ARTICLE

Utilization of loop-mediated isothermal

amplification (LAMP) technology for
detecting White Spot Syndrome Virus
(WSSV) and Vibrio spp. in Litopenaeus
vannamei in selected sites in the
Philippines
Amalea Dulcene D. Nicolasora1, Benedict A. Maralit4, Christopher
Marlowe A. Caipang3, Mudjekeewis D. Santos4, Adelaida Calpe5, and
Mary Beth B. Maningas*1,2
1

Research Center for the Natural and Applied Sciences, Thomas Aquinas Research Complex
Biological Sciences Department, College of Science, University of Santo Tomas, Philippines 1008
3
School of Applied Science, Temasek Polytechnic, Singapore
4
Genetic Fingerprinting Laboratory, National Fisheries Research and Development Institute,
101 Mother Ignacia St. Quezon City 1103 Philippines
5
Philippine Council for Agriculture, Aquatic, and Natural Resources
Research and Development - Department of Science and Technology, Los Banos, Laguna
2

hrimp disease outbreaks in the Philippines remain to

be uncontrollable. This is compounded by the inaccessibility of disease diagnostics to most shrimp
farmers. The loop-mediated isothermal amplification
(LAMP) is a new technology that is used as a practical alternative for rapid detection of viral and bacterial pathogens. The method proves to be rapid, highly sensitive, and costeffective compared to other detection assays. In this study,
LAMP protocols for the detection of the two most common
shrimp pathogens, white spot syndrome virus (WSSV) and Vibrio spp., in the Philippines were developed. A temperature
range of 55C to 68C for WSSV detection and 59C to 67C for
Vibrio spp., and incubation periods of 45 minutes to 1 hour, were
proven to be the suitable conditions for the LAMP assay. Using
*Corresponding author
Email Address: marybethmaningas@yahoo.com
Submitted: August 12, 2013
Revised: June 30, 2014
Accepted: July 4, 2014
Published: September 10, 2014
Editor-in-charge: Eduardo A. Padlan
Vol. 7 | No. 2 | 2014

these conditions, asymptomatic Litopenaeus vannamei samples

from selected sites (Iloilo, Batangas, Bulacan, Laoag, and Leyte)
were tested for WSSV. Samples which indicated WSSV infection were from Iloilo (89.47%), Batangas (30.00%), Bulacan
(43.33%), and Leyte (75.00%), while shrimps from Laoag City
(0.00%) tested negative. Likewise, the occurrence of Vibrio spp.
was determined in shrimps sampled in Pangasinan and six bacterial DNA isolates of Vibrio spp. were identified. Moreover, conventional PCR and microbiological methods were performed
along with the LAMP reaction for comparison and further confirmation. The results showed that the LAMP assay was faster
and 10 times more sensitive than polymerase chain reaction in
detecting WSSV and was more efficient than the traditional
microbiological method in diagnosing vibriosis. Overall, the
results indicated that a LAMP protocol, which is more convenient, highly sensitive, faster, and more practical, has been effectively utilized to detect WSSV and vibriosis in selected Philippine shrimp farms.
KEYWORDS
loop-mediated isothermal amplification, polymerase chain reaction, white spot syndrome virus, Vibrio spp., shrimps

