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Author's Personal Copy Journal of Virological Methods 236 (2016) 62–67

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Development of a rapid diagnostic assay for the detection of tomato chlorotic dwarf viroid based on isothermal reverse-transcription-recombinase polymerase amplification Rosemarie W. Hammond a,∗ , Shulu Zhang b a b

USDA ARS Molecular Plant Pathology Laboratory, Beltsville, MD 20705, United States Agdia Inc., 52642 County Road 1, Elkhart, IN 46514, United States

a b s t r a c t Article history: Received 12 March 2016 Received in revised form 7 June 2016 Accepted 26 June 2016 Available online 15 July 2016 Keywords: AmplifyRP® acceler8TM Tomato Petunia Detection TCDVd Seed Tomato chlorotic dwarf viroid RT-RPA

A molecular diagnostic assay utilizing reverse transcription-recombinase polymerase amplification (RT-RPA) at an isothermal constant temperature of 39 ◦ C and target-specific primers and probe were developed for the rapid, sensitive, and specific detection of tomato chlorotic dwarf viroid (TCDVd) in infected leaf and seed tissues. The performance of the AmplifyRP® Acceler8TM RT-RPA diagnostic assay, utilizing a lateral flow strip contained within an amplicon detection chamber, was evaluated and the results were compared with a standard RT-PCR assay. The AmplifyRP® Acceler8TM assay was specific for TCDVd in leaf and seed tissues, its sensitivity was comparable to conventional RT-PCR in leaf tissues, and it does not require extensive sample purification, specialized equipment, or technical expertise. This is the first report utilizing an RT-RPA assay to detect viroids and the assay can be used both in the laboratory and in the field for TCDVd detection. Published by Elsevier B.V.

Tomato chlorotic dwarf viroid, a member of the genus Pospiviroid in the family Pospiviroidae class of plant pathogens, is a small, covalently-closed, circular, single-stranded RNA that does not encode proteins and that replicates autonomously in infected plants. The genome of tomato chlorotic dwarf viroid (TCDVd) is 360 nucleotides in length and is phylogenetically closely related to, but is distinct from (86–88% sequence homology), potato spindle tuber viroid (PSTVd) (Hammond and Owens, 2006; Di Serio et al., 2014). TCDVd was first reported in natural infections of greenhouse tomato (Solanum lycopersicum L.) in Canada grown from seed imported from the Netherlands through the USA (Singh et al., 1999) and has since been reported in greenhouse tomatoes in the United States (Verhoeven et al., 2004; Ling et al., 2009), France (Candresse et al., 2010), Mexico (Ling and Zhang, 2009), Japan (Matsushita et al., 2008, 2009, 2010), and Norway (Fox et al., 2013), and in potatoes in an experimental field plot in Canada (Bostan et al., 2004). TCDVd has also been found in ornamentals worldwide as symptomless infections in Brugmansia sanguine (Netherlands-Verhoeven et al., 2010), Petunia x hybrida spp. (USA-Verhoeven et al., 2007; UK- James et al.,

∗ Corresponding author. E-mail address: [email protected] (R.W. Hammond). http://dx.doi.org/10.1016/j.jviromet.2016.06.013 0166-0934/Published by Elsevier B.V.

ˇ 2008; Japan-Shiraishi et al., 2013; Cervaná et al., 2011; Finland, ˇ EPPO/OEPP, 2009; Slovenia-Virˇscek Marn and Mavriˇc Pleˇsko, 2010), in trailing verbena in India (Singh et al., 2006), and in Vinca minor in Canada (Singh and Dilworth, 2009). Additional hosts in the Compositae and Solanaceae were found to be susceptible upon inoculation with TCDVd-infected sap (Singh et al., 1999; Matsushita et al., 2009; Matsushita and Tsuda, 2015). The source of these infections may be from contaminated seed or ornamental hosts. TCDVd, in addition to other pospiviroids (Verhoeven et al., 2004), is a serious threat to tomato production. Symptoms in tomato include chlorotic leaves, veinal and petiole necrosis, reduced leaf size, stunting, and small and deformed fruit (Nie, 2012). Symptoms in potato (S. tuberosum L.) include severe dwarfing and upright growth and severe cracking of tubers (Singh et al., 1999, 2010). TCDVd can be easily transmitted by mechanical means through contact and contaminated tools and through vegetative propagation; in greenhouses, TCDVd is spread down the row by mechanical transmission (Ling et al., 2009) and is stable in dried tissues (Matsushita et al., 2009). TCDVd is also spread mechanically by bumblebees (Bombus ignitus) between tomato plants in greenhouses (Matsuura et al., 2010). TCDVd is detected in seeds and is seed transmitted in tomato and the rate of seed and seedto-seedling transmission is variable, ranging from 0% (Koenraadt

