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Molecular detection of nine rice viruses by a reverse-transcription loop-

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Journal of Virological Methods 170 (2010) 90–93

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Protocols

Molecular detection of nine rice viruses by a reverse-transcription

loop-mediated isothermal amplification assay
Dung Tien Le a,b , Osamu Netsu a , Tamaki Uehara-Ichiki a , Takumi Shimizu a ,
Il-Ryong Choi c , Toshihiro Omura a , Takahide Sasaya a,∗
a
Research Team for Vector-Borne Diseases, National Agricultural Research Center, Tsukuba, Ibaraki 305-8666, Japan
b
Department of Plant Molecular Pathology, Agricultural Genetics Institute, Pham-Van-Dong Street, Hanoi, Vietnam
c
Plant Breeding, Genetics, and Biotechnology Division, International Rice Research Institute, DAPO Box 7777, Metro Manila, Philippines

a b s t r a c t

Article history: A reverse-transcription loop-mediated isothermal amplification (RT-LAMP) assay was established for the
Received 5 April 2010 detection of nine viruses from infected rice plants, including rice black-streaked dwarf virus (RBSDV), rice
Received in revised form 31 August 2010 dwarf virus (RDV), rice gall dwarf virus (RGDV), rice ragged stunt virus (RRSV), rice transitory yellowing
Accepted 2 September 2010
virus (RTYV), rice stripe virus (RSV), rice grassy stunt virus (RGSV), rice tungro spherical virus (RTSV), and
Available online 15 September 2010
rice tungro bacilliform virus (RTBV). Virus-specific primer sets were designed from the genome sequences
of these viruses. By the combination of RNA rapid extraction and RT-LAMP, these nine viruses could be
Keywords:
detected within 2 h from infected rice plants. The sensitivities of the assays were either higher than (for
RT-LAMP
Rice virus
RSV, RTBV, and RTYV) or similar (for RDV) to those of one-step RT-PCR. Furthermore, RTBV and RTSV
Detection were detected not only in infected rice plants but also in viruliferous insect vectors. The RT-LAMP assays
may facilitate studies on rice disease epidemiology, outbreak surveillance, and molecular pathology.
© 2010 Elsevier B.V. All rights reserved.

Rice (Oryza sativa L.) is one of the most important crops, with
an annual global production of 650 million tons, of which more
than 90% comes from Asian countries (Zeigler and Barclay, 2008).
Virus diseases of rice are a menace to safe and efficient rice pro-
duction. Of the 15 viruses known to affect rice production, nine
cause severe diseases in Asian countries. All these rice viruses are
transmitted by leafhoppers and planthoppers such as Laodelphax
striatellus, Nephotettix cincticeps, and Nilaparvata lugens. Some of
the viruses multiply in the insects and are transmitted transovari-
ally, which makes their control more difficult (Hibino, 1996). These
insect vectors are distributed widely in Asian countries and they
migrate long distances, even across the ocean (Kishimoto, 1971).
The viruses cause characteristic symptoms on infected rice
plants. Thus, observation of symptoms is a common practical
method for virus detection in the field. However, similar symp-
toms can be developed by different viruses and nonpathogenic
disorders, such as nutritional deficiencies. Excess water, drought, or
Abbreviations: RT-Lamp, reverse transcription-loop-mediated isothermal
amplification; RBSDV, rice black-streaked dwarf virus; RDV, rice dwarf virus; RGDV,
rice gall dwarf virus; RRSV, rice ragged stunt virus; RTYV, rice transitory yellowing
virus; RSV, rice stripe virus; RGSV, rice grassy stunt virus; RTSV, rice tungro spherical
virus; RTBV, rice tungro bacilliform virus.
∗ Corresponding author. Tel.: +81 29 838 8932; fax: +81 29 838 7845.
E-mail address: tsasaya@affrc.go.jp (T. Sasaya).
insect damage can also induce symptoms similar to those caused by
viruses. Furthermore, the symptoms vary depending on rice vari-
eties, rice growth stages, and growth conditions of the rice plants.
Moreover, rice plants may be infected with multiple viruses in the
field. These facts may often make it unreliable to diagnose rice
diseases by symptom observations.
An enzyme-linked immunosorbent assay (ELISA) is a reli-
able method for detecting rice viruses and it is suitable for
high-throughput testing (Takahashi et al., 1991). However, the sen-
sitivity of ELISA may not be high enough to detect viruses in rice
leaves with low virus concentration (Takahashi et al., 1993). Fur-
thermore, ELISA requires a virus-specific antiserum, which is not
easy to obtain. A reverse transcription-polymerase chain reaction
(RT-PCR) assay is more sensitive than ELISA (Takahashi et al., 1993)
and has been used to detect rice viruses (Isogai et al., 2001; Lee et
al., 2005; Cai et al., 2003; Periasamy et al., 2006; Zhang et al., 2008).
However, RT-PCR requires an expensive thermo-cycler and is rela-
tively time-consuming because of its longer amplification time and
an additional step of visualization on agarose gels involving the use
of the mutagen ethidium bromide.
Recent advances in nucleic acid-based diagnosis led to the
invention of a loop-mediated isothermal amplification (LAMP)
assay (Notomi et al., 2000). The LAMP assay employs two pairs of
primers that recognize six regions of a target sequence, and amplify
specifically and isothermally the target sequence, eliminating the
need for thermo-cycling. The LAMP assay has been used to detect

