Implementation of an artificial reaction control in a TaqMan method for PCR detection of <Emphasis Type="Italic">Ralstonia solanacearum</Emphasis> race 3 biovar 2

A previously published TaqMan PCR test for R. solanacearum race 3 biovar 2 was modified to enable both the validation of negative results and the confirmation of positive results in a closed-tube system. Negative results were validated through the use of a reaction control plasmid, designated pRB2C2, which was designed to generate a 94bp product using the same amplimers targeting the primary diagnostic 68bp sequence in R. solanacearum race 3 biovar 2 DNA. SYBR Green was included in the reaction mix to facilitate the identification of post-reaction products using melt peak analysis. The 94bp reaction control had a melt peak temperature of about 90°C, while the diagnostic target amplicon had a melt peak temperature of about 83°C; thus positive results could be easily confirmed and distinguished from the reaction control product. Addition of pRB2C2 at 100 copies per reaction had no effect on the sensitivity of the TaqMan assay for R. solanacearum race 3 biovar 2, and the modified assay successfully detected R. solanacearum race 3 biovar 2 in infected, asymptomatic tomato stems and leaves as well as in potato tubers and stems.