Adaptation of a Phytophthora ramorum real-time polymerase chain reaction assay based on a mitochondrial gene region for use on the Cepheid SmartCycler.

Detection of Phytophthora ramorum in US commercial nurseries has led to a number of quarantine regulations. Methods such as real-time PCR (RT-PCR) provide rapid and reliable detection that can supplement attempts to culture P. ramorum from symptomatic tissue. We adapted and optimized a previously described mitochondrial gene-based RT-PCR assay for use with a Cepheid SmartCycler v.1 and ready-to-use lyophilized PCR beads. The detection limit was 10 fg of P. ramorum genomic DNA. No cross-reactivity was observed on the SmartCycler for seven additional Phytophthora species tested, which included species known to cross-react in other assays as well as recently described species Phytophthora foliorum and P. kernoviae. The SmartCycler assay described here was used to detect P. ramorum in a set of 2008 California field samples with a high degree of accuracy. Introduction When Phytophthora ramorum (Werres, De Cock & Man in’t Veld) sp. nov., cause of significant oak mortality in the coastal forests of California (15,16), was recovered from a California nursery in 2001, it increased concerns that the pathogen could spread to uninfested regions of the USA through movement of infected nursery stock. Despite regulatory efforts in place in California, potentially infected material from a nationwide nursery supplier was shipped to 783 garden centers in 39 states in 2004 (19). Within a few weeks of the shipment, the United States Animal Plant Health Inspection Service (APHIS) increased restrictions (1) and sampling for the national survey to determine the pathogen’s distribution and to prevent its future spread. Both nested PCR and real-time PCR (RT-PCR) have been developed for the detection of P. ramorum (3,6,10,11,12,13,14,18,20,22,23,24). The internal transcribed spacer (ITS) region of the nuclear ribosomal RNA gene has been widely used (8,10,11,12,24), but cross-reactivity has been observed with P. lateralis and/or P. hibernalis in these assays (3,6,10,12,20). The gene sequences of b-tubulin and elicitin can be used for detection (2), but P. lateralis has been shown to cross-react with the elicitin primers (2). The spacer region between coxI and coxII of the mitochondrial genome offers another detection target (13,22,23); this sequence is abundant and variable at the species level (13,22). Using a RT-PCR assay based on this region, no cross-reactivity was observed when tested with 45 other Phytophthora species (22) or when a number of PCR assays for detection of P. ramorum were compared in a multi-laboratory study using DNA from a standardized set of isolates of different Phytophthora species (14). The assay described by Tooley et al. (22) was optimized for the ABI Prism 7700 Sequence Detection System, SDS (Applied Biosystems, Foster City, CA). Here, we report its adaptation to the Cepheid SmartCycler (Cepheid, Sunnyvale, CA). The SmartCycler is widely used by molecular diagnostic laboratories certified as part of the National Plant Diagnostic Network (www.npdn.org), so 13 February 2010 Plant Health Progress successful adaptation of an assay to this platform will contribute to its nationwide applicability. The goals of this study were to optimize assay conditions for the SmartCycler using ready-to-use lyophilized PCR beads, evaluate specificity by testing DNA from Phytophthora species known to crossreact in other PCR assays, and to test recently-described species Phytophthora foliorum (7) and Phytophthora kernoviae (4) to confirm that no cross-reactivity occurs in the assay. Real-time PCR Conditions Specific primers and probes used here were previously designed by Martin et al. (13) and Tooley et al. (22), respectively (Table 1). RT-PCR reactions were performed using the Cepheid SmartCycler v.1 and software 2.0 D (Cepheid, Sunnyvale, CA). Background fluorescence was automatically determined by the analysis software using raw data from cycles 3 to 10. Thresholds were set 10 standard deviations above the background. DNA samples were tested in a total volume of 25 μl containing reconstituted 1X OmniMix HS (Cepheid, Sunnyvale, CA), 1 μM each P. ramorum primer, and 0.15 μM PrFAM probe. Duplex PCR reactions also contained 0.1 μM each plant primer, 0.2 μM Plant CAL Red probe, and an additional 1 μM MgCl and 75 μM each dNTP. Both plant and pathogen probes were diluted and placed at -20°C prior to use. Cycling conditions for all PCR reactions were 95°C for 2 min and 60 cycles of 95°C for 1 s and 60°C for 30 s. Fluorescence was recorded during each annealing/extension step. Detection of CAL Red 610 was recorded through the ROX channel on the SmartCycler without additional calibration. Table 1. Polymerase chain reaction primera and fluorescent probe sequencesb used in the RT-PCR assay for the detection of Phytophthora ramorum on the Cepheid SmartCycler. x Primer sequences were previously described (13). y Probe sequences were previously described (22). Plant CAL Red reporter dye replaced CAL Orange in the previously described probe sequences. z Probes were labeled at the 5’ end with either the fluorescent reporter dye 6carboxyfluorescine-aminohexyl amidite (FAM) or CAL Fluor Red 610 amidite (CAL Red 610) and labeled at the 3’ end with a black hole quencher dye (BHQ, Biosearch Technologies, Novato, CA). Specificity of P. ramorum Primers and Probe Genomic DNA samples from 21 isolates representing eight Phytophthora spp. (Table 2) were tested with RT-PCR. DNA was extracted from lyophilized tissue of P. ramorum, P. cactorum, and P. citricola isolates grown on a liquid synthetic medium (25). A DNeasy Plant Mini kit (Qiagen, Inc.,Valencia, CA) was used to extract DNA from the six P. ramorum isolates. DNA from isolates 384, 385, and 422 was extracted following Goodwin et al. (9). DNA from the remaining species was part of a multi-laboratory study designed to compare different P. ramorum detection assays (14). DNA concentrations for the samples extracted in our laboratory were determined using the NanoDrop ND1000 Spectrophotometer (NanoDrop Technologies, Wilmington, DE). Duplicate RT-PCR reactions for each isolate were tested twice using 100 pg of template DNA along with primers FMPr1a and FMPr7 and the PrFAM probe. Sterile distilled water was used as a negative control. Target Primer/probe Sequence (5’ to 3’ ) Length P. ramorum FMPr-1 GTATTTAAAATCATAGGTGTAATTTG 26 P. ramorum FMPr-7 TGGTTTTTTTAATTTATATTATCAATG 27 P. ramorum PrFAM probe 6-FAM d(CAGATATTAAACAAATTATATATA AAATCAAACAA) BHQ-1 35 Plant FMPl-2 GCGTGGACCTGGAATGACTA 20 Plant FMPl-3 AGGTTGTATTAAAGTTTCGATCG 23