0166-0934/$ – see front matter © 2010 Elsevier B.V. All rights reserved.
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 Author's personal copy

D.T. Le et al. / Journal of Virological Methods 170 (2010) 90–93 91

various viruses, including those infecting plants, such as Japanese
yam mosaic virus (Fukuta et al., 2003), tomato spotted wilt virus
(Fukuta et al., 2004), plum pox virus (Varga and James, 2006) and
peach latent mosaic viroid (Boubourakas et al., 2009). Nevertheless,
there is no report on RT-LAMP assays for rice viruses.
Given the current context of rapid and unpredictable epidemic
outbreaks of rice virus diseases (ProMED-mail, International Soci-
ety for Infectious Diseases, www.promedmail.org), a system for
rapid and accurate diagnosis of the causal viruses in diseased rice
plants and detection of viruses in viruliferous insects is critical for
not only epidemiological studies but also for monitoring and con-
trolling of outbreaks. Such an assay system, once available, has to
be specific, sensitive, and adaptable to detect newly emerged virus
strains. To address these needs, we attempted to establish assays
for the molecular detection of nine major rice viruses that occur in
Asia based on RT-LAMP principles.
The viruses examined by RT-LAMP in this study are rice black-
streaked dwarf virus (RBSDV), rice dwarf virus (RDV), rice gall dwarf
virus (RGDV), rice ragged stunt virus (RRSV), rice transitory yellowing
virus (RTYV), rice stripe virus (RSV), rice grassy stunt virus (RGSV),
rice tungro spherical virus (RTSV), and rice tungro bacilliform virus
(RTBV). The rice viruses were maintained in rice cv. Nipponbare by
inoculating seedlings at the second- to third-leaf stage by virulif-
erous leafhoppers or planthoppers. Infected plants were grown in
an air-conditioned greenhouse as described previously (Takahashi
et al., 1991).
For the design of the RT-LAMP primer sets, it is important
to select the most conserved regions of the nucleotide sequence
for each virus. Nucleotide sequence analysis of strains/isolates of
each virus revealed highly conserved regions in the genes coding
for structural proteins. Thus, RT-LAMP primer sets for detection
of the viruses were designed based on the sequences coding for
structural proteins of each virus (Table 1) with the LAMP Primer
Explorer® web-interface (available at http://primerexplorer.jp/e/).
The bacterial plasmids (Invitrogen, Carlsbad, CA) harboring the
corresponding target viral genomic cDNA were used as positive
controls. Total RNA was extracted from infected rice plants by
the total RNA extraction method described in Wang et al. (1993).
Briefly, 100 mg of rice leaves were ground in 400 ␮l of 0.5 N NaOH
using a multi-bead shaker (Yasuikikai, Osaka, Japan); 10 ␮l of the
resulting solution were diluted quickly with 490 ␮l of 100 mM
Tris–Cl buffer (pH 8) and 1.5 ␮L of the resulting solution were
used in RT-LAMP. RT-LAMP was carried out using a Loopamp® RNA
amplification kit (Eiken Chemicals Co., Tokyo, Japan), in a 25 ␮l
reaction mixture with 20 mM Tris–HCl (pH 8.8), 10 mM KCl, 10 mM
(NH4 )2 SO4 , 8 mM MgSO4 , 0.1% Tween 20, 0.8 M betaine, 0.2 ␮M
each of two outer primers (F3 and B3), and 1.6 ␮M each of two
inner primers (FIP and BIP), 1.4 mM dNTPs, 8 units of Bst DNA
polymerase, 1.25 units of AMV reverse transcriptase, and 1.5 ␮l
of the template RNA preparation. One ␮l of fluorescent detection
reagent (Eiken Chemicals Co.) was also added to each reaction
tube for visualization. The RT-LAMP reaction was carried out

