Mini Kit (Qiagen, Germany). Quality of the extracted DNA was assessed by electrophoresing it in 1% agarose gel, and the DNA was stored at -20◦C.
2.2 Primer designing
Four pairs of primers were designed (Table 1) to simultaneously detect DNA-A, DNA-B, alphasatellite and betasatellite. Complete reference sequences of various distinct species of these components (reported from India) were retrieved from NCBI-GenBank and used for designing the primers. The sequences were aligned using MultAlin software (Corpet, 1988). Conserved regions were analyzed and potential primers were designed keeping in mind the physical properties, internal structures and similar annealing temperatures. OligoCalc (oligonucleotides properties calculator) software (Kibbe, 2007) was used to analyze these properties. Degeneracy was incorporated in the primers wherever required. The primers for mPCR were designed in such a way that the amplicons for each component could differ by at least 100bp, which could be separated easily by electrophoresing them in1% agarose gel. Designed primers were synthesized by Eurofins Genomics India Pvt. Ltd., Bengaluru, India.
2.3 Thermal profiles of simplex PCRs
Simplex PCRs were carried out with the designed component-specific primers in order to optimize the thermal profiles, and to evaluate their specificity. The reactions were carried out in a thermocycler (ThermoFisher Scientific, USA) with a total of 25µl volume containing 2.5µl of total DNA (~ 200ng), 2.5µl of 10X Taq buffer A, 0.5µl of 40mM dNTPs mix, 0.5µl each of 10mM forward and reverse primers, 0.3µl (3U/µl) of Taq DNA Polymerase (Genei, India), and the final volume was makeup with molecular grade water. The parameters used are: initial denaturation at 94ºC for 3 min, followed by 35 cycles of denaturation at 94ºC for 50 sec, annealing from 50ºC to 56ºCfor 30 sec to 1 min, extension at 72ºC for 1 min, and final extension at 72ºC for 7 min. The PCR products were analysed on 1% agarose gel in 1x TAE buffer for 45min at 3V/cm and visualized using EtBr (ethidium bromide) staining. A 100bp DNA ladder (Genei, India) was used as a molecular weight marker.
2.4 Thermal profile of Multiplex PCR
The simplex PCR component-specific primers were used to optimize the mPCR assay. Amplification was carried out in a reaction volume of 50 µl containing: total DNA 5µl (~ 200ng), 10x Taq Buffer A 5µl, 40mM dNTPs mix 1µl, 1µl each of 10mM forward and reverse primers, Taq DNA polymerase 0.6µl (3U/µl), and the final volume was adjusted to 50µl using molecular grade water. Multiplex PCR was performed in a thermocycler (ThermoFisher Scientific, USA) with an initial denaturation step at 94ºC for 3 min, followed by 35 cycles of denaturation at 94ºCfor 50 sec, annealing at 52ºC for 1 min, extension at 72ºC for 1 min, and final extension at 72ºC for 7 min. The PCR products were electrophoresed on 1% agarose gel in 1x TAE buffer for 45min at 3V/cm and visualized using EtBr staining. A 100bp DNA ladder (Genei, India) was used as molecular weight marker.
2.5 Sequencing of the PCR-amplified product
To confirm their identity, the amplicons from selected samples were excised from agarose gel and eluted using QIAquick Gel Extraction Kit (Qiagen, Germany). The eluted DNAs were sequenced by Eurofins Genomics India Pvt. Ltd., Bengaluru, India. The sequences were analysed using BLAST (Altschul et al., 1990) to confirm the specificity of PCR-amplified DNA fragments (data not shown).