Plant Pathology (2013) 62, 1147–1156
Doi: 10.1111/ppa.12017
Fast real-time detection of Potato spindle tuber viroid by RT-LAMP R. Lenarcic†, D. Morisset†*, N. Mehle and M. Ravnikar na pot 111, 1000, Ljubljana, Slovenia National Institute of Biology, Vec
This paper reports the development of a single tube, real-time, reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay for detecting Potato spindle tuber viroid (PSTVd), one of the quarantine pathogens of potato in Europe and North America. The method enables detection of a broad range of PSTVd isolates, and is about 10 times more sensitive than the conventional reverse transcription polymerase chain reaction (RT-PCR) assay. Its benefits are not only its speed (15–25 min to obtain results) and cost effectiveness (resulting from time saved as well as cheaper consumables), but also its demonstrated ability to be performed in the field, using portable instruments. Keywords: Isothermal amplification, melting curve, potato, PSTVd, RT-LAMP, viroid
Introduction Potato (Solanum tuberosum) is grown in more than 100 countries under temperate, subtropical and tropical conditions. Its adaptability to different growth conditions, as well as its nutritive qualities, have resulted in very wide adoption, ranking it as the world’s fourth most important food crop after maize, wheat and rice. However, it is vulnerable to a number of pests and diseases. One of these, Potato spindle tuber viroid (PSTVd), belonging to the Pospiviroidae family, is a small, 356- to 361-nucleotide circular RNA with a very stable secondary structure (Gross et al., 1978). It was discovered in 1971 as the causative agent of potato spindle tuber disease (Diener, 1971). Disease symptoms differ significantly depending on PSTVd strain and host cultivar (Schnolzer et al., 1985). The most obvious visible symptom is spindly foliage with a clockwise phyllotaxy. Foliage is often darker green than normal and slightly rugous, and the infected plants are stunted. Tubers are smaller, often elongated and misshapen. Potato spindle tuber viroid can cause severe economic losses in potato (Pfannenstiel & Slack, 1980), by reducing the size and number of tubers. It is listed as a quarantine pest in North America and by the European and Mediterranean Plant Protection Organization (EPPO, http:// www.eppo.int/). Apart from potato, its main economically important host is tomato (Solanum lycopersicum), but it has also been reported in wild Solanum spp., avocado (Querci et al., 1995), pepino (Puchta et al., 1990) and sweet potato. Moreover, in the past few years, there
*E-mail:
[email protected] † These authors contributed equally to this work.
Published online 22 November 2012 ª 2012 British Society for Plant Pathology
has been an increased number of reports of PSTVd infections in ornamental species, which are mostly latent and symptomless but play an important role in spreading the infection to potato and tomato. It remains unclear how viroids cause diseases in plants, although it is most commonly accepted that it happens via an RNA-silencing mechanism. Viroid-specific siRNAs that can act as endogenous miRNAs may base-pair with host mRNAs, blocking normal gene expression and inducing disease (Wang et al., 2004; Daros et al., 2006; Owens, 2007; Navarro et al., 2009). Apart from vegetative propagation, the most common mechanism of PSTVd transmission is mechanical, by direct contact of non-infected with infected plants or with agricultural machinery, although other mechanisms of transmission have been suggested. Wild plants that are economically unimportant and show no symptoms of PSTVd infection could be an important reservoir for the spread of PSTVd (Martı´nez-Soriano et al., 1996). Unlike viruses, PSTVd RNA does not code for proteins, which limits the detection methods that can be used to identify the pathogen. Diagnostic discrimination between viroids is challenging because of the relatively short PSTVd nucleotide sequence (356–361 nucleotides) and the high level of homology between different pospiviroids. Diagnostic methods for PSTVd detection were initially based on polyacrylamide gel electrophoresis PAGE (Morris & Wright, 1975). Methods based on hybridization that enable large-scale screening (Owens & Diener, 1981), and various PCR-based methods combined with reverse transcription (RT) later became available (Shamloul et al., 1997; Weidemann & Buchta, 1998; Verhoeven et al., 2004). Nowadays, a combined RT and real-time PCR single-tube test, that provides high sensitivity and specificity (Boonham et al., 2004), is widely used for routine detection of PSTVd. Despite its benefits and increased popularity, real-time RT-PCR technology has the disadvantage of requiring 1147
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expensive equipment for thermal cycling and fluorescence determination, and cannot be used in the field because of the lack of convenient portable instruments. Moreover, the real-time RT-PCR technique is often sensitive to inhibitors present in plant extracts (Boonham et al., 2004). A recently developed loop-mediated isothermal amplification (LAMP) method (Notomi et al., 2000) is less sensitive to inhibitors (Francois et al., 2011) and, thanks to its isothermal nature, has the potential to be deployed in the field. Because of its speed, robustness and simplicity of use LAMP is gaining popularity in diagnostics in human medicine (Parida et al., 2008) and, more recently, also in plant health (Kubota et al., 2008; Tomlinson et al., 2010; Buhlmann et al., 2012). An RTLAMP assay for PSTVd detection has been reported, although it takes 1 h for completion and does not allow for rapid and simple determination of the final results, which makes it unsuitable for in-field testing. This paper reports the development of a rapid method for detecting PSTVd, based on RT-LAMP. Amplification is followed in real-time and the specificity of the final product is confirmed by its melting temperature. Specificity and sensitivity have been determined and compared to those of real-time and conventional RT-PCR.
Materials and methods Tissue and viroid collection Potato plants Potato plants 15 cm high were infected mechanically with PSTVd (isolate NIB V 190) as follows. Approximately 20 mg lyophilized plant material infected with PSTVd was ground with a mortar and pestle in 5 mL freshly prepared inoculation buffer (19 mL 02 M NaH2PO4, 81 mL 02 M Na2HPO4, 90 mL dH2O and 2 g polyvinylpyrrolidone MW 10 000). Three lower, fully expanded leaves were dusted with carborundum and inoculated mechanically with the PSTVd-containing buffer suspension. The inoculated plants were grown in a quarantine greenhouse at 75% humidity, under a regime of 14 h light (3000 lux) at 25°C and 10 h darkness at 20°C. From each of the 16 inoculated plants, three upper leaves (not in contact with leaves used for inoculation) were collected 5 weeks after inoculation. Small pieces of these leaves (c. 1 cm2) were homogenized and used as infected plant material for total plant RNA extraction. Potato plants were left in a quarantine greenhouse for another 2 months under the same growth conditions described above, then tubers were collected. Total plant RNA was then extracted from the tuber upper eye tissue.