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INTRODUCTION
Shrimp is one of the most popular seafood in the world.
Annual global production of shrimps reaches up to 4 million
metric tons, one-third of which comes from the shrimp culture
industry (FAO 2010). In most aquaculture-producing countries
in Asia, shrimp is considered an economic asset (Caipang and
Aguana 2011). The prevalence of shrimp pathogens, however,
has contributed to a significant loss in production.
In the Philippines, farmed shrimps have great potential for
global economic profit. The country was a top shrimp exporter in
the early 1990s, earning approximately US$ 300,000,000 during
its peak. Similar to the global conditions, disease problems in the
late 1990s caused a considerable decline in the countrys shrimp
output (FAO 2006) and until now the industry is still trying ways
to recover its great loss.
Diseases due to luminous Vibrio spp. and the white spot
syndrome virus (WSSV) are regarded as the two major causes of
decline in shrimp production. However, established protocols for
diagnosis and preventive measures for infection that are adapted
to the Philippine setting are still lacking. Early disease diagnosis
must be carried out so that immediate mitigating measures could
be undertaken to prevent massive losses due to heavy mortality.
The development of new methods for early pathogen detection
can be an effective means of prevention. Since small-scale farms
constitute a big bulk of the Philippine shrimp industry, a lowcost, rapid, and simple on-site disease diagnostic protocol needs
to be established.
To date, various molecular techniques have been developed
to detect viral and bacterial pathogens. Conventional polymerase
chain reaction (PCR) and reverse transcription polymerase chain
reaction (RT-PCR) are the most popular and widely used methods. However, several disadvantages, such as the need for an
expensive thermal cycler, insufficient specificity, and low amplification are observed for these techniques (Mori et al. 2001).
Cost effectiveness and practicality are the major concerns in
creating protocols that can improve the quality of shrimp products. In the Philippines, methods for prevention and early detection of pathogens in shrimp aquaculture have already been designed. PCR (Tapay et al. 1999, Maralit et al. 2011, Caipang and
Aguana 2011, Alenton and Maningas 2011), multiplex PCR
(Castroverde et al. 2006), pathogenicity test, histopathology, and
traditional microbiological techniques (Lavilla-Pitogo et al.
1998, de la Pea et al. 2007) are the commonly used methods for
disease diagnosis in shrimp in the country. However, expensive
and sophisticated equipment are required for the implementation
of such techniques. They are laborious and difficult to apply
during on-site testing. In addition, these procedures are quite
inaccessible to a large number of small-scale shrimp farmers
because they do not possess the financial capabilities that are
required for such assays.
Notomi et al. (2000) developed a technology called loopmediated isothermal amplification (LAMP) that can detect a
wide array of pathogens. This assay allows amplification of nu310

cleic acid with high specificity under isothermal conditions. It

can amplify target sequences up to 10 9 copies at 60C to 65C
incubation in an hour or less. It relies on the strand displacement
activity of Bst polymerase and a set of four specially designed
primers that recognize six independent target sequences (Notomi
et al. 2000). LAMP reaction proceeds at isothermal conditions
that can be performed using only a water bath or heater block to
maintain the required temperature, essentially eliminating the
need for an expensive thermocycler, making it a practical and
low-cost diagnostic tool.
Given that shrimp aquaculture is faced with the constant
threat of bacterial and viral pathogens, an early detection technique with the possibility of on-site application is necessary.
LAMP is a potential molecular-diseases diagnostic tool for
shrimp farms in the Philippines, since it is both effective and
practical. Thus, this study aims to develop a LAMP protocol that
will be beneficial, low-cost, and accessible for on-site application in most shrimp farms. This work makes use of the set of
primers designed by Maralit et al. (2012) and Xu et al. (2012)
which were tested at a wider temperature range. The LAMP
primers were utilized for WSSV and Vibrio spp. detection on
selected sites in the Philippines.
METHODOLOGY
Shrimp Collection
Litopenaeus vannamei were purchased from wet markets
and collected from shrimp farms at randomly selected sites in the
Philippines. The shrimp farms and the origin of the shrimps
bought from wet markets are identified in Table 1. For the following sites (Iloilo, Batangas, Bulacan, and Leyte), 15 shrimps
were collected for WSSV detection and for Bolinao, Pangasinan,
15 shrimps were tested for Vibrio infection. For each site, 2
shrimps were separated for species identification. Samples were
stored in an ice bucket and were transported to the Thomas
Aquinas Research Complex of the University of Santo Tomas.
Sampled shrimps were stored in a -80C freezer until the commencement of the experiment.
Table 1. Sampling Sites of the Study
Site
Batangas
Nasugbu
Calatagan

