Vol.3, No.7, 447-452 (2011)
Copyright © 2011 SciRes. Openly accessible at http://www.scirp.org/journal/HEALTH/
Detection of nucleic acid of classical swine fever virus
by reverse transcription loop-mediated isothermal
amplification (RT-LAMP)
Kanokwan W ongsawa t1, Tararaj Dharakul2, Phairot Narat3, Jundee Rabablert 1*
1Department of Biology, Faculty of Science, Silpakorn University, Nakhon Pathom, Thailand;
*Correspondence author: jundee@su.ac.th
2Department of Immunology, Faculty of Medicine, Siriraj Hospital Mahidol University, Bangkok, Thailand;
3Bangkok Agro-Industrial Products Public Co., Ltd., Bangkok, Thailand.
Received 2 May 2011; revised 31 May 2011; accepted 27 June 2011.
Classical swine fever virus (CSFV) is the causa-
tive agent of Classical swine fever which is a
highly contagious disease affecting swine and
resulting in severe economic losses. In this
study, we developed reverse transcription loop-
mediated isothermal amplification (RT-LAMP)
assay targ eting t he 5’UTR gene for the detecti on
of CSFV. This amplification method can be ob-
tained in 1 h under isothermal conditions (65°C)
employing a set of six specific primers mixtures.
Amplification product was visualized by using
hydroxynaphthol blue (HNB) dye and agarose
gel electrophoresis. The sensitivity was 100 copy
numbers. No cross-reactivity related to Japa-
nese encephalitis virus (JEV) and porcine re-
productive and respiratory syndrome virus (PR-
RSV) was demonstrated. The results demon-
strated that the RT-LAMP assay is a useful tool
for the rapid and sensitive for CSFV detection in
Keywords: Classical Swine Fever Virus (CSFV);
RT-LAMP; Hydroxynaphthol Blue Dye
Classical swine fever (CSF) is a highly contagious
disease affecting swine, resulting in severe economic
losses [1]. Classical swine fever virus (CSFV), the caus-
ative agent of CSF is a member of the genus Pesti- virus
within the Family Flav iviridae [2]. CSFV is a small,
enveloped virus with a 12.5 kb; positive sin- gle-str-
anded RNA genome containing a single, large open
reading frame (ORF) flanked bytwo highly conserved
untranslated regions (UTR) at the 5’ and 3’ends. The
5’UTR functions as an internal ribosomal entry site for
translation initiation of the pre-polyprotein and genome
replication [3]. The host range of CSFV is narrow; this
virus is restricted to its natural hosts, domestic pigs and
feral pigs. The control of CSF is based on stamping out
policies and/or on vaccination. However, failed immu-
nization against CSF has been reported, mainly due to
interference from maternal antibodies and other infec-
tions [4,5].
In Thailand, CSF was first reported in 1950 at
Bangkhen area in Bangkok. It was declared a notifiable
disease in 1954 up to date; it has gradually become en-
zootic. [6]. The disease is generally subject to statutory
control, involving slaughter of affected pig herds and
restrictions on movements of pigs and pig products from
affected regions [2]. Although eradicated from many
countries, CSF continues to cause serious problems in
different parts of the world [7]. Rapid and accurate di-
agnosis is the key factor for the control of CSF. The di-
agnosis based on clinical signs is often difficult, because
clinical signs are rather variable and may be mistaken
for other febrile and/or haemorrhagic diseases of pigs,
essentially African swine fever [8]. Rapid, accurate, and
pre-clinical laboratory diagnosis of CSFV is therefore a
matter of urgency in order to prevent and control the
epidemics. Current laboratory diagnoses of CSFV rely
on virus isolation, serological methods, detection of an-
tigen and nucleic acid amplification [9-12]. The PCR-
base procedures are generally considered to be the most
sensitive in vitro method for detecting CSFV infection.
However, these techniques require centralized laboratory
facilities and clinical specimen submissions.
