Real-time PCR assay for rapid, efficient and accurate detection of Paramyrothecium roridum a leaf spot pathogen of Gossypium species
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Here we report a highly sensitive real-time PCR (qPCR) assay to detect Paramyrothecium roridum from pure culture and infected samples of cotton plants. A specific set of primer pair pMyro F/R is designed to target the 185 bp ITS region of rDNA of Paramyrothecium roridum species and validated using qPCR. The fluorescence signals were detected above the baseline threshold from samples containing Paramyrothecium roridum DNA, whereas other samples did not produce any fluorescence or produced fluorescence which did not reach detection threshold values. A single dissociation peak of increased fluorescence was obtained for the specific primers at 92.2 °C melting temperature. The limit of detection using SYBR Green dye in this assay was up to 0.1 pg per µL of DNA from pure culture of P. roridum. The assay is accurate, sensitive, less laborious and time saving for detection of P. roridum in infected tissues of cotton.
KeywordsSYBR® Green Paramyrothecium roridum Leaf spot Cotton Internal transcribed spacer
The authors gratefully acknowledge financial support under ICAR-Technology Mission on Cotton and DBT Grant No. BT/PR3900/GBD/27/386/2012.
Compliance with ethical standards
Conflict of interest
The authors declare that they do not have any conflict of interest.
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