Development of a multiplex PCR assay for detection and identificationof a chickpea phytoplasma strain utilizing 16S rRNA and imp genesspecific primers

A multiplex PCR assay was developed by optimizing reaction components and cycles for the detection of a phytoplasma associated with the chickpea phyllody disease. Three sets of primers corresponding to 16S rRNA universal primer pair P1/P7, nested primer pair R16F2n/R16R2 and imp gene specific primers (IMPIIF2/IMPIIR1) were used together. Different concentrations of the PCR components such as primers, template DNA and PCR annealing temperature were optimized for amplification of phytoplasma DNA in a multiplex assay. Expected length fragments of 1.8 kb (P1/P7), 1.2 kb (R16F2n/R16R2) and 717 bp (IMPIIF2/IMPIIR1) were amplified from the symptomatic tissue. The developed multiplex PCR protocol provided a sensitive, rapid and cheap assay in identifying the phytoplasma associated with the chickpea phyllody disease. The phytoplasma was identified as a strain enclosed in the 16SrII-D subgroup based on 16S rRNA and imp genes amplifed both by PCR and the multiplex PCR assay developed.