Loop-mediated isothermal amplification (LAMP) is a
nucleic acid amplification method developed by Notomi
et al. [13] The benefits of LAMP compared with other
nucleic acid amplification techniques is an easy opera-
K. Wongsaw at et al. / Health 3 (2011) 447-452
Copyright © 2011 SciRes. Openly accessible at http://www.scirp.org/journal/HEALTH/
tion, no need for special equipment, superior sensitivity
and speed, low contamination risk, and suitability for
high-throughput DNA detection. Additionally, LAMP
products can be observed by the naked eye when a white
precipitate of magnesium pyrophosphate is present in the
reaction mixture [14]. However, this detection is limited
when the turbidity of reaction is low. To increase the
sensitivity, LAMP products stained with ethidium bro-
mide were used [15]. Unfortunately, the product of de-
tection system with ethidium bromide has several limita-
tions, such as generation of hazardous waste and less
sensitivity than that of SYBR Green. Therefore, many
investigators have developed RT-LAMP to be visualized
by naked eye and with SYBR Green for DNA detection
Reverse transcription loop-mediated isothermal am-
plification (RT-LAMP) was used as tool for amplifica-
tion and has emerged as a powerful gene amplification
tool due to its simplicity, speed, specificity and cost-
effectiveness. This technique is being used increasingly
for rapid detection and typing of emerging viruses such
as Severe acute respiratory syndrome coronavirus [17],
Japanese encephalitis virus [18], Pseudorabies virus [19]
and Classical swine fever virus [20]. Rapid and cost-
effective RT-LAMP assays for the pre-clinical detection
of CSFV visualized directly with the naked eye by addi-
tion of SYBR Green have been described [20-22]. SYBR
Green is an intercalating agent binding with double-
stranded DNA; thus a disadvantage of SYBR Green is
that it is equally incorporated into every amplicon. Should
unspecific sequences be amplified, the measured signal
would correspond to both non-specific and specific
products [23]. It has been reported that hydroxynaphthol
blue (HNB) was used as a colorimetric indicator for the
titration of calcium ion and magnesium ion [24]. In ad-
dition, the sensitivity of LAMP assay using HNB was
equivalent to that of the assay using SYBR Green [25].
Therefore, this colorimetric assay is suitable not only for
laboratory research but also for clinical diagnoses of
many infectious diseases.
In the present study, we developed RT-LAMP deter-
mined by hydroxynaphthol blue (HNB) dye-mediated
visualization using the naked eye. The high sensitivity
and specificity of the RT-LAMP reaction were due to
continuous amplification under isothermal conditions.
This assay employed six primers that recognized eight
distinct regions of the 5’UTR gene from CSFV.
2.1. Design of CSFV-Specific Primers
Nucleic acid sequences of different CSFV were ob-
tained from GenBank and aligned with using the CLU-
STALW multiple sequence alignment programs. The
conserved fragment was chosen to be the target region,
which was used to design CSFV primers for RT-LAMP
and RT-PCR by the Primer Explorer V4 software pro-
gram and Primer3 Input (version 0.4.0) program, re-
specttively. All oligonucleotide primers were custom-
ary synthesized by Bio Basic Inc. (East Markham, On-
tario, Canada).
2.2. Viruses and Vaccines
The CSFV Bangkhen strain as reference strain, ob-
tained from Department of Livestock Development,
Ministry of Agriculture and Cooperatives, was used as
the viral nucleic acid/positive standard in the assay sys-
tem employed in this study. The other strains included
Japanese encephalitis virus (JEV), cultured at Depart-
ment of Biology, Faculty of Science, Silpakorn Univer-
sity. Hog-cholera tissue culture live vaccine (LPC-PRK
strain) was purchased from Formosa Biomedical Inc.
(Taiwan) and provided as 10 vaccine doses per vial of
which each dose contained tissue culture of Hog-cholera
virus fluid at least 103.5 RID50 (Rabbit Infectious Doses).
Porcine reproductive and respiratory syndrome virus
(PRRSV) modified live vaccine was purchased from
USA. All viruses and vaccines were identified by con-
ventional RT-PCR.
2.3. RNA Extraction
The spleen from swine infected with CSFV Bangkhen
strain was harvested and homogenized with PBS pH 7.4.
The cell pellet was centrifuged at 400 g for 10 min at
4°C. Total RNA was extracted from the suspension with
QIAmp viral RNA mini kit (Qiagen, Germany) accord-
ing to the manufacturer’s instruction. After elution, the
RNA sample was stored at 80˚C until required.