Table 1
Primer sets of RT-LAMP for detection of rice viruses.

Virus (Family/genera) Accession The targeted genomic Primer sequences (5 –3 )
numbers segment or gene

RBSDV (Reoviridae/Fijivirus) GU322365 Segment 10 F3: 5 -CCCCAGAGACTTTCCGATAC-3

B3: 5 -GGTCTTTAAGTTGCGTGATGT-3
FIP: 5 -CGTGGGGTGCTTTGACAATGTTTCAACCGACCAACAATCACTC-3
BIP: 5 -TCGCAAACATTTGTTGACCCGACGGTAAAGTGCTAGTTTCTACG-3

RDV (Reoviridae/Phytoreovirus) D13773 Segment 8 F3: 5 -ATTCCAGCCGGGGCATAT-3

B3: 5 -CCCACCACCAAGTGAGAAC-3
FIP: 5 -AACGCCAGCTATTGTCGTTCCAGGGCATCAGTGCTAAGTGT-3
BIP: 5 -CTACTGCAACTGCCGCAGACGTCCGTTTGGACAGGGAGG-3

RGDV (Reoviridae/Phytoreovirus) D13410 Segment 8 F3: 5 -AATCAGATTGCGCGCTTC-3

B3: 5 -TTTTCGGGATGCAAATGG-3
FIP: 5 -CCTGATTAGCTGGCATATATTGCCTAATTTTTAGTCAGTCGATGAACAC-3
BIP: 5 -TTGCTTTTGTATCACCCTGGTACGCAGGGGTTGGTTAAACG-3

RRSV (Reoviridae/Oryzavirus) AF486811 Segment 8 F3: 5 -GACTAGGGATGTGCGTTC-3

B3: 5 -TGTAATCGACGTTCGCTC-3
FIP: 5 -TGTTATTCTGCCTTGTTCTTTCAACTTCTGATTTGATTGTTTTGAGCA-3
BIP: 5 -TCGACTTGGTTTAGCCAAGATG-TTGTTCAGTGATGATTCGC-3

RTYV (Rhabdoviridae/Nucleorhabdovirus) AB011257 Nucleoprotein gene F3: 5 -GGACGACCATCAAGACAGC-3

B3: 5 -GCAACAGGTGTACCACTGTA-3
FIP: 5 -GCCCCTGAGGTTGCATGCTATCACAACACTTTCAGCGAGACA-3
BIP: 5 -TGGCAGCACCCCTTTGTTGGCATCAGTTGACGGAGCGG-3

RSV (–/Tenuivirus) DQ333944 RNA3 F3: 5 -GTGACCTTTGCTGGTCAGAT-3

B3: 5 -ACCGAGGACACTATCCCAT-3
FIP: 5 -GGCCAGTGTGTCACCACCTTGGCTATGATGCTGCAACTCT-3
BIP: 5 -GAGAGGCACTGGCTTTGTGAGACCAAGGTTGAAGCCTCTGTG-3

RGSV (–/Tenuivirus) AB000403 RNA5 F3: 5 -AAAGACCAACTCAGAGGCA-3

B3: 5 -TCTAGAGCAGTTTCCTGTAGTC-3
FIP: 5 -CTGACTTAGTGTGGACACTGTGCTTTTGTGTTACCAAGTCTGTGTG-3
BIP: 5 -CACTGCATGGGTTTTGTCAACCTGGAGATCATCCTTCTACCAGCT-3