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et al., 2009; Matsushita et al., 2011), 0.8% (Candresse et al., 2010), to 80% (Singh and Dilworth, 2009) in a test of 4000, 120, 2500, and 50 seedlings, respectively. The most effective control of pospiviroid diseases includes prevention by using viroid-free planting materials and working under strict hygiene conditions (EFSA Panel on Plant Health, 2011). This may include testing for pospiviroids in seeds and propagation materials at pre-entry (certified viroid-free), point of entry, postentry in propagation stocks, surveillance, cultural practices and hygiene practices that minimize spread (Matsuura, 2012; EFSA Panel on Plant Health, 2011). Methods for viroid detection include biological indexing (Raymer and O’Brien, 1962), polyacrylamide gel electrophoresis (Schumacher et al., 1986), nucleic acid hybridization (Owens and Diener, 1981; reverse transcription-polymerase chain reaction (RTPCR) and real-time RT-PCR assays (Bostan et al., 2004; Botermans et al., 2013). Using a combination of these methods for detection, successful management and eradication of PSTVd in potato in the USA and Canada has been achieved (De Boer and DeHaan, 2005; Sun et al., 2004). Testing for viroids by hybridization, microarrays, RT-PCR, and real-time RT-PCR requires the purification of total nucleic acids, lengthy test times, technical expertise, expensive thermal cycling equipment for amplification reactions, and post amplification analysis of amplicons by gel electrophoresis or specialized laboratory equipment (Singh and Crowley, 1985; Bostan et al., 2004; Candresse et al., 2010; Matsushita et al., 2010; Zhang et al., 2013). In this study, we describe the development of a rapid, specific, sensitive, and easy to perform test for TCDVd infection in leaf and seed tissues, based on reverse transcription-recombinase polymerase amplification (RT-RPA) technology that can be used in field and laboratory conditions. To aid in the design of the primers and probe for the RT-RPA assay, the complete genome sequences of TCDVd isolates from several different countries and the type isolates of all the pospiviroids were aligned using the CLUSTAL 2.1 program (see Fig. 1). A target region in TCDVd was identified between nucleotide (nt) position 113 and 340 and two primers and an internal probe were selected from the sequence-conserved regions of TCDVd as required for the RT-RPA. The two selected primers, TCDVd FP3 (5 ACTGGCAAAAGGCGGCAGGGAGCTTGTGGA-3 ) and 5 end biotinlabeled TCDVd RP4 (5 -AGGGCTAAACACCCTCGCCCCGGTAGCAGC3 ), yield an amplicon size of 228 base pairs (bp). The internal probe was selected between nt 210 to nt 260 of TCDVd and labeled with FAM at its 5 end (Fig. 1). The 5 portion of the probe, upon cleavage by the endonuclease, works with the RP4 primers to produce a dually (FAM/Biotin) labeled amplicon of 131 bp. This dually labeled amplicon is then captured and visualized on an immunostrip contained inside the Amplicon Detection Chamber (ADC98800, Agdia) through a lateral flow strip assay. The results are recorded 20 min after the flow begins. If both a test line and a control line are shown on the strip, the test is positive. If only a control line, which is the internal test standard, is seen the test is considered negative. Tomato (S. lycopersicum cv. Rutgers) seedlings were mechanically inoculated at the cotyledon stage (4 biological replicas per viroid strain) to obtain viroid-infected leaf material. Briefly, two leaflets were dusted with carborundum and inoculated with dried infected material, homogenized in 50 mM KH2 PO4 , pH 7.0 buffer, of the following pospiviroid species: TCDVd (from infected petunia), potato spindle tuber viroid (PSTVd), tomato apical stunt viroid (TASVd), and tomato planta macho viroid (TPMVd) at the Beltsville laboratory. For comparison, replicates of mock-inoculated plants were prepared at the same time. Following inoculation, plants were maintained in a contained greenhouse under high temperature and long day conditions (30 ◦ C, 16 h light and 8 h dark) to favor viroid replication, symptom development and tomato fruit production.

