Studies on Parameters Influencing the Performance of Reverse Transcriptase Polymerase Chain Reaction (RT-PCR) in Detecting Prunus Necrotic Ringpot Virus (PNRSV)

M. Usta, H.M. Sipahioglu, B. Polat

Abstract


In order to have a more detailed understanding of the various factors influencing a reverse transcriptase
polymerase chain reaction (RT-PCR), a number of important parameters such as Mg+2, primer, enzyme concentration
and others were optimized for the detection of Prunus necrotic ringspot virus (PNRSV). Using a PNRSV isolate with
a pair of primers, complementary DNA of viral genome as template, and an appropriate enzyme together with magnesium
chloride, the following optimal conditions were identified: primer concentration between 0.2 and 0.0002 pmol
µl-1 and 0.06–2 units µl-1 for Taq DNA polymerase enzyme for a 50 µl reaction volume when other parameters were
optimum; magnesium chloride concentration less than 2.5 mM; dNTP concentration between 1 and 10 mM. The
optimum cDNA amount should be ~360 ng for a 50 µl reaction mixture. When these optimized concentrations and/or
values of the main PCR parameters were brought together for a new RT-PCR, a clear and a reliable PNRSV detection
having no background was performed from both growth-chamber and field-grown PNRSV-infected plants.

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DOI: http://dx.doi.org/10.14601/Phytopathol_Mediterr-1790



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