2.4. RT-PCR
The amplification 5’UTR gene of CSFV was per-
formed by using a QIAGEN OneStep RT-PCR kit (Qia-
gen, Germany) according to the manufacturer’s in-
struction. The thermal profile of RT-PCR was 50˚C for
30 min and 95˚C for 15 min, followed by 30 cycles of
94˚C for 1 min, 52˚C for 1 min, and 72˚C for 1 min and
a final extension cycle at 72˚C for 10 min. After ampli-
fication, the PCR products were stored overnight at 2˚C
8˚C until required and were analyzed by 1% of agarose
gel electrophoresis.
2.5. Recombinant Plasmid Construction
The 445-bp amplicon was purified using a QIAquick
Gel Extraction kit (Qiagen, Germany) and cloned into
K. Wongsaw at et al. / Health 3 (2011) 447-452
Copyright © 2011 SciRes. Openly accessible at http://www.scirp.org/journal/HEALTH/
the pGEM-T easy vector system I (Promega, Madison,
Wis.) according to the manufacturer’s specifications.
The recombinant plasmid was transformed into E. coli
DH5α cells followed by blue–white colony selection.
The white colonies were picked and inoculated into LB
broth and incubated at 37˚C overnight with horizontal
shaking. Plasmid DNA was extracted from the culture
with the QIAprep Spin Miniprep kit (Qiagen, Germany)
as followed manufacture’s instructions and checked for
DNA insertion by a vector-specific restriction enzyme
digestion (EcoRI and NcoI). The digested products were
UV visualized by 1% of agarose gel electrophoresis after
stained with ethidium bromide. Plasmid was diluted to
determine the sensitivity of RT-LAMP assays.
2.6. RT-LAMP Assay
The RT-LAMP reaction was carried out in a total vol-
ume of 25 μL, with mixture of 1×Thermo buffer (New
England Biolabs Inc., Beverly, MA, USA) contained 20 mM
Tris-HCl (pH 8.8), 10 mM KCl, 10 mM (NH4)2SO4, 8 mM
MgSO4 and 0.1% Triton X-100, 0.8 M betaine (Sig-
ma-Aldrich, USA), 1.0 mM dNTP mix (Promega, Mad-
ison, WI, USA), 8 U Bst DNA polymerase (large frag-
ment; New England Biolabs Inc., Beverly, MA, USA),
5 U AMV reverse transcriptase (Promega, Madison, WI,
USA), 0.2 μM each of primers CSUF3 and CSUB3,
1.6 μM each of primers CSUFIP and CSUBIP, 8 μM
each of primers ULF and ULB, 4.5 μL of template RNA.
The mixture was incubated at 65˚C for 60 min, and then
heated at 80˚C for 3 min to stop the reaction.
2.7. Monitoring of RT-LAMP Amplification
1) Analyzation by agarose gel eletrophoresis. After
amplification, the 2 μL aliquots of RT-LAMP products
were UV visualized by 1% of agarose gel electrophore-
sis after stained with ethidium bromide
2) Visualiza ti on by th e n ak ed eye. The inspection for
amplification was also performed through observations
of color change following the addition of 120 μM hy-
droxynaphthol blue (HNB) dye (Sigma-Aldrich, USA)
to the tube. The positive amplification was indicated by
a color change from violet to sky blue. Negative ampli-
fication was retained of violet.
2.8. Sensitivity of CSF V RT-LAMP Assay
The sensitivity was determined by testing serial
10-fold dilutions of a cloned target. The template over a
range of 106 to 10 copy numbers was obtained. The
RNase free water was used as the negative control. The
RT-LAMP amplification product was analyzed by aga-
rose gel eletrophoresis and naked eye.
2.9. Specificity of CSFV RT-LAMP Assay
RNA was extracted from Hog-cholera live vaccine, JE
virus and modified live PRRS vaccine with QIAmp viral
RNA mini kit (Qiagen), according to the manufacturer’s
instruction. After elution, RNA samples were used as
template in the specificity test of this assay. Hog-cholera
live vaccine was used as positive control. The RNase
free water was used as the negative control. After ampli-
fication, the RT-LAMP product was analyzed by agarose
gel eletrophoresis and naked eye.