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Seeds were extracted from tomato fruit by incubation of ripe fruit pulp for three days at room temperature to allow fermentation, followed by extensive washing of the seeds with water, and air drying for 3 days. In addition, petunia plants infected with TCDVd were maintained at the two locations and other host plants infected with TCDVd, PSTVd, TASVd, TPMVd, citrus exocortis viroid (CEVd), chrysanthemum stunt viroid (CSVd), Mexican papita viroid (MPVd), Columnea latent viroid (CLVd), and pepper chat fruit viroid (PCFVd) maintained at Agdia, Inc. were used for these studies. Purified RNAs was prepared from 100 mg samples of leaves of viroid-infected and mock inoculated (healthy) tomato (four weeks post-inoculation) or petunia plants that had been inoculated several months earlier. Alternatively, ten seeds harvested from the fruits of viroid-infected tomatoes were used for RNA extraction. Total RNA was extracted using TriReagent (Molecular Research Center.; Cincinnati, OH) according to the manufacturer’s instructions. Tissue samples were dusted with carborundum before extraction, and RNA concentrations and quality were determined by optical density at 260 nm using a Nanodrop 8000 Spectrophotometer (Nanodrop; Wilmington, DE). Isolated RNA samples were dissolved in nuclease-free water and stored at −20 ◦ C. To specifically amplify the target region in TCDVd using primers TCDVd FP3 and TCDVd RP4, designed for use in the Amplify RP assay 1 ␮L of purified RNA from TCDVd-infected petunia was combined in a final volume of 50 ␮L with 1X reaction buffer, 1.5 mM MgCl2 , 0.2 mM of each of four dNTPs, 1 ␮L of each primer at 20 pmol/␮L, 0.5 ␮L enzyme mix, 5 mM DTT, and 20 units of RNase Out Recombinant Ribonuclease Inhibitor (Invitrogen; Carlsbad, CA) using the Titan One tube RT-PCR system (Roche Molecular Biochemicals; Chicago, IL). The cycling parameters were: 50 ◦ C for 30 min; 95 ◦ C for 5 min; followed by 35 cycles of 95 ◦ C, 45 s, 50 ◦ C, 1 min, 68 ◦ C, 2 min, and finally 68 ◦ C for 7 min. The RT-PCR products were analyzed by electrophoresis through a 1.5% TBE agarose gel, stained with SYBR safe DNA gel stain (Invitrogen) and visualized on a UV-transilluminator. The TriDyeTM 100 bp DNA Ladder (New England Biolabs, Ipswich, MA) was used to estimate PCR product sizes. The primer sets were tested against RNAs extracted from PSTVd-, TASVd-, TPMVd-, and TCDV- infected leaf tissues, and tomato seeds harvested from tomato plants infected with PSTVd, TASVd, TPMVd, and TCDVd. The experiments were repeated in triplicate. Once amplified, TCDVd DNA fragments (228 bp) were cloned into the vector pCRTM 4-TOPO ® (Invitrogen). Three plasmid clones were sequenced and BLAST analysis revealed 100% sequence identity with TCDVd GenBank Accessions EF582392 (UK/tomato), DQ859013 (USA/petunia), and AB329668 (Japan/tomato). The sensitivity of the RT-RPA assay was evaluated using purified, in vitro-generated transcripts and crude extracts. TCDVd transcripts were generated by SP6 transcription of linearized plasmid DNA template using the mMessage mMachine SP6 kit (Ambion, Inc., Austin, TX) using manufacturer’s instructions. RT-PCR reactions were performed using with primers TCDVd FP3 and TCDVd RP4 using the Takara PrimeScriptTM One Step RT-PCR Kit Ver. 2 (Takara Bio Inc., Shiga, Japan) in a reaction containing 1X buffer, 0.5 ␮L enzyme mix, 0.4 ␮M final concentration of each primer, and 1 ␮L of RNA (transcripts, crude extracts or total RNAs purified from viroidinfected plants) in a final volume of 12.5 ␮L. The cycling conditions were 50◦ 30 min, 94◦ for 2 min, followed by 25 cycles of 94◦ 30 s, 68◦ 60 s. The reagent mixture for the AmplifyRP® Acceler8TM (Agdia, Elkhart, IN) was lyophilized into a powder in a 200 ␮L PCR tube and was designated the AmplifyRP® Acceler8TM Reaction Pellet (abbreviated ‘pellet’) (ACP65500; Agdia). The pellet contained all of the reaction components, including TCDVd-specific primers (TCDVd FP3 and TCDVd RP4) and an internal probe, TCDVdP1. To prepare total sample nucleic acids, 300 mg of fresh, 30 mg of dried leaf tissue, or seeds (10 seeds = 27 mg; as described in the