Tomato plants Using the same inoculation procedure as for potato plants, 2week-old tomato plants, about 10 cm high, were inoculated with PSTVd (isolate NIB V 190) and incubated for 5 weeks at 75% humidity under a regime of 14 h light (3000 lux) at 25°C and 10 h darkness at 20°C. From each of the 24 inoculated plants, three upper leaves (not in contact with the leaves used for inoculation) were collected and cut into small pieces (c. 1 cm2), homogenized and used as infected plant material for total plant RNA extraction.
Viroid collection Viroid isolates were obtained from a collection at the Food and Environment Research Agency (Fera, UK), and from the National Plant Protection Organization of the Netherlands (Table 1). Most of the viroids were isolated from lyophilized plant material. PSTVd isolates were obtained from tomato (leaves), Petunia (leaves), Solanum jasminoides (leaves) and Solanum rantonettii (leaves). Isolates of Tomato apical stunt viroid (TASVd), Citrus exocortis viroid (CEVd) and Columnea latent viroid (CLVd) were obtained from tomato (leaves); Tomato chlorotic dwarf viroid (TCDVd) isolates were obtained from tomato (leaves) or Petunia; Chrysanthemum stunt viroid (CSVd) isolates were obtained from senetti or Chrysanthemum leaves; no data were available on the origin of the infected plant material in the case of isolates of Peach latent mosaic viroid (PLMVd), Eggplant latent viroid (ELVd), Avocado sunblotch viroid (ASBVd) and Hop latent viroid (HLVd). Additionally, two PSTVd isolates were obtained from fresh plant tissue of potato (tubers and leaves), and all Tomato planta macho viroid (TPMVd) isolates were obtained from fresh tomato leaves.
RNA extraction Total RNA was extracted from fresh leaves or tuber tissue (200 mg), or from lyophilized plant material (20 mg), using the RNeasy Plant Mini Kit (QIAGEN), following the manufacturer’s recommendations with minor modifications: mercaptoethanol was omitted from the procedure, and RNAse-free water at 65°C was added to the QIAGEN column where it was incubated for 5 min before final elution (2 9 50 lL). In order to control the RNA extraction procedure, each sample was tested by real-time RT-PCR using the cytochrome oxidase (COX) gene-specific assay (Weller et al., 2000). For each of the extraction series, an extraction blank control containing only the buffer was tested in parallel with samples at all steps of extraction. The analytical sensitivity of the RT-LAMP assays was determined using a dilution series of PSTVd-infected material in noninfected homogenized tomato material. Samples of 04 g pooled plant leaves infected with PSTVd NIB V 190, and 1 g pooled non-infected plant leaves were homogenized in 18 mL and 45 mL RLT buffer (QIAGEN), respectively. Homogenized material was then centrifuged for 1 min at 4500 g, and a 10 9 dilution series of infected material prepared using the noninfected homogenized tomato material as diluent. Total plant RNA was then extracted from 540 lL of each prepared dilution, and used in tests to assess the analytical sensitivity of the RT-LAMP assays under the reaction conditions described below.
Design of RT-LAMP primers Sequences from the 173 PSTVd isolates available in the NCBI database at the time of this study were aligned using the MUSCLE alignment algorithm and MEGA 5 software (Tamura et al., 2011) (Fig. S1). From this alignment, a consensus sequence was determined and compared to retrieved sequences of viroids belonging to other species of the genus Pospiviroid: TCDVd, Mexican papita viroid (MPVd), TPMVd, CSVd, CEVd, TASVd and CLVd. The most highly conserved and PSTVd-specific sequence within the consensus was then used for primer design. Three sets of primers for the PSTVd-specific LAMP reaction, each comprising six primers (external primers: F3 NIB PSTVd and B3 NIB PSTVd, internal primers: FIP NIB PSTVd and BIP NIB PSTVd,
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Table 1 Results of analyses performed on viroid isolates with RT-LAMP, Tsutsumi RT-LAMP and real-time RT-PCR assays
Viroida PSTVd PSTVd PSTVd PSTVd PSTVd PSTVd PSTVd PSTVd PSTVd PSTVd PSTVd PSTVd PSTVd PSTVd PSTVd PSTVd TPMVd TPMVd TPMVd (MPVd)* TCDVd TCDVd TCDVd TCDVd TCDVd TCDVd TCDVd TCDVd TCDVd TCDVd TCDVd TCDVd TASVd TASVd TASVd TASVd TASVd TASVd TASVd CSVd CSVd CSVd CSVd CEVd CEVd CEVd CLVd CLVd CLVd CLVd CLVd CLVd CLVd CLVd CLVd CLVd CLVd CLVd
Isolate (original name; accession no.)b NIB NIB NIB NIB NIB NIB NIB NIB NIB NIB NIB NIB NIB NIB NIB NIB NIB NIB NIB
V V V V V V V V V V V V V V V V V V V
190 (S1; EF92393)1 95 (CSL, York, UK)2 213 (N)1 214 (Howell)1 217 (20623859)2 218 (UK isolate)2 219 (20707189)2 220 (20700546)2 221 (UK isolate)2 222 (20709024)2 223 (6/3/2007)2 225 (20709917)2 227 (20709644)2 228 (20709918)2 229 (20709915)2 230 (29/6/07)2 215 (3601768)1 216 (3289954; K00817)1 267 (OG1; L78454)1
