Arch Virol DOI 10.1007/s00705-014-2161-9 BRIEF REPORT Multiplex RT-PCR detection of three common viruses infecting orchids Raymond N. Ali • Alison L. Dann • Peter A. Cross Calum R. Wilson • Received: 19 February 2014 / Accepted: 23 June 2014 Ó Springer-Verlag Wien 2014 Abstract A multiplex reverse transcription polymerase chain reaction (RT-PCR) assay was developed for simultaneous detection of three orchid viruses: cymbidium mosaic virus (CymMV), odontoglossum ringspot virus (ORSV), and orchid fleck virus (OFV). Primers were used to amplify nucleocapsid protein gene fragments of 845 bp (ORSV), 505 bp (CymMV) and 160 bp (OFV). A 60-bp amplicon of plant glyceraldehyde-3-phophate dehydrogenase mRNA was included as an internal control against false negatives. The assay was validated against 31 collected plants from six orchid genera and compared with results obtained by transmission electron microscopy (TEM). The RT-PCR assay proved more sensitive than TEM for detection of OFV. Keywords Orchid fleck virus
Cymbidium mosaic virus
Odontoglossum ringspot virus
Multiplex RT-PCR Electronic supplementary material The online version of this article (doi:10.1007/s00705-014-2161-9) contains supplementary material, which is available to authorized users. R. N. Ali
C. R. Wilson (&) Tasmanian Institute of Agriculture, University of Tasmania, New Town Research Laboratories, 13 St. John’s Avenue, New Town, TAS 7008, Australia e-mail: Calum.Wilson@utas.edu.au A. L. Dann
P. A. Cross Department of Primary Industry, Parks, Water and Environment, New Town Research Laboratories, 13 St. John’s Avenue, New Town, TAS 7008, Australia Introduction Orchids are an important part of the global floriculture trade in cut flowers and potted plants. Viruses infecting orchids reduce plant vigour and often affect flower and foliage quality and plant value [1]. Cymbidium mosaic virus (CymMV; genus Potexvirus), odontoglossum ringspot virus (ORSV; genus Tobamovirus) and orchid fleck virus (OFV; proposed genus Dichorhabdovirus) occur globally in cultivated orchids [2–4]. Symptoms of viral infection in orchids are diverse and can vary substantially between genera and even between individual plants of the same species [4]. Multiple virus infections and variations in environmental conditions can also alter symptomology [4]. CymMV and ORSV are transmitted by propagation methods and horticultural tools, while OFV is transmitted persistently by false spider mites [4–6]. All three viruses spread through nurseries and private collections. Diagnosis is critical for effective control so infected plants may be destroyed and propagation material sourced from virus-free germplasm [1, 4]. CymMV and ORSV are readily detected by commercially available immunoassays, transmission electron microscopy (TEM), and reverse transcription polymerase chain reaction (RT-PCR), where both single and duplex detection systems have been developed [4, 7–10]. Recently reverse-transcriptase loopmediated isothermal amplification (RT-LAMP) [11] and fibre optic particle plasmon resonance (FOPPR) immunosensor [12] techniques have been adapted for detection of CymMV or CymMV and ORSV, with some advantages over traditional testing methods. In contrast, commercial sources of antisera for OFV are not available, and TEM can be problematic as the relatively unstable OFV particles are less distinct and present at lower titre than the other two viruses. Development of RT-LAMP and FOPPR assays has 123 R. N. Ali et al. not yet been attempted for OFV. RT-PCR assays for detection of OFV have been developed [2, 3] but are not widely used, as testing for several viruses would require multiple tests. Multiplex PCR can be used for the simultaneous detection of several viruses and is highly sensitive and cost effective [13]. We describe a multiplex RT-PCR assay optimized for simultaneous detection of OFV, CymMV and ORSV in orchids. Materials and methods Leaves of Cymbidium (21 samples), Dendrobium (1 sample) Oncidium (1 sample) and Phalaenopsis (2 samples) orchid species showing symptoms suggestive of virus infection were obtained from Australian collections in Tasmania, New South Wales and Victoria submitted for laboratory testing (Supplementary Fig. S1). Infections with ORSV, CymMV and/or OFV were determined by TEM examination. Four samples (Cymbidium, Dendrobium, Masdevallia, and Oncidium) lacking obvious symptoms of virus infection were also collected and confirmed to be free of ORSV, CymMV and OFV by TEM. Two further samples, one each from Masdevallia and Brassia species with possible symptoms of virus infection, were collected from the Royal Tasmanian Botanical Gardens orchid collection but were not tested by TEM (Supplementary Fig. S1). All leaves were stored at -80 °C for later use. TEM examinations were conducted by macerating a 2to 3-mm square section of symptomatic tissue in a drop of 2 % ammonium molybdate, pH 6.5, on a glass slide. Sap and stain mixture was transferred to a carbon-coated Parlodion support film 400 mesh grid and allowed