Plant Pathology (2012)
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
has been an increased number of reports of PSTVd infections in ornamental species, which are mostly latent and
*E-mail: [email protected]
†
These authors contributed equally to this work.
ª 2012 British Society for Plant Pathology
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
(Martı́nez-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
expensive equipment for thermal cycling and fluores1
2
R. Lenarcic et al.
cence 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 RT-LAMP 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,
Plant Pathology (2012)
Potato spindle tuber viroid detection by RT-LAMP
3
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)
Plant Pathology (2012)
4
R. Lenarcic et al.
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).
Plant Pathology (2012)
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
5
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.
Plant Pathology (2012)
6
R. Lenarcic et al.
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 (Martı́nez-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 (2012)
Potato spindle tuber viroid detection by RT-LAMP
7
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 (2012)
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
8
R. Lenarcic et al.
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 (2012)
Potato spindle tuber viroid detection by RT-LAMP
9
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 (2012)
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).
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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 (2012)