NIB V 191 (22006456)1 NIB V 236 (20707216)2 NIB V 237 (20708587)2 NIB V 238 (20707218)2 NIB V 239 (20707876)2 NIB V 240 (20707416)2 NIB V 241 (20707874)2 NIB V 242 (20709821)2 NIB V 243 (20707416)2 NIB V 244 (20709822)2 NIB V 245 (22006456)2 NIB V 246 (20707864)2 NIB V 195 (CSL 20519794, Senegal)2 NIB V 211 (2112010gg0 20/10/09)2 NIB V 231 (2010990; DQ144506)2 NIB V 232 (3153272)2 NIB V 233 (2010gg0 20/10/09)2 NIB V 234 (3264933)2 NIB V 235 (2010gg0 10/12/09)2 NIB V 196 (CLS, Hollandsk isolat)2 NIB V 247 (20903856)2 NIB V 248 (20821085)2 NIB V 249 (20821084)2 NIB V 192 (89002600)1 NIB V 250 (20811884)2 NIB V 251 (20522610)2 NIB V 193 (9389007481)1 NIB V 210 (20801688)2 NIB V 252 (20721428)2 NIB V 253 (20800356)2 NIB V 254 (CE Room)2 NIB V 255 (20712353)2 NIB V 256 (20712666)2 NIB–V 257 (20712664)2 NIB V 258 (20712665)2 NIB V 259 (20710779)2 NIB V 260 (20712353)2 NIB V 261 (20712675)2
Plant material
Tissue
RT-LAMPc
Tsutsumi RT-LAMPd
Real-time RT-PCRe
Tomato/potato Tomato Potato Potato nd nd Petunia nd Tomato Solanum jasminoides nd S. jasminoides white S. jasminoides white Solanum rantonetti S. rantonetti S. jasminoides Tomato Tomato Tomato
Tuber and leaves Leaves Tuber Tuber Leaves Leaves Leaves Leaves Leaves Leaves Leaves Leaves Leaves Leaves Leaves Leaves Leaves Leaves Leaves
+ + + + + + + + + + + + + + + + + + +
+ + – + + + – + + + + + + + + + – – –
+ + + + + + + + + + + + + + + + + + +
(2216) (2723) (1909) (2094) (1629) (1631) (1877) (1737) (1651) (197) (1446) (1725) (1555) (2488) (1909) (2016) (2041) (2391) (2092)
nd Petunia Tomato Petunia Petunia Petunia Petunia Petunia Petunia Petunia nd Petunia nd nd Tomato Tomato nd Tomato nd nd Senetti Chrysanthemum Chrysanthemum nd Tomato nd nd nd Tomato Tomato Tomato Tomato Tomato Tomato Tomato Tomato Tomato Tomato
Leaves Leaves Leaves Leaves Leaves Leaves Leaves Leaves Leaves Leaves Leaves Leaves Leaves Leaves Leaves Leaves Leaves Leaves Leaves Leaves Leaves Leaves Leaves Leaves Leaves Leaves Leaves Leaves Leaves Leaves Leaves Leaves Leaves Leaves Leaves Leaves Leaves Leaves
– + + – – – – + – – – – – – – – – – – – + – – – – – – – – + – – – – – – – –
+ + + + + + + + + + + + + – – + – – + – – – – + – + + – – – – – – – – – – –
(2518) (1900) (1556) (2179) (2553) (1860) (2176) (1601) (2206) (1936) (1755) (2251) (3898)
(1436) (2374) (1740) (1328) (1750) (1755) (2102) (1302) (1907) (1353) (1907) (1248) (1208) (1509) (1355) (1327) (1409) (1804) (1610)
(2446) (2390)
(2343)
(2253)**
(2496)**
(2167) (328) (1812) (3110) (2299) (2115) (2300) (2378) (2309) (2249) (2214) (2937) (2693) (2867)
– – + (3256) – – + (3307) – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – –
(3994)
(3738)
(3636) (3843) (3807)
(continued)
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Table 1 (continued)
Viroida
Isolate (original name; accession no.)b
CLVd HLVd PLMVd PLMVd PLMVd ELVd ASBVd
NIB NIB NIB NIB NIB NIB NIB
V V V V V V V
262 212 209 263 264 207 208
(07481)2 (20802479)2 (1110-01A4 VABN29)2 (17/7/08)2 (GF05 1922-0183)2 (20811683)2 (20811686)2
Plant material
Tissue
RT-LAMPc
Tsutsumi RT-LAMPd
Real-time RT-PCRe
nd nd nd nd nd nd nd
Leaves Leaves Leaves Leaves Leaves Leaves Leaves
– – – – – – –
– – – – – – –
– – – – – – –
a
PSTVd, Potato spindle tuber viroid; TPMVd, Tomato planta macho viroid; TCDVd, Tomato chlorotic dwarf viroid; TASVd, Tomato apical stunt viroid; CSVd, Chrysanthemum stunt viroid; CEVd, Citrus exocortis viroid; CLVd, Columnea latent viroid; HLVd, Hop latent viroid; PLMVd, Peach latent mosaic viroid; ELVd, Eggplant latent viroid; ASBVd, Avocado sunblotch viroid. *Isolate NIB V 267 was originally described as Mexican papita viroid (MPVd) but has been proposed to be taxonomically grouped as TPMVd (Verhoeven et al., 2011). b Isolate provided by: 1NPPO, National Plant Protection Organization, Netherlands; 2Fera, Food and Environment Research Agency, UK. c RT-LAMP: reactions were performed for a duration of 25 min. Numbers in brackets indicate time when signal occurred. **Melting temperature significantly different from the acceptance criteria for positive PSTVd signal. d Tsutsumi-RT-LAMP (modified from Tsutsumi et al., 2010): reactions were performed for a duration of 35 min. Numbers in brackets indicate time when signal occurred. e Real-time RT-PCR (Boonham et al., 2004): numbers in brackets correspond to quantification cycle (Cq) values. nd, no data available; –, negative result (absence of signal); +, positive result (presence of signal).
and loop primers: LF NIB PSTVd and LB NIB PSTVd), were designed based on the strategy described by Notomi et al. (2000) and using LAMP DESIGNER software (Premier Biosoft). Primers were synthesized at Eurofins MWG Operon. The specificity of designed primers was further confirmed by using the BLAST algorithm (standard nucleotide BLAST available at http://blast.ncbi. nlm.nih.gov/Blast.cgi) and compared first against all available sequences, and then against all available sequences excluding PSTVd.
