Studies on parameters influencing the performance of reverse transcriptase polymerase chain reaction (RT-PCR) in detecting Prunus necrotic ringpot virus (PNRSV)

Usta M., Sıpahıoglu H. M., Polat B.

Phytopathologia Mediterranea, vol.44, no.2, pp.189-194, 2005 (SCI-Expanded) identifier identifier

  • Publication Type: Article / Article
  • Volume: 44 Issue: 2
  • Publication Date: 2005
  • Journal Name: Phytopathologia Mediterranea
  • Journal Indexes: Science Citation Index Expanded (SCI-EXPANDED)
  • Page Numbers: pp.189-194
  • Van Yüzüncü Yıl University Affiliated: Yes

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.