Date

Sample

October
November

2011
2012

Farm sample
Market sample

Hagonoy, Bulacan

October

2012

Farm sample and

Market sample

San Jose, Leyte

February

2012

Market sample

Iloilo

August

2012

Farm sample
Farm sample
Farm sample

Laoag City, Ilocos Sur

December

2012

Market sample

Bolinao, Pangasinan

February

2013

Market Sample

Barkatok
Dagat
Roxas

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Vol. 7 | No. 2 | 2014

Table 2. LAMP Primers

Primer Name
WSSV
C-FIP
C-BIP
C-F3
C-B3
WSSV-FIP
WSSV-BIP
WSSV-F3
WSSV-B3
Vibrio
Vibrio-FIP
Vibrio-BIP
Vibrio-F3
Vibrio-B3

Sequence (5 3)
ACC CAA TGT ATG TGA CCA GCC -TTTT- GGA GGA GGT ACA TCC ACT
ACA CTG GGT ACA GAT CAG GGA A -TTTT- ATT CAG ACC GCC CGT TAA
GAG GAG GGT ACG GCA ATA
CAA GGA TTC AAA ATT TAC TGT GG

Maralit et al.
(2012)

GGG TCG TCG AAT GTT GCC CA -TTTT- GCC TAC GCA CCA ATC TGT G
AAA GGA CAA TCC CTC TCC TGC G -TTTT- AGA ACG GAA GAA ACT GCC TT
ACG GAG GAC CCA AAT CGA ACG GAG GAC CCA AAT CGA
GCC TCT GCA ACA TCC TTT CC

Kono et al.
(2004)

CGG CTG CTG GCA CGG AGT - TTTT-GCA TTA TTT GAC GTT AGC GAC AGA AG
GAG CGT TAA TCG GAA TTA CTG GGC -TTTT- CCG GGC TTT CAC ATC TGA CTT AAC
CAG TCG TGA GGA AGG TGG TGT
CTA GTC TGC CAG TTT CAA ATG CT

DNA Extraction for WSSV Detection

Tissue samples from gills, muscles, and intestines of 15
shrimps were dissected and stored separately in 1.5 mL sterile
microcentrifuge tubes with appropriate labels. DNA was extracted from tissue samples using a Wizard Genomic DNA Purification Kit (Promega) following the manufacturers protocol.
The DNA extracts were used for LAMP and PCR detection.
Bacterial DNA Isolation for Vibrio spp.
Fifteen shrimp samples were subjected to bacterial colony
isolation. Dissected intestines of each shrimp were suspended in
a 500 L 0.9% natural sodium saline solution followed by homogenization. The lysate was streaked on a Thiosulfate Citrate
Bile Salt (TCBS) agar plate and incubated for 24 to 48 hours at
25C until bacterial growth was observed.
Suspected Vibrio spp. colonies were subcultured and purified using MacConkey agar plates and incubated for 24 to 48
hour at 25C. Pure isolates were cultured in 8 ml Tryptic Soy
Broth media for 18 24 hours at 25C and DNA extraction was
conducted using a Wizard Genomic DNA Purification Kit
(Promega) following the manufacturers protocol. Isolated DNA
was subjected to PCR and LAMP detection using published Vibrio spp. primers and 16s rRNA bacterial primers.
Molecular Analysis
LAMP Detection
The methodology that was used for LAMP detection was adapted from Maralit et al. (2012) with minor
modifications. LAMP assay was carried out on a total of 25
L reaction volume containing 2.0 pmol of each published FIP and BIP inner primers (see Table 2), 0.2 pmol of -F3
and -B3 outer primers (see Table 2), 12.5 L of 2X LAMP
reaction buffer (40mM TrisHCl, 20mM KCl, 16mM
MgSO4, 20mM (NH4)2SO4, 0.1% Triton-X, 1.6M Betaine
and 0.25 mM dNTPs each), 1 L of target DNA, and 2.3 L
of distilled water. The mixture was initially incubated at 95
C for 5 minutes and then chilled on ice for 2 minutes. Subsequently, 0.3 U/ml of Bst DNA polymerase was added. The
mixture was then incubated for 60 minutes at 55C to 68C
for WSSV and 59C to 67C for Vibrio spp.; the reaction
was terminated at 80C for 10 minutes. Positive controls
used were WSSV DNA template for viral detection and Vibrio vulnificus DNA isolate for bacterial detection. Reaction
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Sources