RT-LAMP reactions RT-LAMP reactions were performed in single tubes in a 25-lL total reaction volume containing a sample of 2 lL RNA, 125 lL Isothermal Master Mix (Optigene Ltd.), 025 U AMV reverse transcriptase and 1 lL provided buffer (Finnzymes). LAMP primers for PSTVd specific amplification were added to the reaction mixture at the following final concentrations: external F3 NIB PSTVd (5′-AAAAAGGACGGTGGGGAG-3′) and B3 NIB PSTVd (5′-CCCCGAAGCAAGTAAGATAG-3′) primers at 02 lM, internal FIP NIB PSTVd (5′-GGAAGGACACCCGAA GAAAGGGCCGACAGGAGTAATTCC-3′) and BIP NIB PSTVd (5′-GCTGTCGCTTCGGCTACTACAGAAAAAGCGGTTCTCG G-3′) primers at 1 lM and loop primers LF NIB PSTVd (5′GGTGAAAACCCTGTTTCGG-3′) and LB NIB PSTVd (5′CGGTGGAAACAACTGAAGC-3′) at 1 lM, respectively. Singletube RT-LAMP reactions were performed at 65°C for 25 min with the additional step of melting temperature determination carried out in a SmartCycler (Cepheid). Additionally, in order to assess the robustness of the RT-LAMP assay, reactions were carried out using two instruments: Genie II (Optigene Ltd.) and ABI PRISM 7900 HT Sequence Detection System (Applied Biosystems). LAMP products are concatemers of a target specific sequence (Notomi et al., 2000). The melting temperature (Tm) is the temperature at which double-stranded DNA product dissociates into
single strands. Tm is specific to a given LAMP amplicon under given reaction conditions and differs between amplicons according to their nucleotide composition. In addition to monitoring the increase of fluorescence in positive samples, melting curve analysis was used in all instruments to further verify the positive samples obtained with the RT-LAMP assays.
Tsutsumi RT-LAMP reactions The performance of the PSTVd-RT-LAMP assay was compared with that of another PSTVd-RT-LAMP assay (Tsutsumi et al., 2010; hereafter referred to as the Tsutsumi RT-LAMP). This assay was tested here using the originally described conditions with some modifications – reactions were performed in a single tube in a total volume of 25 lL containing 2 lL RNA sample, 125 lL Isothermal Master Mix (Optigene Ltd.), 025 U AMV reverse transcriptase and 1 lL provided buffer (Finnzymes). Primers were mixed and added to the reaction mixture to a final concentration as originally described. Single-tube RT-LAMP reactions were performed at 65°C for 35 min. For detection of LAMP product, amplification was followed in real-time using a SmartCycler (Cepheid) instead of measuring turbidity as originally described by Tsutsumi et al. (2010), or in the Genie II (Optigene Ltd.).
Real-time RT-PCR The performance of the RT-LAMP assay (defined by its sensitivity and specificity) was compared with that of a single-step realtime RT-PCR assay specific to PSTVd, performed as described by Boonham et al. (2004) using the AgPath-IDTM One-step RTPCR kit (Ambion). The test was carried out in triplicate on an ABI PRISM 7900 HT Sequence Detection System (Applied Biosystems). The results were analysed using SDS v. 2.2 software (Applied Biosystems).
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Potato spindle tuber viroid detection by RT-LAMP
RT-PCR The one-step RT-PCR test was performed with two primer sets as described (Verhoeven et al., 2004) using the QIAGEN OneStep RT-PCR Kit. Agarose gel electrophoresis and ethidium bromide were used to visualize the RT-PCR products. This assay was used to compare the RT-LAMP assays’ sensitivity parameters.
Validation of the RT-LAMP reaction RT-LAMP reactions for both assays were performed using 2 lL total plant extract RNA, as described above. The specificity of the RT-LAMP assays was evaluated once in each of the following instruments: SmartCycler (Cepheid) and ABI PRISM 7900 HT Sequence Detection System (Applied Biosystems) for all viroids, and additionally in the Genie II (Optigene Ltd.) for PSTVd isolates only. Analytical sensitivity was evaluated three times independently in a SmartCycler (Cepheid).
Results The performance of the RT-LAMP assay was evaluated using total plant RNA extracts from different plant tissues (leaves and tubers) originating from various host
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plants (potato, tomato, Petunia, S. jasminoides, S. rantonettii, senetti and Chrysanthemum). A cytochrome oxidase (COX) gene-specific real-time RT-PCR assay was used as the endogenous control (Weller et al., 2000).
In silico analysis The sequences of 173 PSTVd isolates were aligned and the consensus sequence used for LAMP primer design. A total of three sets of LAMP primers satisfying LAMP acceptance criteria were designed. However, after analysing the specificity of the designed primer sets in silico, only one primer set was found to be satisfactory, as based on the acceptance criteria (rating) of the software used for the design. Annealing positions of this chosen primer set on the PSTVd RNA are shown in Figure 1. All six primers annealed to the regions specific for PSTVd. The in silico specificity study showed only a few possible mismatches on some PSTVd isolates, occurring only in the internal parts of the primers (Fig. S1). No information exists regarding the effect of internal mismatch on LAMP performance. However, it was assumed that such a mismatch would have a very limited impact
Figure 1 Alignment of consensus sequences of Potato spindle tuber viroid (PSTVd), Mexican papita viroid (MPVd), Tomato planta macho viroid (TPMVd) and Tomato chlorotic dwarf viroid (TCDVd), and of RT-LAMP primer sequences. The internal FIB NIB PSTVd primer consists of the reverse sequence of F1 NIB PSTVd followed by that of F2 NIB PSTVd. The internal BIP NIB PSTVd primer consists of the B1 NIB PSTVd sequence followed by that of the reverse B2 NIB PSTVd sequence. Mismatches to the primer sequences are indicated by shading.
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on LAMP performance, as is the case in real-time PCR assays (Kwok et al., 1990; Bru et al., 2008; Ghedira et al., 2009; Suech et al., 2009). Potential cross-reactivity with non-target viroids in the RT-LAMP assay was investigated in silico. According to this, TCDVd, TPMVd and MPVd, which are all highly homologous to PSTVd, each had the potential to give a signal when tested with the RT-LAMP assay (Fig. 1; Fig. S2).
