Computational protocol: Molecular Diagnostic Tools for Detection and Differentiation of Phytoplasmas Based on Chaperonin-60 Reveal Differences in Host Plant Infection Patterns

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Protocol publication

[…] Sequences for all amplification primers and hybridization probes along with optimized amplification conditions are shown in . Primers for phytoplasma PCR amplification were based on the cpn60 UT primer annealing sites , but were adapted to phytoplasma sequences using full-length cpn60 genes from public databases (www.cpndb.ca and www.ncbi.nlm.nih.gov). One set of primers (H279p/H280p) was based on 18 full-length cpn60 sequences, which primarily represented the AY group. A second set of PCR primers (D0317/D0318) was designed based on the full-length cpn60 reported in the Peanut Witches’ Broom (PnWB) Phytoplasma genome sequence reported by Chung et al. . Hybridization probes for the fluorescent microsphere detection assay were designed using PrimerPlex v2.62 (Premier Biosoft, Palo Alto, CA, USA). [...] The diagnostic sensitivity and specificity, along with 95% confidence intervals, were calculated using 192 DNA extracts from field-collected plant tissues according to standard methods . The results of 16S–23S rRNA locus-targeted PCR amplified with P1/Tint were used as the gold standard to call positive and negative samples. The limit of detection (LOD) of each assay was determined by probit analysis of 8 replicates each of a dilution series of non-linearized plasmid DNA containing 107–101 copies/reaction. Plasmids were diluted in 10 mM Tris-Cl, pH 8.0 containing 5 ng/ml yeast tRNA and stored in DNA low-bind tubes (Eppendorf). Probit analysis was conducted using SPSS (IBM Corp. Released 2010. IBM SPSS Statistics for Windows, Version 21.0) and the LOD was specified as the copy number that was predicted to yield a positive result in 95% of assays performed, as specified by Bustin et al. . Plasmid templates for the LOD assays were as follows: for H279p/H280p, AY-OY-M; for D0317/D0318, FD; for P1/Tint, PCR product from infected flax plants cloned into pGEM-T Easy. [...] Sequences were aligned using ClustalW and analyzed using the Maximum Likelihood method based on the Tamura-Nei model . Trees were bootstrapped 1000 times. Analysis was conducted in MEGA5 . […]

Pipeline specifications

Software tools SPSS, Clustal W, MEGA
Applications Miscellaneous, Phylogenetics
Organisms Brassica napus, Camelina sativa