Xu et al.
(2012)

mixtures without DNA template and non-Vibrio isolates (for

Vibrio spp. detection) were used as negative controls.
LAMP products were centrifuged briefly in a tabletop spinner and were run on a 2% agarose gel electrophoresis for
confirmation. Further visualization was done by the addition
of 4 L of Sybr Safe stain (diluted 1000 times) to the LAMP
products and illumination under UV light for color intensity
observation.
PCR Detection
Detection with PCR was carried out with a 10 L
reaction mixture containing 1 unit of Taq polymerase, 0.6
M of each published forward and reverse primers, 0.2 M
dNTP solution mix, 1 L 10x buffer with 20 mM MgCl2, and
1 L of DNA extract. Positive controls used were WSSV
DNA template and Vibrio vulnificus DNA isolate, while a
reaction mixture without any DNA template was used as a
negative control. Thermal cycling conditions were: initial
denaturation temperature of 95C for 5 minutes, followed by
30 cycles of denaturation temperature of 95C for 30 seconds, annealing at 55C for 30 seconds, and extension at 72
C for 30 seconds. Final DNA extension was at 72C for 10
minutes, then 4C was set as the holding temperature for
storage. PCR products were viewed using agarose gel electrophoresis at 1% gel concentration. The primers used for
PCR detection of WSSV and Vibrio spp. are shown in Table
3.
Table 3. PCR Primers
Primer Name

Sequence (5 3)

WSSV
WVF
WVR

GTA CGG CAA TAC TGG AGG AGG

GGA GAT GTG TAA GAT GGA CAA GG

Vibrio
Vibrio-F3
Vibrio-B3

CAG TCG TGA GGA AGG TGG TGT

CTA GTC TGC CAG TTT CAA ATG CT

16s
16s-F (27F)
16s-R (1492R)

AGA GTT TGA TCM TGG CTC AG

ACC TTG TTA CGA CTT

Amplicon size/
Source
200 bp
Maralit et al.
(2011)
200 bp
Xu et al. (2012)
1500 bp
Lane et al. (1991)
Universal primers

Agarose Gel Electrophoresis

Agarose gel electrophoresis was carried out for the
LAMP reaction by using 2% agarose gel with 3L ethidium
bromide (per 100 ml) for staining. Five microliters of

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LAMP products with 2 mL of 6X loading dye were loaded

in each well. For the PCR detection, 1% agarose gel stained
with 3 L ethidium bromide loaded with 3 L PCR products
and 1 L 6x loading dye was used. 1X TAE served as the
running buffer and 25 minutes was the running time for
LAMP products, while 20 minutes running time was used
for PCR products.
Data Interpretation and Calculations
The percentage of occurrence of WSSV and Vibrio spp. in
the shrimps collected from the selected sites was evaluated as the
number of shrimps found positive for WSSV or Vibrio spp., divided by the total number of shrimps collected, multiplied by
100.
RESULTS AND DISCUSSION
LAMP Primer Selection and Optimization
To establish a LAMP protocol for the WSSV and Vibrio
spp. detections, primers that produced positive LAMP reactions
using WSSV and Vibrio spp. isolates from the Philippines were
selected and optimized at different temperature conditions.