Diagnostic and analytical specificity The analytical specificity of the RT-LAMP assay was tested experimentally against a collection of 16 PSTVd isolates and 42 isolates belonging to six other species of the genus Pospiviroid: TPMVd (three isolates), TCDVd (12 isolates), TASVd (seven isolates), CSVd (four isolates), CEVd (three isolates) and CLVd (13 isolates). Also, cross-reactivity was tested with one isolate of HLVd of the genus Cocadviroid from the family Pospiviroidae and with five isolates from three genera of the family Avsunviroidae: PLMVd (three isolates), ELVd (one isolate) and ASBVd (one isolate) (Table 1).
Coverage Both real-time RT-PCR and RT-LAMP assays detected all 16 tested PSTVd isolates. Tsutsumi RT-LAMP detected 14 out of the 16 PSTVd isolates.
Specificity The RT-LAMP assay gave a positive signal with all three tested TPMVd isolates that were closely related to PSTVd (Martı´nez-Soriano et al., 1996). These experimental results confirmed the in silico analyses that showed that the NIB PSTVd primers annealed to both viroids’ RNAs. Also, four of the 12 TCDVd isolates were detected. Based on the overall homology between both viroids, partial cross-reactivity was expected, although in silico analysis showed some mismatches. Positive results were also obtained with one of the four isolates of CSVd and one of 13 isolates of CLVd. In both cases the signals were late (after 22 min) and the in silico analysis showed that only the FL NIB PSTVd and BL NIB PSTVd primers were closely similar to these viroid sequences. The B2 sequence of the BIP NIB PSTVd primer also showed some similarities at its 5′ end. Therefore, the observed positive signals obtained with CSVd and CLVd might be the result of products of unspecific amplification driven mainly by the loop primers (Table 1). The specificity of the RT-LAMP assay was compared to that of the single-step real-time RT-PCR assay. This showed that both methods detected PSTVd and TPMVd, because all isolates gave positive signals. However, the RT-LAMP assay appeared to be more specific in the case of these two viroids. It only detected a few TCDVd isolates (similar Tm to that for PSTVd) with a late signal, while all TCDVd isolates were detected by real-time RT-
PCR with low quantification cycle (Cq) values (early signals) (Boonham et al., 2004). Also, the RT-LAMP assay did not show cross-reactivity with TASVd isolates, unlike the RT-PCR assay, which gave late signals (Cq > 35) with three out of the seven isolates. Both methods showed occasional cross-reactivity with other viroids (Table 1), although the signals were late (after 225 min in RT-LAMP, and >35 Cq in real-time RT-PCR). Moreover, in the case of the RT-LAMP assay, these signals could be distinguished from the specific PSTVd signal by melting temperature analysis (see below). In the case of the real-time RT-PCR assay, with its very high sensitivity as a given, one cannot exclude the possibility that the late signals observed with some CLVd, CEVd and TASVd isolates were caused by the low titres of PSTVd in the sample. The specificity of the RT-LAMP assay was also compared to that of Tsutsumi RT-LAMP, which was able to detect only 14 of the 16 PSTVd isolates, two of them only with late signals (after 31 min). When testing other viroids, the Tsutsumi RT-LAMP assay detected two of the 12 TCDVd isolates (Table 1), both with very late signals (after 325 min). In addition to specificity tests, potato leaf extracts of different cultivars were investigated as negative control samples in the RT-LAMP assay in order to exclude any possible cross-reactivity with non-infected plant material. Total plant RNA extracts from non-infected healthy potato cvs Ulster, Sante, Nadine, Nadine H, Igor, Desiree, Carlingford, King Edward and Pentlands were tested. None showed a positive signal. Furthermore, potato leaf extracts from the following potato cultivars infected with Potato virus Y (PVY) were also tested: King Edward, Pentland and Igor all infected with PVYNTN; Pentland infected with PVYW; and Pentland infected with PVYO. None of the PVY-infected extracts showed any cross-reactivity with RT-LAMP.
Melting curve analysis To confirm the specificity of the RT-LAMP amplification product, and to distinguish between true positive reactions and results from potential cross-reactivity, melting curve analysis was performed using the same devices as for real-time detection of the RT-LAMP reactions. Under RT-LAMP constant reaction conditions, the Tm of the PSTVd amplicon was found to range between 916°C and 923°C on the SmartCycler apparatus (Fig. 2), and between 906°C and 916°C on the Genie II apparatus (data not shown). This small variability in Tm values may be caused by the sequence variability observed within the PSTVd sequences. TPMVd shows a slightly lower Tm (908°C) than PSTVd (920°C) using the SmartCycler apparatus. It was therefore concluded that the three tested TPMVd isolates could be distinguished from PSTVd by their melting curves. TCDVd, which is occasionally detected with RTLAMP (but also with Tsutsumi RT-LAMP and real-time RT-PCR), could not be distinguished from PSTVd by Tm Plant Pathology (2013) 62, 1147–1156
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Figure 2 Melting temperatures of the RT-LAMP products of Potato spindle tuber viroid (PSTVd), Tomato chlorotic dwarf viroid (TCDVd), Tomato planta macho viroid (TPMVd), Chrysanthemum stunt viroid (CSVd) and Columnea latent viroid (CLVd) isolates, measured on the SmartCycler apparatus (Cepheid). The boxes of the box-plots represent upper and lower quartiles of the data around the median value, the whiskers represent the maximum and minimum values. For CSVd and CLVd, only one isolate of each viroid gave signal.
(Fig. 2), presumably because of the sequence similarity observed during the in silico specificity study (Fig. 1; Fig. S2). On the other hand, the melting temperatures observed for RT-LAMP products for one isolate of CLVd and one isolate of CSVd were below 90°C on the SmartCycler apparatus. Signals obtained from these viroids could therefore be readily distinguished from PSTVd-positive signals (Fig. 2).