3.1. Designing the CSFV-Specific Primers
In this study, the 5’UTR gene which showed highly
conserved regions was selected as the target sequence
for designing primers RT-LAMP and RT-PCR. The RT-
LAMP primers, a set of six primers comprising two out-
er primers were described as being forward outer primer
(CSUF3) and backward outer primer (CSUB3). The in-
ner primers were described as being forward inner pri-
mer (CSUFIP) and backward inner primer (CSUBIP).
Furthermore, two loop primers, viz, forward loop primer
(ULF) and backward loop primer (ULB) that recognized
eight distinct regions on the target sequence. The primers
were selected based on criteria described previously by
Notomi et al. [13]. The RT-PCR primer comprised of
forward (CSU1) and reverse (CSU2) primers.
3.2. RT-PCR Product
The RT-PCR product was generated by employing the
CSU1 and CSU2 primers targeting the 5’UTR gene of
CSFV. The target was amplified by using one step
RT-PCR, reverses transcription and PCR carried out se-
quentially in the same tube. After amplification, the PCR
product was analyzed by agarose gel eletrophoresis. For
positive, the RT-PCR product size was 445 bp (Figure 1).
Figure 1. The size of recombinant plasmid compared
with plasmid control after being cut with restriction
enzymes as observed from gel electrophoresis relied.
K. Wongsaw at et al. / Health 3 (2011) 447-452
Copyright © 2011 SciRes. Openly accessible at http://www.scirp.org/journal/HEALTH/
3.3. Recombinant Plasmid Construction
The white colonies were picked and detected by re-
striction enzyme digestion NcoI or EcoRI. The recombi-
nant plasmid was digested with NcoI for 1 site cutting.
The DNA pattern was shown one band at size of ap-
proximately 3460 bp (molecular weight of CSFV-5’UTR
DNA and pGEM-T easy vector were 445 bp and 3015 bp,
respectively). The recombinant plasmid was digested
with EcoRI for 2 sites cutting. The DNA patterns were
shown two bands of approximately 450 bp and 3000 bp
(5’UTR DNA and pGEM-T easy vector, respectively).
The assay detected CSFV revealed molecular weight
size was 445 bp, as observed from gel electrophoresis
(Figure 1).
3.4. Sensitivity of RT-LAMP Assay
The sensitivity of RT-LAMP assay for the detection of
CSFV was determined with serial dilutions of a recom-
binant plasmid. The template was ranging from 106 to 10
copy numbers. The assay detected at least 100 copy
numbers, showing the characteristic ladder-like pattern
in the gel (Figure 2) and visualized directly with the
naked eye by addition of hydroxynaphthol blue (HNB)
dye, showing color changes from violet to sky blue
(Figure 3). For negative control, the RNase free water
did not show either ladder-like pattern in the gel (Figure
2) or color change of HNB dye (Figure 3).
Figure 2. Sensitivity of LAMP was analyzed by agarose
gel electrophoresis. Lane M: 100 bp DNA ladder, Lane
1-6: Serial 10-fold dilutions of recombinant plasmid
106 to 10 copies/tube, respectively and Lane 7: RNase
free wat er (negative control).
Figure 3. Sensitivity of LAMP was analyzed by visual-
izetion HNB dye. Tube 1-6: Serial 10-fold dilutions of
recombinant plasmid 106 to 10 copies/tube, respectively
and Tube 7: RNase free water (negative control).
3.5. Specificity of CSFV RT-LAMP Assay
Analytical cross-reaction of CSFV RT-LAMP assay
with other pig disease viruses consisted of JEV and
PRRSV. Viral RNA was used as template in this speci-
ficity test. The cross-reactions of RT-LAMP with RNA
of JEV and PRRSV were carried out. In this study, the
RT-LAMP assay detected CSFV from a hog-cholera live
vaccine, revealing the characteristic ladder-like pattern
in the gel (Figure 4) and color changes from violet to
sky blue after the addition of HNB dye (Figure 5). No
ladder-like pattern or color changes of HNB dye was
observed for JEV, PRRSV and RNase free water. This
result revealed that the RT-LAMP assay has a high speci-
ficity for CSFV.
Classical swine fever virus (CSFV), a member of the
Pestivirus genus causes a highly contagious febrile dis-
ease worldwide. Outbreaks of CSF cause heavy losses in
pig production and severely hamper the international
trade in livestock [1]. Previous studies have been re-
ported that CSFV and PRRSV may cause disease with
similar clinical symptoms [26]. Additionally, CSFV
co-infection with PRRSV played an important role in
Figure 4. Specificity of RT-LAMP products
visualized with agarose gel electrophoresis.