to air dry. Each sample was viewed with a Phillips 201 TEM (20,0009) for 3-5 minutes covering c. 50 fields of view. Total RNA was extracted from 50 mg of orchid leaves using a PowerPlantÒ RNA Isolation Kit (MO-BIO, Carlsbad, CA, USA) according to the manufacturer’s instructions and eluted into a final volume of 100 lL. Total RNA content was estimated by fluorescence spectroscopy (Qubit 2.0 flurometer; Life Technologies, Carlsbad, CA, USA). First-strand cDNA was synthesized using a Tetro cDNA synthesis kit (Bioline, Taunton, MA, USA) in 20-lL reaction mixtures containing 2 lL of RNA extract, following the manufacturer’s instructions, and stored at -20 °C. Virus-species-specific primers were designed from conserved regions within the nucleocapsid protein gene identified by Clustal W alignments of sequences in the GenBank database using the PrimerSelect module of DNASTAR 5.01 (DNASTAR, Inc., Madison, WI, USA). For OFV primers, 2-4 degeneracies were required to match sequences of the two known strains [2, 14]. The primer 123 pairs used were ORSV (ORSV-F, 50 -ATTTAAGCT CGGCTTGGGCT-30 ; ORSV-R, 50 -CTACCCGAGGTAA GGGGGAA-30 ; amplicon size 845 bp), CyMV (CyMV-F, 50 -ACCCCACTTCTGCACCAAAA-30 ; CyMV-R, 50 -CC GTACTTCCCGATCGAGTG-30 ; amplicon size 505 bp); OFV (OFV-F, 50 -GRCTKGCWGCGGAGGCWGAC-30 ; OFV-R, 50 -CTGGCGGAWGGKGGTGTGAACAG-30 ; R = A/G, K = G/T, W = A/T; amplicon size, 160 bp). A primer pair designed from conserved regions of an alignment of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA sequences from diverse plant species was selected as an RT-PCR internal control (GAPDH-F, 50 AAGGACTGGAGGGGTGGAAG-30 ; GADPH-R, 50 -AGCTCCAGTGCTGCTAGGAA-30 ; amplicon size 60 bp). All primer pairs had similar annealing temperatures (57.263.8 °C) and were not predicted to form secondary structures or primer dimers. The uniqueness of the primer sequences was confirmed using the NCBI Basic Local Alignment Search Tool. Each primer pair was tested in isolation with its complementary virus cDNA template (from Cymbidium samples shown to contain individual virus infections by TEM) to confirm amplification of the expected gene product. Primer pairs were then tested in a multiplex RT-PCR against all combinations of virus cDNA templates. Single and multiplex RT-PCR reaction mixtures contained 1 lL of cDNA (either single or pooled templates), 10 lL of 109 HotstarTaq Plus Master Mix (QIAGEN), 1 lL or 0.5 lL of each (10 lM) forward and reverse primer with RNase-free water added to a final volume of 20 lL. Amplifications were performed in an Eppendorf Mastercycler Gradient 5331 (Hamburg, Germany). A series of optimization steps were performed to determine the most appropriate annealing temperature and number of cycles and concentration of each primer pair. The optimal annealing temperature in the single and multiplex assays was determined by gradient PCR using single incremental temperature units from 50.0 °C to 70.5 °C. Concentrations of the individual primer pairs within the multiplex RT-PCR were then varied in order to obtain a relatively even amplification of all products. Cycling conditions for both single and multiplex RT-PCR consisted of initial heat activation at 95 °C for 5 minutes, followed by 30 to 40 cycles of denaturation at 95 °C for 1 minute, primer annealing (50.0 °C to 70.5 °C for the gradient run and 60 °C for all subsequent assays) for 1 minute, and primer extension at 72 °C for 1 minute, terminating in final extension at 72 °C for 10 minutes. Reaction products (6 lL) were separated by 1.5 % agarose gel electrophoresis in Tris-acetate-EDTA buffer (pH 8.0) and visualized with 0.1 lL SYBRÒ Safe DNA gel stain (Life Technologies) per mL. A non-template control and cDNA from a healthy plant were used as negative controls. Multiplex RT-PCR detection of viruses in orchids Multiplex RT-PCR reactions were used to test the sensitivity of virus detection using tenfold dilutions of cDNA virus templates. First, a mixture of equal dilutions of all three virus cDNA templates (from 100 to 10-3) was tested, and then dilutions of individual virus cDNA (100 to 10-3) mixed with undiluted cDNA of the other two viruses to simulate detection efficiency if one virus was present at low titre within a sample. The optimized multiplex RTPCR assay was then validated against 31 orchid samples from six different genera (Cymbidium, Brassia, Dendrobium, Phalaenopsis, Masdevallia, and Oncidium), 29 of which had been previously assessed for ORSV, CymMV and OFV infection by TEM. viral pathogens and the internal control were detected in assays corresponding to each viral cDNA mix. Amplicons for all viruses were visually discernable in dilutions of the three viral cDNAs up to 10-3 (Fig. 1). Dilution of single-virus cDNAs within a mix in which the other two remained undiluted allowed detection of ORSV and CymMV up to 10-2 and OFV up to 10-3 (Fig. 1). The multiplex RT-PCR assay successfully detected infections of OFV, CymMV and/or ORSV or the lack of infection in 31 samples from six different orchid