Diagnostic and analytical sensitivity The sensitivity of the RT-LAMP assay was compared to that of an RT-PCR assay for general pospiviroid detection (Verhoeven et al., 2004) and to that of a one-step real-time RT-PCR assay (Boonham et al., 2004; Table 2). The analytical sensitivity of the RT-LAMP PSTVd assay was determined by 10-fold dilution of total plant RNA from PSTVd (isolate NIB V 190)-infected plants. The number of PSTVd RNA copies in the samples was estimated based on the known sensitivity of the one-step real-time RT-PCR assay (Boonham et al., 2004), which is considered to be between one and 10 copies of PSTVd RNA at the last level at which signal is observed. The analytical sensitivity of the RT-LAMP PSTVd assay was estimated to be between 100 and 1000 viroid copies per reaction, a 10-fold greater sensitivity than that of the RT-PCR (Verhoeven et al., 2004) and 100 times lower than real-time RT-PCR assays (Boonham et al., 2004) (Table 2). The RT-LAMP assay was also compared with the recently published Tsutsumi RT-LAMP assay. This assay was initially developed for turbiditybased detection and was adapted here for real-time detection using the same apparatus and chemistry as for the RT-LAMP assay. The latter showed a 10-fold greater sensitivity than the Tsutsumi RT-LAMP assay (Table 2).
Time of positivity in LAMP assays Positivity in a reaction is expressed by the time of positivity (tp) value, i.e. the amplification time at which the Plant Pathology (2013) 62, 1147–1156
fluorescence second derivative reached its peak above the baseline value. The majority of PSTVd isolates tested with the RT-LAMP assay gave values in the range of 13 –19 min, apart from two isolates that showed a time of positivity greater than 20 min (Table 1). In the case of isolate NIB V 95, the late positive signal (2374 min) may have been the result of the low concentration of the PSTVd isolate in this sample (50–100 times lower than that of the NIB V 190 isolate), as confirmed with the real-time RT-PCR assay. Also, one cannot exclude a possible sequence effect on the efficiency of the LAMP assay. Based on these results, it was estimated that a 25-min reaction time is sufficient to detect all PSTVd isolates with the RT-LAMP assay. The speed of the RT-LAMP assay was compared to that of the Tsutsumi RT-LAMP assay. The latter has been reported to give signals for positive samples (tp) in less than 60 min for reactions performed in 80 min. For the purpose of comparing the two LAMP assays, the Tsutsumi RT-LAMP assay was performed under the same original reaction conditions, except that the Isothermal Master Mix (Optigene Ltd.) was used instead of the in-house reaction mix used in the original study. The detection method also differed in the assay amplification step, which was followed by increase of fluorescence in real-time, instead of by turbidity as proposed initially. With the new optimized set-up, the Tsutsumi RT-LAMP assay performed faster, with a reaction time of only 35 min for detection of PSTVd isolates (Table 1). The sensitivity of the RT-LAMP assay proved to be 10-fold better and the time required about 10 min less than for the optimized Tsutsumi RT-LAMP assay (Fig. 3). Moreover, comparison of the shapes of the amplification curves suggested that the efficiency of amplification was greater in the RT-LAMP assay.
Discussion In the domain of plant protection, there is an urgent demand for quick and reliable in-field, first-line pathogen detection in order to reduce the time needed for plant testing as well as the costly consequences of possible
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Table 2 Comparison of the sensitivity of different detection methods for Potato spindle tuber viroid (PSTVd) Total plant RNA dilution 1 1 1 1 1 1 1 1
9 9 9 9 9 9 9 9
10 10 10 10 10 10 10 10
2 3 4 5 6 7 8 9
Estimated PSTVd RNA copy number
Real-time RT-PCR (Cq)a
RT-LAMP (tp) (this study)b
Tsutsumi RT-LAMP assay (tp)c
RTPCRd
10 000–100 000 1000–10 000 100–1000 10–100 1–10 0 0 0
+(236 +(276 +(309 +(339 +(376
+(130 01) +(141 04) +(155 02)*
+(262 11) +(285 18)
+ +
02) 05) 01) 02) 03)
a
Based on Boonham et al. (2004). Cq (quantification cycle): real-time PCR cycle at which fluorescence exceeded the threshold value. An average Cq value of triplicates with a corresponding standard deviation is given for each dilution. b tp (time of positivity): amplification time at which the fluorescence second derivative reached its peak above the baseline value. The average tp value represents tp values of three independent runs. *Average tp value calculated on the basis of two independent runs because of an outlier in a third run. c Based on Tsutsumi et al. (2010). d Based on Verhoeven et al. (2004). –, negative result (absence of signal); +, positive result (presence of signal).
