Computational protocol: Development of Multiplex PCR for Simultaneous Detection of Citrus Viruses and the Incidence of Citrus Viral Diseases in Late-Maturity Citrus Trees in Jeju Island

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Protocol publication

[…] To sequence the PP2 gene for the polyprotein of the SDV or CiMV, general procedures for plasmid DNA purification, cloning, and transformation were used, according to each of manufacturer’s recommendations. The amplified DNA fragments were recovered from agarose gels and purified using the AccuPrep Gel Purification Kit (catalog no. K-3035; Bioneer) and then cloned and transformed using the Topo TA Cloning Kit (Invitrogen). Sequencing grade plasmid DNA was obtained using the AccuPrep Nano-Plus Plasmid Mini Extraction Kit (catalog no. K-3111; Bioneer). For each isolate, DNA was sequenced in both directions using M13 forward and SP6 reverse primers for three subclones. Sequencing was done automatically using dyelabeled dideoxy nucleotides and DNA polymerase in an Applied Biosystems 3730XL DNA sequencer (Applied Biosystems, Foster City, CA, USA). The consensus sequence for the three subclones was used for phylogenetic analysis. Sequence alignments were done with the Clustal X 2.0 program (). The region corresponding to base pairs 2,287 to 5,230 of CiMV genomic RNA, segment RNA 2, complete sequence (Gen-Bank AB465581.1), and that corresponding to base pairs 2,235 to 5,311 of SDV genomic RNA (RNA2) for polyprotein, complete cds (GenBank AB009959.2) were used as reference for sequence analysis.The nucleotide sequences were aligned using Clustal W and pairwise sequence comparisons, and phylogenetic analysis was performed with MEGA 6 (). Neighbor-joining trees were constructed according to the maximum-likelihood method in MEGA 6. A heuristic search employing 1,000 random stepwise addition replicates and all gaps and missing data were completely deleted. Bootstrap values were based on 1,000 repetitions. […]

Pipeline specifications

Software tools Clustal W, MEGA
Application Phylogenetics
Organisms Satsuma dwarf virus, Citrus tristeza virus
Diseases Virus Diseases, HIV Infections