genera (Fig. 2). Prior TEM examination of 29 of these samples confirmed the presence of CymMV (3 samples), ORSV (12 samples) and OFV (15 samples) but failed to detect OFV in three samples with co-infections with CymMV or ORSV (Fig. 2). Results Discussion In individual RT-PCR assays, ORSV, CymMV, OFV and GAPDH primers produced visible amplicons over a wide range of annealing temperatures (50.0 °C–68.1 °C, 50.0 °C–63.5 °C, 50.0 °C–70.5 °C, and 50.0 °C–70.5 °C, respectively). In multiplex RT-PCR, the use of equimolar concentrations of all primer pairs (0.5 lM) resulted in suppression of CymMV at an annealing temperature below 55.5 °C, with no amplification above 66.0 °C and with OFV and GAPDH amplicons being considerably more intense than others. Reducing the concentration of both OFV and GAPDH primers to 0.25 lM within the multiplex reaction gave more even amplification of all amplicons. Detection of OFV, CymMV and ORSV by multiplex RTPCR was deemed optimal at an annealing temperature of 60 °C for 1 minute with 35 amplification cycles. The appropriate-sized amplicons corresponding to the three We describe the development and optimization of a multiplex RT-PCR assay for simultaneous detection of CymMV, ORSV and OFV. Single infection and co-infections with these three viruses were detected from multiple orchid genera. Multiplex RT-PCR enables rapid detection of multiple viruses, removing the need for several individual tests [15, 16]. The development of a multiplex RT-PCR assay requires a systematic approach to determine optimal reaction conditions [17, 18]. Designing primers that target conserved regions of the viral genome enables detection of multiple strains or isolates despite natural intraspecific sequence variability [17]. Here, redundant nucleotides in OFV primers allowed for detection of the known OFV variants [2, 14]. Sequence analysis of the OFV isolates tested here showed that all belong to the most common Fig. 1 Sensitivity of the multiplex RT-PCR for detection of ORSV, CymMV and OFV. Lanes 1-3 show amplification of individual virus products. Lanes 6-18 show the capacity of the assay to detect virus- specific amplicons following 100 to 10-3 dilutions of cDNA templates. Lanes 1, 5 and 20, 50-bp DNA size marker; lane 19, notemplate control 123 R. N. Ali et al. Fig. 2 Orchid leaf samples (n = 27) originating from collections around Australia were tested by multiplex RT-PCR and TEM for ORSV, CymMV and OFV (lanes 2-32). Labels above the figure denote infection results determined by RT-PCR, and results of TEM examination are indicated by ? (presence) or – (absence) beneath the figure. ? indicates that the sample was not tested by TEM. (cy, Cymbidium; on, Oncidium; de, Dendrobium; ph, Phalaenopsis; ma, Masdevallia; Br, Brassia species). * indicates that co-infection with OFV was not detected by TEM. Lanes 1 and 34, 50-bp DNA size marker; lane 33, no-template control strain [2], and testing of the assay with further variants may be warranted. Primer analysis can predict incompatible primer interactions, such as primer self-annealing, selflooping and annealing between forward and reverse primers [19]. However, empirical testing is necessary for efficient amplification of target products in the multiplex reaction [20]. In particular, inappropriate ratios of primers may reduce amplification or cause nonspecific amplification of target templates [20]. The primers used in this study reliably produced amplicons of the appropriate size for each virus target without extraneous banding at the annealing temperature used. However, a reduction in the concentration of both OFV and GAPDH primers was required for optimal detection as equimolar primer concentrations resulted in preferential amplification of the two shortest fragments. Previous studies have reported similar preferential amplification of short fragments [19, 21]. This is the first RT-PCR assay that simultaneously detects OFV, CymMV, and ORSV. This is important because the absence of an efficient single assay for all three viruses may have meant that OFV infections have gone undetected [10]. Routine testing relying on single assays for CymMV and ORSV (immunoassay or RT-PCR) will not detect OFV, and direct examination of the leaf sap by TEM can miss OFV infections, as the particles are less distinct than CymMV and ORSV and generally in low abundance. This was demonstrated in this study with routine TEM examinations failing to detect OFV in three samples that were co-infected with CymMV or ORSV. Routine and sensitive detection of important orchid viruses is critical for managing virus spread and gaining a better understanding of their epidemiology. Multiplex RTPCR provides a rapid sensitive assay for several viruses simultaneously and offers significant advantages over many traditional assays. Limitations of the test include potential detection failures if new virus variants are encountered with significant sequence changes at the site of primer binding. 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