delay during shipment of materials or harvesting of crops. Although efforts have been made to decrease the size of the instrumentation (Mumford et al., 2006), the portability of real-time PCR instrumentation continues to be an issue, and the routine use of real-time PCR for infield testing remains questionable. Recently, the possibility of DNA-based in-field testing was demonstrated, based on a simple real-time LAMP reaction preparation (enzyme mix, primer mix, water and DNA) and making use of simple, portable equipment (Bekele et al., 2011). These authors verified that LAMP assays are less sensitive to inhibitors usually affecting PCR amplification. These observations make LAMP technology a good candidate for the development of assays to be deployed for on-site plant health testing. The one-step RT-LAMP assay reported in this work is proposed as a simple first-line screening of potato PSTVd RNA, allowing real-time detection of this quarantine pathogen. The assay displays performance characteristics suitable for diagnostic as well as research use. Its specificity is comparable to that of the one-step real-time RTPCR PSTVd assay (Boonham et al., 2004) and superior to that of the previously described Tsutsumi RT-LAMP assay (Tsutsumi et al., 2010), and is appropriate for PSTVd detection. The few cross-reactions in the PSTVd RT-LAMP assay observed when testing different viroids (TCDVd, TPMVd, CSVd and CLVd) pose no problem, because the signals obtained are late and most of these viroids (with the exception of TCDVd) can be distinguished from PSTVd by melting curve analysis. Moreover, all these plant pathogens occur on plant hosts other than potato, and are therefore not expected in potato samples. Additionally, PSTVd is the only viroid known to infect cultivated species of potato naturally. Therefore, a positive result obtained when testing potato samples with the proposed RT-LAMP assay for the PSTVd viroid (with an amplicon Tm in the acceptable range) is directly indicative of infection with this quaran-
tine pathogen. Such samples should be considered for further confirmation testing. The lower sensitivity of the newly developed assay compared to that of real-time RT-PCR (Boonham et al., 2004) is not a limitation for its use as a first-line screening assay, because infected plants usually contain a high titre of PSTVd copies, far above its observed detection limit. Moreover, the proposed RT-LAMP assay shows better sensitivity than the conventional RT-PCR assay for pospiviroids (Verhoeven et al., 2004), and than the recently described PSTVd-specific LAMP assay (Tsutsumi et al., 2010). The RT-LAMP assay was developed and validated on the portable SmartCycler system. Its robustness was tested on another portable system that can easily be deployed in the field (Genie II), and on a bench real-time PCR (ABI PRISM 7900 HT) thermocycler. Similar performance in terms of specificity and sensitivity (data not shown) was observed. Moreover, comparable results were obtained when testing the RT-LAMP assay at 60°C and at 65°C. Signals appeared about 2 min later when assays were performed at 60°C rather than at 65°C, and the melting temperature of the amplification product was in the same range at both reaction temperatures (data not shown). The robustness toward a device change ensures more flexibility of the RT-LAMP assay deployment in different laboratories and national plant protection organizations. The robustness to small differences in amplification temperature is a valuable advantage for infield use, where it could be more difficult to control reaction conditions. The newly developed RT-LAMP assay can be used to detect PSTVd in potato tuber and leaf tissues with the same accuracy (Table 1). It also enables detection of the target pathogen in other plants and plant materials such as tomato, and the ornamental plants S. jasminoides, S. rantonettii and Petunia (Table 1). This last element of robustness to plant species and tissues adds to the flexibility of use of the assay. Plant Pathology (2013) 62, 1147–1156
Potato spindle tuber viroid detection by RT-LAMP
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Figure 3 Comparison of the sensitivity and speed of RT-LAMP and Tsutsumi RT-LAMP assays. Tested samples: total plant RNA dilutions of 1 9 10–2, 1 9 10–3, 1 9 10–4 and no-template controls (NTC). RT-LAMP was completed in 30 min, while the Tsutsumi RT-LAMP assay was completed in 40 min.
In addition to its fit-for-purpose performance characteristics, the proposed assay and its underlying detection platform possess further advantages over currently used methods. The first advantage is the speed with which results are obtained. The assay provides results on the presence or absence of the target pathogen in a maximum of 25 min (+5 min for Tm analysis where required), compared to the 60 min required with another recently reported PSTVdspecific RT-LAMP assay (Tsutsumi et al., 2010). The RTLAMP assay is even more competitive when compared with the c. 25 h required for the pospiviroid-specific RTPCR assay (excluding gel electrophoresis for detection) (Verhoeven et al., 2004), and when compared to the c. 80 min required for the one-step real-time RT-PCR assay for specific PSTVd detection (Boonham et al., 2004). The second advantage of the proposed real-time RTLAMP is the simple interpretation of the final results, obtained by observing the amplification curve, compared with that for the Tsutsumi RT-LAMP assay, originally based on turbidity measurement, and that of conventional RT-PCR, based on gel electrophoresis analysis. The third advantage is that it is carried out entirely in a single reaction tube, significantly reducing the risk of carry-over contamination, one of the main hazards when performing in-field testing. In conclusion, the proposed RT-LAMP assay offers a rapid, robust and reliable first-line PSTVd screening method in potato tubers or plants. Because of its simplicity, it can be performed in laboratories, in the field or at any crop trade entry point (e.g. ports or airports). The speed, lower screening time (compared to the real-time RT-PCR or conventional RT-PCR assays) and cheaper reagents and consumables (compared to those for realtime RT-PCR) all contribute significantly to reducing the cost of testing. Moreover, the assay could facilitate crop Plant Pathology (2013) 62, 1147–1156
trade by significantly reducing the time necessary to obtain test results on the presence or absence of the quarantine pathogen in traded crops. In the case of a positive result being obtained, PSTVd infection of the potato material can be confirmed in plant protection laboratories by conventional methods.
Acknowledgements We thank Dr Neil Boonham, Tom Nixon, Samantha Bennett and Adrian Fox from the Food and Environment Research Agency (Fera) in York, UK and Dr J. Th. J. (Ko) Verhoeven from the National Plant Protection Organization of the Netherlands for providing us with a valuable collection of viroid-infected material. We also thank Dr Steen Lykke Nielsen from Aarhus University, Denmark for clarifying the origin of certain isolates. We thank Neza Turnsek from the National Institute of Biology in Ljubjana, Slovenia, for providing PSTVd-negative potato extracts. We thank Dr Natasa Petrovic and Professor Roger Pain for reviewing the manuscript. This work was carried out under the sponsorship of the EU Framework 7 Programme (FP7-KBBE-2009-3) project 245047 (Q-DETECT – Developing Quarantine Pest Detection Methods for Use by National Plant Protection Organizations (NPPO) and Inspection Services).
References Bekele B, Hodgetts J, Tomlinson J et al., 2011. Use of a real-time LAMP isothermal assay for detecting 16SrII and XII phytoplasmas in fruit and weeds of the Ethiopian Rift Valley. Plant Pathology 60, 345–55. Boonham N, Gonzalez Perez L, Mendez MS et al., 2004. Development of a real-time RT-PCR assay for the detection of Potato spindle tuber viroid. Journal of Virological Methods 116, 139–46. Bru D, Martin-Laurent F, Philippot L, 2008. Quantification of the detrimental effect of a single primer-template mismatch by real-time