Figure 1. LAMP detection at 65C for 1 hour incubation period.

WSSV positive templates were utilized. Primer set C (Maralit et
al. 2012) and WSSV primer (Kono et. al. 2004) were being tested
for primer selection for WSSV detection.

For WSSV detection, LAMP primers by Kono et al. (2004)

and Maralit et al. (2012) were utilized (see Fig. 1). The primer
set C of Maralit et al. (2012) yielded better results at 65C incubation for 60 minutes. However, the published primers of Kono
et al. (2004) gave false positive results in the LAMP assay due to
the appearance of ladder-like patterns in the negative control.
This finding indicates the tendency of the primers to interact
with one another without the presence of a DNA template. Thus,
the primer set C designed by Maralit et al. (2012) was chosen to
be utilized for LAMP detection of WSSV on L. vannamei samples from the selected sites in the Philippines.

The LAMP reaction was carried out at 55C, 63C, and

68C for 60 minutes using primer set C and WSSV DNA templates in order to determine the optimal temperature (Fig. 2).
LAMP products were formed under those three conditions. The
best result was obtained at 65C incubation for 60 minutes (see
Fig. 1).
Reference sequences on which the primer design was based
were also considered in the selection of primers. The WSSV
sequence generated from the work of van Hulten et al. (2001)
was the only reference sequence of Kono et al. (2004). However,
primer set C was designed by Maralit et al. (2012) using PCR
primers based on the conserved sequence of WSSV isolates from
China, Taiwan, and Thailand, which also showed homology to
WSSV DNA fragments sequenced from the Philippine isolate.
This study validates the study made by Maralit et al. (2012) and
shows that primer set C is the most suitable set of primers for
WSSV LAMP detection in the Philippine setting.
Published LAMP and PCR primers for identification of Vibrio spp. were mostly specific only to a certain strain. A universal primer for the genus has not been established, although the
recent work of Xu et al. (2012) suggested a universal LAMP
primer for pathogenic Vibrio spp. The LAMP primer set used in
that study was designed from conserved regions of 16s rRNA
specific to genus Vibrio and was determined to be specific to
pathogenic Vibrio spp. The LAMP primer set was tested on 30
strains of nine Vibrio species and 21 related non-Vibrio microorganism strains as controls. A positive LAMP reaction was observed in the 30 pathogenic Vibrio strains, while no crossreaction was found on the control organisms, suggesting the
specificity of the primers only to pathogenic Vibrio spp. (Xu et
al. 2012). That led us to select and test those primers for the
study of the suspected Vibrio bacterial DNA isolates.
Using Vibrio vulnificus DNA as positive control, the optimum temperature of the selected primer was determined using
the range of 55C to 75C conditions. As shown in Figure 3, the
laddering pattern indicated a positive LAMP reaction. Products
were generated at 59C, 63C, and 67C and no product was
formed at 55C, 57C, 71C, 73.5C, and 75C (see Fig. 3).
Thus LAMP detection was determined to be viable only in the
59C to 67C temperature set-ups.

Figure 3. Optimization of Vibrio spp. to varying temperature conditions. M - marker, A - 75C, B - 73.5C, C - 71C, D - 67.2C, E
- 62.5C, F - 58.9C, G - 56.5C, H - 55C, (-) - negative control.

Figure 2. Optimization of primer set C at varying temperature

conditions (55C, 63C, and 68C) using WSSV DNA templates.
312

Specificity was evaluated using different bacterial DNA

isolates and the findings in Figure 4 confirmed that the published
Vibrio spp. primer was specific only to Vibrio species (Fig. 4,F).
No bands or ladder-like patterns were produced on non-Vibrio
bacterial DNA isolates.