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Fig. 1. Multiple alignment of all reported TCDVd nucleotide sequences (GenBank Accession numbers AB329668, DQ859013, EF582392, AY372399, KJ817885, NC 000885, FJ822877, JQ975098, EU729744, HG739070, GQ169709, KF683201, GQ131572, KF683201, GQ131572) and representative pospiviroid sequences (NC 002030, PSTVd), NC 001558 (TPMVd), NC 003637 (MPVd), NC 003538 (CLVd), NC 003613 (IrVd 1), NC 001553 (TASVd), NC 001464 (CEVd), NC 002015 (CSVd), and NC 011590 (PCFVd) from GenBank using Clustal 2.1. Numbers on the right indicate the nucleotide position number. The location of the FP3 and RP4 primers and the probe described in the text are indicated and designated by green in TCDVd isolates and blue in other pospiviroids. Dashes represent gaps; stars below are nucleotides that are conserved in all isolates aligned in this analysis;//represents introduce gap in sequence. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig. 2. Comparison of the sensitivity of TCDVd-detection by AmplifyRP® Acceler8TM assay (panel A) and RT-PCR (panel B) on purified transcripts. Serial dilutions of purified transcripts were made in water. 1. 100 pg, 2. 10 pg, 3. 1.0 pg, 4. 100 fg, 5. 10, 6. 1.0 fg, 7. 0.1 fg, 8. water control. A 5 ␮L aliquot of the RT-PCR reaction was electrophoresed on a 1.5% agarose gel. The test and control lines are indicated by arrows on the left in panel A. M = TriDyeTM 100 bp DNA marker (New England Biolabs, Ipswich, MA).

text) were ground in sample mesh bag containing 3 ml of GEB3 buffer (ACC00360; Agdia). Ten ␮L of the PD1 Pellet Diluent (PD1; ACC00480; Agdia) was added to a 0.2 mL microcentrifuge tube containing the reaction pellet; one ␮L of the sample extract (or 1 ␮L of

purified RNA) was immediately added to the rehydrated reaction pellet and mixed with a vortex mixer. The tubes were incubated at 39 ◦ C for 15 min. The tubes were briefly centrifuged. A foil pouch containing the Amplicon Detection Chamber (ADC98800; Agdia)

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Fig. 3. Comparison of the specificity of TCDVd-detection on crude plant extracts using the AmplifyRP® Acceler8TM (panel A) and RT-PCR (panel B) assays. Detection of TCDVd in 5-fold serial dilutions (made in a healthy plant extract) of a Petunia plant crude extract by AmplifyRP® Acceler8® (panel A) for TCDVd and by RT-PCR (panel B). A 5 ␮L aliquot of the RT-PCR reaction was electrophoresed on a 1.5% agarose gel. 1. 1x (1:10 ratio extract), 2. 5x, 3. 25x, 4. 125x, 5. 625x, 6. 3,125x, 7. 15,625x, 8. 1x, healthy plant extract. The test and control lines are indicated by arrows on the left in panel A. M = TriDyeTM 100 bp DNA marker (New England Biolabs). (Panel C) Detection of TCDVd with AmplifyRP® Acceler8® for TCDVd in the pure total RNA of plants infected by one of the pospiviroids. 1. CEVd (A33791), 2. CLVd (A60248), 3. CSVd (A43286), 4. MPVd (A40540), 5. PCFVd (A47257), 6. PSTVd (A47615), 7. TASVd (A42822), 8. TCDVd (A57963), 9. TPMVd (A33795), and 10. No RNA control. The test and control lines are indicated by arrows on the left.

Fig. 4. Evaluation of the ability of the AmplifyRP® Acceler8TM assay to specifically detect TCDVd in tomato seed extracts. Panel A. Crude sap from undiluted extracts of 10 seeds obtained from the fruits of either healthy or viroid-infected plants. 1. Healthy, 2. TCDVd, 3. PSTVd, 4. TASVd, and 5. TPMVd. Panel B. Crude sap undiluted extract of 10 infected seeds obtained from TCDVd-infected tomato fruits (N), followed by 1:10, 1:100, 1:1000 dilutions made with a healthy seed extract. The test and control lines are indicated by arrows on the left.

was opened. The unopened reaction tube was added to the reaction apparatus and the apparatus was snapped shut. The reaction apparatus was added to the detection chamber and the results of the lateral flow assay were interpreted within 20 min incubation at room temperature. Buffer, negative, and positive controls were included for each set of reactions. Results were recorded visually and by photography (see Fig. 2A). This assay was repeated in triplicate for each sample. The relative detection sensitivities for TCDVd in the AmplifyRP® Acceler8TM RT-RPA and RT-PCR assays were compared for pure RNA transcripts and crude extracts generated as described above. For pure transcripts, the sensitivity for detection of TCDVd was 1 pg

