Multiplex real-time polymerase chain reaction (PCR) for detection of Phytophthora ramorum, the causal agent of sudden oak death

Abstract

Since 1995, Phytophthora ramorum has been causing sudden oak death in California and Oregon, affecting tens of thousands of oak trees over large areas and infecting more than 100 other plant species. Quarantine measures are in effect, and regulatory agencies need to detect this organism in a timely and efficient manner. Various molecular assays have been developed over the past few years, and redundancy using multiple gene regions of P. ramorum was shown to increase the reliability in detecting the pathogen. However, such multi-gene assays require different polymerase chain reactions (PCRs) to test a single sample. To improve P. ramorum detection, three different TaqMan assays were multiplexed with a fourth TaqMan specific to the Phytophthora genus in a single reaction. A second multiplex TaqMan PCR assay to detect oomycetes and give a positive PCR reaction in the presence of plant DNA was also designed and tested in conjunction with the P. ramorum internal transcribed spacer (ITS) and Phytophthora genus TaqMan assays. These assays were tested on different Phytophthora species and were verified on two different sets of field samples previously assayed by other laboratories. These were obtained from multiple field hosts infected by various Phytophthora species, and the DNA from one set was extracted from ELISA lysates. All known P. ramorum samples from pure cultures or field samples were detected using these multiplex real-time PCR assays. In general, TaqMan multiplex assays showed lower detection sensitivity than single separated reactions. However, the multiplex assays still detected P. ramorum accurately while decreasing the cost and increasing throughput.

Keywords:

• molecular diagnostic
• multiplex PCR assay
• invasive pathogens