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WSSV Detection
To determine the sensitivity of LAMP compared to the conventional PCR method, WSSV detection using both methods
was performed. Positive WSSV DNA template was serially diluted ten-fold. Results showed that LAMP, which detected the
virus at 10-2 dilution with the concentration of 8.2 ng/l, is ten
(10) times more sensitive than conventional PCR, which was
able to produce a band only down to 10 -1 dilution with a concentration of 84.6 ng/ml (see Fig. 5). Sybr Safe staining indicated
that positive samples have greater intensity and were seen
brighter than the negative control (see Fig. 6).

Figure 4. Primer specificity. LAMP primer set by Xu et al. (2012)

specific to pathogenic Vibrio spp. was tested for specificity using
non-Vibrio bacterial DNA isolates and Vibirio vulnificus DNA isolates. M - marker; A, B, C, D, E, and G non-Vibrio Bacterial DNA;
F - Vibrio vulnificus DNA, H - negative control.

Temperature conditions wherein the selected primers were

found to yield positive LAMP reactions were determined. Since
the LAMP assay works under isothermal conditions, varying
temperatures for incubation were utilized. The tested temperature range (55C - 68C) for the WSSV LAMP assay was found
to be lower by five degrees and higher by 3 degrees than the
published protocols of Notomi et al. (2000), Kono et al. (2004),
and Maralit et al. (2012), which were limited only to 60C 65C incubation for an hour or less. Our results indicate that the
formation of LAMP products is possible at a temperature lower
than 60C. However, the best product to be amplified was
formed at 65C and 60 minutes incubation, which are the suggested and established optimum parameters for WSSV LAMP
detection (Maralit et al. 2012). According to Notomi et al.
(2000), specificity increases at higher temperatures and that well
-formed bands likewise indicate optimum conditions.
Our optimization of Vibrio spp. primers produced results
comparable to those observed in the study of Xu et al. (2012),
where LAMP products were formed at 58C to 66C; and 62C
for 60 minutes was the optimum condition. An almost similar
viable temperature range of 59C to 67C, and an optimum condition of 63C for 60 minutes, were obtained in our study.
Figure 5. Comparison
of LAMP and PCR
detection using
ten-fold serial dilution
of positive WSSV
DNA templates.

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Figure 6. Sybr Safe staining of LAMP products for WSSV detection. Confirmation of WSSV positive samples by the addition of
Sybr Safe stain on LAMP products and visualized under UV illuminator. 1, 3, and 5 WSSV positive samples, 4 and 2 negative control.

This result coincides with the findings of Yano et al. (2007)

and Wang et al. (2008) for detection of E. coli and Salmonella.
Also, Kono et al. (2004) showed that the LAMP assay detected
WSSV DNA templates as low as 1 fg concentration, which is ten
times more sensitive than nested PCR whose detection limit is
10 fg. Thus, LAMP has been proven to have high amplification
efficiency, making it highly sensitive. Since, the reaction works
under isothermal conditions, time loss during thermal change
does not occur (Ren et al. 2008). Moreover, the high specificity
of LAMP can also be attributed to its use of a set of four primers
which targets six distinct regions in the DNA template, while
PCR only targets a single region in the gene of interest (Notomi
et al. 2000).
WSSV LAMP detection was conducted on shrimps from the
selected regions of the Philippines. Asymptomatic L. vannamei
samples collected from the wet markets of various sampling sites
indicated positivity to WSSV infection. Shrimps from Iloilo
(89.47%), Batangas (30.00%), Bulacan (43.33%), and Leyte
(75.00%) were found to be WSSV-infected, while shrimps from
Laoag City tested negative for WSSV. A recent record of occurrence of WSSV is from the study of Maralit et al. (2011) where

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Figure 9. PCR detection results of Vibrio spp. using the five

suspected Vibrio bacterial DNA isolates. 200 bp amplicons were
produced for isolates identified as genus Vibrio spp. M marker
and V1 V5 suspected Vibrio bacterial DNA isolates.