1156
R. Lenarcic et al.
PCR using the 16S rRNA gene as an example. Applied and Environmental Microbiology 74, 1660–3. Buhlmann A, Pothier JF, Tomlinson JA et al., 2012. Genomics-informed design of loop-mediated isothermal amplification for detection of phytopathogenic Xanthomonas arboricola pv. pruni at the intraspecific level. Plant Pathology 62, 475–484. Daros JA, Elena SF, Flores R, 2006. Viroids: an Ariadne’s thread into the RNA labyrinth. EMBO Reporter 7, 593–8. Diener TO, 1971. Potato spindle tuber “virus”: IV. A replicating, low molecular weight RNA. Virology 45, 411–28. Francois P, Tangomo M, Hibbs J et al., 2011. Robustness of a loopmediated isothermal amplification reaction for diagnostic applications. FEMS Immunology and Medical Microbiology 62, 41–8. Ghedira R, Papazova N, Vuylsteke M, Ruttink T, Taverniers I, De Loose M, 2009. Assessment of primer/template mismatch effects on real-time PCR amplification of target taxa for GMO quantification. Journal of Agricultural and Food Chemistry 57, 9370–7. Gross HJ, Domdey H, Lossow C et al., 1978. Nucleotide sequence and secondary structure of potato spindle tuber viroid. Nature 273, 203–8. Kubota R, Vine BG, Alvarez AM, Jenkins DM, 2008. Detection of Ralstonia solanacearum by loop-mediated isothermal amplification. Phytopathology 98, 1045–51. Kwok S, Kellogg DE, McKinney N et al., 1990. Effects of primertemplate mismatches on the polymerase chain reaction: human immunodeficiency virus type 1 model studies. Nucleic Acids Research 18, 999–1005. Martı´nez-Soriano JP, Galindo-Alonso J, Maroon CJM, Yucel I, Smith DR, Diener TO, 1996. Mexican papita viroid: putative ancestor of crop viroids. Proceedings of the National Academy of Sciences, USA 93, 9397–401. Morris TJ, Wright NS, 1975. Detection on polyacrylamide-gel of a diagnostic nucleic acid from tissue infected with potato spindle tuber viroid. American Potato Journal 52, 57–63. Mumford R, Boonham N, Tomlinson J, Barker I, 2006. Advances in molecular phytodiagnostics – new solutions for old problems. European Journal of Plant Pathology 116, 1–19. Navarro B, Pantaleo V, Gisel A et al., 2009. Deep sequencing of viroid-derived small RNAs from grapevine provides new insights on the role of RNA silencing in plant–viroid interaction. PLoS ONE 4, e7686. Notomi T, Okayama H, Masubuchi H et al., 2000. Loop-mediated isothermal amplification of DNA. Nucleic Acids Reseach 28, e63. Owens RA, 2007. Potato spindle tuber viroid: the simplicity paradox resolved? Molecular Plant Pathology 8, 549–60. Owens RA, Diener TO, 1981. Sensitive and rapid diagnosis of potato spindle tuber viroid disease by nucleic acid hybridization. Science 213, 670–2. Parida M, Sannarangaiah S, Dash PK, Rao PVL, Morita K, 2008. Loop mediated isothermal amplification (LAMP): a new generation of innovative gene amplification technique; perspectives in clinical diagnosis of infectious diseases. Reviews in Medical Virology 18, 407– 21. Pfannenstiel MA, Slack SA, 1980. Response of potato cultivars to infection by the potato spindle tuber viroid. Phytopathology 70, 922– 6. Puchta H, Herold T, Verhoeven K et al., 1990. A new strain of potato spindle tuber viroid (Pstvd-N) exhibits major sequence differences as
compared to all other Pstvd strains sequenced so far. Plant Molecular Biology 15, 509–11. Querci M, Owens RA, Vargas C, Salazar LF, 1995. Detection of potato spindle tuber viroid in avocado growing in Peru. Plant Disease 79, 196–202. Schnolzer M, Haas B, Ramm K, Hofmann H, Sanger HL, 1985. Correlation between structure and pathogenicity of potato spindle tuber viroid (PSTV). EMBO Journal 4, 2181–90. Shamloul AM, Hadidi A, Zhu SF, Singh RP, Sagredo B, 1997. Sensitive detection of potato spindle tuber viroid using RT-PCR and identification of a viroid variant naturally infecting pepino plants. Canadian Journal of Plant Pathology 19, 89–96. Suech B, Flekna G, Wagner M, Hein I, 2009. Studying the effect of single mismatches in primer and probe binding regions on amplification curves and quantification in real-time PCR. Journal of Microbiological Methods 76, 316–9. Tamura K, Peterson D, Peterson N, Stecher G, Nei M, Kumar S, 2011. MEGA5: molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods. Molecular Biology and Evolution 28, 2731–9. Tomlinson JA, Dickinson MJ, Boonham N, 2010. Detection of Botrytis cinerea by loop-mediated isothermal amplification. Letters in Applied Microbiology 51, 650–7. Tsutsumi N, Yanagisawa H, Fujiwara Y, Ohara T, 2010. Detection of potato spindle tuber viroid by reverse transcription loop-mediated isothermal amplification. Research Bulletin of the Plant Protection Service Japan 46, 61–7. Verhoeven JTJ, Jansen CCC, Willemen TM, Kox LFF, Owens RA, Roenhorst JW, 2004. Natural infections of tomato by Citrus exocortis viroid, Columnea latent viroid, Potato spindle tuber viroid and Tomato chlorotic dwarf viroid. European Journal of Plant Pathology 110, 823–31. Verhoeven JTJ, Roenhorst JW, Owens RA, 2011. Mexican papita viroid and tomato planta macho viroid belong to a single species in the genus. Pospiviroid. Archives of Virology 156, 1433–7. Wang MB, Bian XY, Wu LM et al., 2004. On the role of RNA silencing in the pathogenicity and evolution of viroids and viral satellites. Proceedings of the National Academy of Sciences, USA 101, 3275–80. Weidemann HL, Buchta U, 1998. A simple and rapid method for the detection of potato spindle tuber viroid (PSTVd) by RT-PCR. Potato Research 41, 1–8. Weller SA, Elphinstone JG, Smith NC, Boonham N, Stead DE, 2000. Detection of Ralstonia solanacearum strains with a quantitative, multiplex, real-time, fluorogenic PCR (TaqMan) assay. Applied and Environmental Microbiology 66, 2853–8.
Supporting Information Additional Supporting Information may be found in the online version of this article. Figure S1 Alignment of all available PSTVd sequences and designed primers for here described RT-LAMP. Figure S2 Alignment of PSTVd consensus sequence with viroids that can potentially cross-react in the here described RT-LAMP: TPMVd, MPVd and TCDVd. Primer positions are shown at the bottom of the alignment.
Plant Pathology (2013) 62, 1147–1156