RTSV (Sequivridae/Waikavirus) GU723290 ORF1 F3: 5 -CCGTACTGTGCAAGAACAGA-3

B3: 5 -GCTCTTGATGTCATCCGCG-3
FIB: 5 -GGCACCGCTACGCAAATCAAGTCCCAAAGGCTTATGCGTCTA-3
BIP: 5 -TTGTCTCGATCGCTGGGGGAGTCACTCACTGAGCCACATT-3

RTBV (Caulimoviridae/Tungrovirus) D10774 ORF3 F3: 5 -ACTCTTTGATAGACTACCAGAAG-3

B3: 5 -GGATTTTTCGTTTCTTATAATCTCC-3
FIP: 5 -GCTATTCCTATTCCTGCTTCATAGGGGAAAGGTAGTAAAAGCGGA-3
BIP: 5 -CATGGATGAGAGCAAAATGCATTAAGATCTACAGAATGCTAAGGATG-3
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92 D.T. Le et al. / Journal of Virological Methods 170 (2010) 90–93

Fig. 1. Specificity of RT-LAMP assays for the detection of nine rice viruses from infected rice plants. (A)–(I): RT-LAMP assay for rice black-streaked dwarf virus (RBSDV), rice
dwarf virus (RDV), rice gall dwarf virus (RGDV), rice ragged stunt virus (RRSV), rice transitory yellowing virus (RTYV), rice stripe virus (RSV), rice grassy stunt virus (RGSV), rice
tungro spherical virus (RTSV), and rice tungro bacilliform virus (RTBV) as targeted viruses. The RT-LAMP reaction mixtures were amplified using RNA preparations from healthy
rice plants (tube 1), rice plants infected with non-targeted viruses (tubes 2–4), and rice plant infected with targeted viruses (tube 5). The plasmids harboring target viral
cDNA were used as positive controls (tube 6).