PCR was also conducted on suspected Vibrio spp. isolates

using specific Vibrio primers, and a 200 bp amplicon was amplified from the samples (see Fig. 9).
Figure 7. Prevalence of WSSV among selected sites in the
Philippines. Litopenaeus vannamei from five randomly selected
regions (Leyte, Iloilo, Batangas, Bulacan, and Laoag City) in the
Philippines were tested for the presence or absence of WSSV
using LAMP assay.

PCR was the method used for detection. In that study, the same
species of shrimp (L. vannamei) was used and two sites were
confirmed to be WSSV-positive, namely Zambales and General
Santos City, while two other sites, Batangas and Capiz, were
found to be negative. In our study, results verified one region as
WSSV-free (Laoag City), three new regions as WSSV-infected
(Iloilo, Bulacan, and Leyte); those three and the Batangas region,
which was previously shown to be WSSV-free, are now added to
the list of infected areas.
Vibrio spp. Detection
To assess the feasibility of LAMP as a practical tool for
Vibrio spp. detection, it was compared to the conventional methods for bacterial detection. The TCBS (Fig. 8,B) agar plates utilized in the experiment, likewise, indicated growth of suspected
Vibrio spp. colonies, which were observed as yellow-, green-, or
blue- to green-centered colonies. The Triple Sugar Iron Agar
(TSIA) biochemical test and API NE kit (bioMerieux, Inc.) identification were conducted for bacterial characterization, wherein
24 to 48 hours incubation was needed to produce results. As seen
in Figure 8, the identification of bacteria using standard microbiological methods took 2-3 days or more.

Specific primers for Vibrio spp. designed from the 16s

rRNA gene conservative sequence of genus Vibrio were used for
LAMP detection. The LAMP assay was carried out on the suspected Vibrio isolates and ladder-like patterns were observed,
which confirmed that the samples belong to the genus Vibrio
(see Fig. 10). The LAMP reaction was conducted using the optimized condition of 60C to 63C for 60 minutes. Sybr Safe stain
was added to the LAMP products and positive samples showed
more intense color under UV illumination.

Figure 10. (Left) LAMP detection using six suspected Vibrio spp.
isolates and (Right) Sybr Safe staining of LAMP products for Vibrio spp. detection. M-marker, V1 V6 suspected Vibrio isolates,
and (-) negative control.

The detection of Vibrio spp. is commonly performed by

isolation on a selective agar medium followed by biochemical
and serological testing (Harwood et al. 2004), which is arduous,
time-consuming, and requires more than three days to finish. In
contrast, LAMP offers a cost-effective and faster bacterial identification using primers designed using conserved regions of 16s
rRNA specific to genus Vibrio (Xu et al. 2012).
Both LAMP and PCR were performed on suspected bacterial isolates from Pangasinan and six isolates were confirmed as
Vibrio spp. genera based on the two methods. The performance
of LAMP and PCR on bacterial DNA isolates suggests that biochemical and serological testing for bacterial detection could be
replaced by LAMP and PCR methods. Nonetheless, the requirement of thermal cycling equipment would discourage the use of
PCR also as a suggested method for a low-cost diagnostic tool.

Figure 8. Standard micriobiological techniques for Vibrio spp.

bacterial identification was performed simultaneously with molecular analysis. A TSIA Biochemical test, B - TCBS selective
media, C - API NE kit.
314

LAMP Product Visualization

Two methods of Sybr Safe staining were done in the study.
In Figure 6, the leftmost image was obtained when the staining
was done simultaneously with the incubation. In this process, the
stain was added in the LAMP reaction mixture before incubation
and the results showed that positive LAMP products were indicated by the turbidity of the mixture, while the negative control
was observed as a clear solution. Moreover, tubes were viewed

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Figure 11. Confirmation of positive LAMP products through staining, (leftmost viral detection, middle bacterial detection) and precipitate observation, (rightmost). (+) positive control w/ WSSV DNA template, (-) negative control w/o DNA template.