Lane M: 100 bp DNA ladder, Lane 1: CSFV,
Lane 2: JEV, Lane 3: PRRSV and Lane 4:
RNase free water.
Figure 5. Specificity of RT-LAMP products
visualized with HNB dye. Tube 1: CSFV, Tube
2: JEV, Tube 3: PRRSV and Tube 4: RNase
free water.
K. Wongsaw at et al. / Health 3 (2011) 447-452
Copyright © 2011 SciRes. Openly accessible at http://www.scirp.org/journal/HEALTH/
reproducing typical post weaning multisystemic wasting
syndrome [27]. Taxonomically, JEV belongs to the same
Flaviviridae family as CSFV [20]. Therefore, the devel-
opment of a simple and rapid diagnostic tool that can
detect CSFV and differentiae it from PRRSV in the same
samples would be of significant importance in the epi-
demiologic surveillance and the prediction of severity of
economically important viral diseases in swine herds.
It is very important to find a conserved nucleic acid
fragment to design specific RT-LAMP primers. In this
study, the nucleic acid sequence of 33 CSFV in Gen-
Bank was analyzed with the CLUSTALW multiple se-
quence alignment programs. The most conserved of 445
bp was found in the 5’ untranslated gene which appeared
highly conserved. The CSFV primers for RT-LAMP and
RT-PCR targeting the conserved sequence of 5’ untrans-
lated genes were designed successfully with the Primer
Explorer V4 software program and Primer3 Input (ver-
sion 0.4.0) program, respectively. The RT-LAMP assay
is a simple diagnostic tool in which the reaction is car-
ried out in a single tube by a mixing of the buffer, prim-
ers, reverse transcriptase, and DNA polymerase, and
incubating the mixture at 65˚C for 60 min. Besides, the
higher amplification efficiency of the RT-LAMP reaction
yields a large amount of a by-product, pyrophosphate
ion, leading to white precipitate of magnesium pyro-
phosphate in the reaction mixture. Since the increase in
the turbidity of the reaction mixture according to the
production of precipitate correlates with the amount of
the DNA synthesized, monitoring of the RT-LAMP reac-
tion can be achieved with the naked eye or in a real-time
with a turbidimeter [14]. Another useful feature was that
RT-LAMP products could be directly observed by the
addition of hydroxynaphthol blue (HNB) dye to the am-
plified products. Therefore, this technique is effective
due to the high specificity and amplification efficiency,
and may facilitate the application of RT-LAMP, espe-
cially in the field.
The results revealed that RT-LAMP assay detected at
least 100 copy numbers of recombinant plasmid. Fur-
thermore, the specificity of CSFV RT-LAMP did not
show cross-reactivity with JEV and PRRSV, suggesting
that this method is highly specific among the viral
strains used in this study. The higher sensitivity and spe-
cificity of the RT-LAMP reaction were attributed to its
continuous amplification under the isothermal condi-
tions employing six primers that recognized eight dis-
tinct regions of the target.
Our studies presented the developed RT-LAMP assay
is an extremely rapid, cost-effective, sensitive, and spe-
cific method for the detection of CSFV RNA. The me-
thod requires only simple conditions and less time to
obtain a result using the HNB dye, compared with the
traditional gel electrophoresis. Therefore, the assay is
more suitable for use under field conditions for rapid
diagnosis of CSFV, which would allow emergency con-
trol measures to be implemented to prevent spread of
We are thank Department of Biology Faculty of Sci-
ence Silpakorn University and Department of Immunol-
ogy Faculty of Medicine Siriraj Hospital Mahidol Uni-
versity, Omnoisophonchanupathom school, The Institute
for the Promotion of Teaching Science and Technology,
Thailand Research Fund (TRF no. DIG 5180004), Par-
tial fund from MRG-WII525S092 and RGP 2552-06 for
the financial supports, Department of Livestock Devel-
opment, Ministry of Agriculture and Cooperatives for
CSFV Bangkhen strain. We also thank to Dr. Pahol Ko-
siyachinda for reviewing the manuscript, Miss Siri-
porn Kaewklom for photographing.
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