at 63 ◦ C for 60 min; heating at 80 ◦ C for 5 min terminated the PCR. RT-PCR was carried out using the One-step RT-PCR version
reaction. 2 kit (Takara Bio Inc., Shiga, Japan) and the amplification condi-
RT-LAMP procedures with the eight primer sets for detection tions were set as suggested in the manufacturer’s instructions with
of the eight individual viruses, except for RBSDV, at conditions of 30 cycles of 15 s at 94 ◦ C, 15 s at 55 ◦ C, 1 min at 68 ◦ C, and finally
63 ◦ C within 60 min were established (Fig. 1). The primer set for an extension time of 5 min at 68 ◦ C. The F3 and B3 primers of
detection of RBSDV required a temperature of 61 ◦ C for optimal the respective RT-LAMP primer sets (Table 1) were used as virus-
amplification. All primer sets detected consistently the presence of specific primers for amplification of virus genome by PCR. The
the corresponding virus RNA from the RNA extracts of infected rice resultant RT-LAMP and RT-PCR products were analyzed by agarose
plants in a similar fashion as detected from the plasmids harboring gel electrophoresis. As shown in Fig. 2, the sensitivity of RT-LAMP
target sequences (Fig. 1, tube 6). The assays appeared to be specific assays for respective viral detection was higher than or at least sim-
as no amplification occurred in samples with RNA extracts from ilar to that of RT-PCR assays (Fig. 2). By RT-LAMP assays, RDV, RSV,
either healthy rice plants (Fig. 1, tube 1) or rice plants infected with and RTYV were detected in the RNA preparations from infected rice
the non-targeted viruses (Fig. 1, tubes 2–4). For example, when the plants diluted up to 105 -fold. The sensitivity of RT-LAMP was sim-
primer set designed to detect RBSDV was used for the RT-LAMP ilar to that of the RT-PCR assay for RDV, and it was higher than
assay, the virus genome was amplified from the RNA preparation that of the RT-PCR assays for RSV and RTYV. The detection thresh-
of RBSDV-infected rice plants, but it was not detected from plants old for RTBV by the RT-LAMP assay was 104 -fold dilution, while
infected with its related viruses, RDV, RGDV, and RRSV (Fig. 1A).
Similarly, when the primer sets for detection of RSV and RGSV
were used, only the targeted virus genome was amplified from
rice plants infected with the corresponding virus (Fig. 1F and G).
The positive controls are important and essential for the diagno-
sis of rice viruses. Usually, virus-infected rice plants are used as
positive controls. Unfortunately, not all virus-infected rice plants
are available at the agricultural institutes and plant quarantine sta-
tions where diagnosis is actually taking place. The RT-LAMP assay
demonstrated that the plasmids harboring a virus target sequence
could be used as positive controls and seemed to eliminate the
need for verified infected rice plants as positive controls. Further-
more, it will be easier to share resources in the form of plasmids
as positive controls with places where verified infected rice is
unavailable.
To compare the sensitivity of RT-LAMP for viral detection with Fig. 2. Sensitivity of RT-LAMP (top) and RT-PCR (bottom) using serial dilutions of
that of conventional RT-PCR, total RNA preparations from infected RNA preparations from RDV (A), RTYV (B), RSV (C) and RTBV (D) infected rice plants
as templates. 3 ␮l of reaction mixture was electrophoresed on a 2% agarose gel. Lane
rice plants were diluted serially up to 106 -fold and the same amount
1: RNA preparations from healthy rice plants; lanes 2–8: 100 –106 dilutions of RNA
of diluted samples was used for RT-LAMP and conventional RT- preparations from infected rice plants.
 Author's personal copy
D.T. Le et al. / Journal of Virological Methods 170 (2010) 90–93
that of the RT-PCR assay was 103 -fold dilution of the same RNA
preparations.
Among the nine rice viruses used in this study, seven, except
for RTBV and RTSV, were detectable from individual viruliferous
insects owing to their multiplication in vector insects by ELISA
(Takahashi et al., 1991). However, RTBV and RTSV were unde-
tectable practically by ELISA or RT-PCR from the viruliferous insects,
probably because the viruses do not multiply in the vector insect
(Takahashi et al., 1993). The detection of these viruses from virulif-
erous insects by RT-LAMP was attempted. Virus-free insects were
allowed to acquire RTBV and RTSV over 48 h on tungro-infected
rice plants presenting obvious symptoms. Individual insects were
then collected and total RNA was extracted according to the pro-
tocol reported by Cai et al. (2003). In three repeated experiments,
RTBV and RTSV were detected in 87.5 ± 12.5% and 18.8 ± 6.2% of
green leafhopper insects by RT-LAMP, respectively. On the other
hand, neither virus was detected by RT-LAMP from the insects
that had been exposed to healthy rice plants. These results sug-
gest that RT-LAMP is useful for a routine diagnosis of RTBV from
the viruliferous insects and may be applicable for forecasting the
tungro disease in regions of South and Southeast Asia where tun-
gro disease is a serious problem inasmuch as its main agent is RTBV
(Hibino, 1996).
Another advantage of the RT-LAMP assay for rice viruses over
ELISA is its rapid adaptability. It is known that tungro virus strains
are diverse, which may make it difficult to detect some tungro virus
strains by serological methods (Azzam et al., 1999). A primer set
designed on the basis of an RTSV strain (GenBank accession num-
ber M95497) reported in 1995 did not work to detect viral RNA by
RT-LAMP using total RNA extracted from RTSV-infected rice plants
that were collected recently from rice fields. Thus, the nucleotide
sequence of the region of the recently collected RTSV strain was
analyzed (GenBank accession number GU723290). The nucleotide
sequences between the two strains shared only 86.5% in identity.
By using the specific primer sets designed on the basis of the newly
analyzed nucleotide sequence, the RTSV strain was detected clearly
by RT-LAMP. This procedure took 2 weeks. Thus, the RT-LAMP
assay can be easily and rapidly adapted for the detection of newly
emerged virus strains.
In conclusion, the RT-LAMP assay for detecting nine rice viruses,
including eight RNA viruses and one DNA virus, was established
in this study. The RT-LAMP method was specific and sensitive, and
can be rapidly adapted to detect various virus strains. The RT-LAMP
method for rice viruses can therefore be adopted in broad areas of
not only diagnosis but also epidemiology and molecular pathology
studies of rice viruses.
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Acknowledgement
Dung Tien Le was supported by a postdoctoral fellowship for
foreign researchers from the Japan Society for the Promotion of
Sciences. This work was supported by a Basic Research Activities
for Innovative Biosciences grant from the Bio-oriented Technology
Research Advancement Institution (PROBRAIN). The authors wish
to thank Ms. Stephanie K. Dalquist for correcting English usage in
this manuscript.
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