only under white light, which suggests a very practical technique

for confirmation of LAMP products. The middle image in Figure
6 was from the established process of staining, which is the addition of the Sybr Safe stain after the LAMP reaction. The positive
LAMP products by this process exhibited a more intense and
brighter image under UV illumination compared to the negative
control. Further, shown in the rightmost figure is the appearance
of precipitate, which was observed in LAMP products with
higher DNA yield.
SUMMARY AND CONCLUSION
In conclusion, the results presented in this study suggest that
LAMP can be used in the Philippine setting as a practical alternative to conventional PCR and standard microbiological techniques. The temperature range of 55C to 68C for WSSV detection and 59C to 67 for Vibrio spp., and incubation periods of
60 minutes to 1 hour, were proven to be viable conditions for the
LAMP assay, wherein all reactions were incubated initially at
95C for 5 minutes and terminated at 80C for 10 minutes.
The LAMP assay was able to detect WSSV from L. vannamei collected from the four selected sites (Leyte - 75.00%,
Batangas - 30.00%, Iloilo - 89.47%, and Bulacan - 43.33%),
while samples collected from Laoag City, Ilocos Sur, tested
negative. However, six bacterial isolates were confirmed as Vibrio spp. from the shrimps sampled from Bolinao, Pangasinan.
Conventional PCR and microbiological methods were performed along with LAMP for comparison and further confirmation. WSSV detection using the LAMP assay was 10 times more
sensitive than PCR, and bacterial identification of Vibrio spp.
through the LAMP reaction was less time-consuming than the
traditional microbiological methods. Visualization of LAMP
products using Sybr Safe stain also offers an advantage, since it
could eliminate the commonly used agarose gel electrophoresis
which is somewhat impractical. Thus, our results suggest that a
LAMP protocol is more convenient, highly sensitive, and more
rapid than the established methods for disease diagnostics. Moreover, the system is cheaper and practical since it does not require
an expensive PCR machine, which can benefit the small-scale
shrimp industry.
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The optimum LAMP conditions that were determined and

tested in this study could be readily applied, or adapted, on-site.
Testing the foregoing LAMP protocols for pathogen detection on
small-scale shrimp farms is a primary step in the adoption of
LAMP technology in the Philippines.
ACKNOWLEDGEMENTS
This research was supported in part by the project,
Biotechnology for Shrimp: Utilization of Molecular Technologies to Elucidate Shrimp Immunity and Develop Disease Diagnostics, funded by the Department of Science and Technology.
Special thanks to the Thomas Aquinas Research Complex, University of Santo Tomas, for housing the project. Special thanks
also to the laboratory technician Charles Gerald Ganal, and project staff members Erica M. Ocampo, Patrick Ellis Go, Ricardo
S. Balog, Paul Rodrigo F. Cordero, Donna May de la Cruz-Papa,
Research Assistants of the NFRDI - Genetic Fingerprinting
Laboratory (GFL), and the graduate students of The UST Graduate School, Ma. Sheila de Jesus, Reuben Jerome Atayde, Ritchie
Gorospe, Irma Dabu, and Vivian Villegas, who have helped in
this study.
CONFLICTS OF INTEREST
There is no conflict of interest among authors, institutions
and individuals mentioned above in the conduct of this study and
the preparation and submission of this manuscript.
CONTRIBUTIONS OF INDIVIDUAL AUTHORS
Dr. Mary Beth B. Maningas conceived and designed the
experiments. Amalea Dulcene D. Nicolasora performed the experiments. Both worked together on the data analysis and writing of the manuscript. Dr. Maningas and Benedict A. Maralit
designed and developed the primers. Dr. Mudjekeewis D. Santos
and Dr. Christopher Marlowe A. Caipang provided technical
inputs as well as laboratory resources, and Dr. Adelaida Calpe
served as a consultant for the project.

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Philippine Science Letters

Vol. 7 | No. 1 | 2014