in both assays (Fig. 2A and B). In crude extracts of TCDVd-infected petunia leaf tissues, the RT-RPA assay detected TCDVd in a 25 fold dilution of the original crude extract (Fig. 3A) and was equivalent to detection of the same dilution standard RT-PCR assay (Fig. 3B). When the crude sap of 10 seeds obtained from TCDVd-infected tomato fruit was processed using the AmplifyRP® Acceler8TM RTRPA assay, TCDVd could be detected in the 1:10 dilution (Fig. 4A). To determine the specificity for detection of TCDVd using the AmplifyRP® Acceler8TM RT-RPA assays, the assay was evaluated against a selection of tomato-infecting pospiviroids. In triplicate assays of purified RNAs from tomato leaf tissue samples, the specificity of this assay for TCDVd in tomato leaf tissues was confirmed

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(Fig. 3C). When undiluted extracts of seeds (ten) obtained from the fruits of tomatoes were tested after grinding in 3.0 mL GEB, the results revealed the detection of TCDVd and no cross-reaction with seed extracts from tomato plants infected with TASVd, TPMVd, or PSTVd (Fig. 4B). In addition, previously frozen extracts and extracts of dried tissue (30 mg/3 mL GEB3) yielded the same results (data not shown). There were no reactions to healthy tissue. The results show that the TCDVd AmplifyRP® Acceler8TM assay is specific for TCDVd. To our knowledge, this is the first report of the use of the AmplifyRP® Acceler8TM RT-RPA assay for the detection of viroid RNAs, and specifically, for the detection of TCDVd in infected leaf and seed tissues. The AmplifyRP® Acceler8TM RT-RPA assay, an isothermal RNA/DNA detection platform uses recombinase polymerase amplification technology (Piepenburg et al., 2006) and is an attractive method for leaf and seed testing for TCDVd in ornamentals and crop plants as it can specifically detect TCDVd in crude extracts of leaf and seed tissues. The contained assay limits introduction of contamination and reduces false positives. Similar to the isothermal Amplify RP assays developed for detection of plum pox virus (Zhang et al., 2014), Little cherry virus 2 (Mekuria et al., 2014) and Fusarium oxysporum (Doan et al., 2014) the assay has the advantage of other technologies in that a single constant temperature is used for amplification, the assay is sensitive so samples can be pooled. As viroids do not encode proteins, a simple immunostrip assay is not an option for detection. As the AmplifyRP® Acceler8TM assay includes an antibody to a fluorophore (FAM) attached to a viroidspecific probe, an antibody-based assay that is specific, sensitive and easy to perform was developed. In our studies, the assay based on TCDVd-specific amplification primers and probe, revealed specificity of detection in tomato and petunia leaf and tomato seed tissues. In addition, the one-step plant extraction buffer eliminates the need for RNA purification and the color indicator lines of the test and control on the lateral flow strip test were obvious and easy to distinguish (Figs. 2–4). The sensitivity of detection of TCDVd in crude extracts and purified RNAs of leaf tissues was equivalent using the RT-RPA assay (Fig. 2 and 3), indicating that RT-RPA has no inhibition from the crude extracts which is often the case for PCR. However, when compared to conventional RT-PCR results (Fig. 3D), the detection level of the RT-RPA assay was slightly less sensitive when the pure RNA was used as a template. This is similar to the sensitivity equivalence of the RT-RPA assay to real-time PCR noted for single-stranded RNA viral molecules (Zhang et al., 2014). When the assay was tested on TCDVd in vitro, single-stranded transcripts, the sensitivity was 100 fg-1 pg of pure RNA. To achieve international harmonization for the detection of pospiviroids in tomato and pepper seeds, the Netherlands Inspection Service for Horticulture has developed a reference protocol for a real-time RT-PCR assay to detect eight pospiviroids) (http://www.naktuinbouw.nl/sites/naktuinbouw.eu/file/ 20140801Tomatopopsiviroids.pdf) and the protocol was verified for detection of PSTVd and TCDVd in tomato seeds, with a sensitivity of detection of a single infected seed in a subsample of 1000 seeds; however there was a weak cross-reaction with TPMVd and a strong cross-reaction with MPVd, closely related to TPMVd (Verhoeven et al., 2011; Bakker et al., 2015). In our test, we did not see cross reaction to TPMVd. In addition, samples can be pooled prior to the assay (eg. 1 infected sample/100 for leaf tissue). For reliable and sensitive in tomato seeds, 1 infected seed/100 seeds could be detected (data not shown). Development of more inclusive pospiviroid test and increasing the sensitivity of detection of TCDVd in seeds using the TCDVd-specific test are the focus of continuing studies in our laboratories.

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