Biotechnological

Communication

Biosci. Biotech. Res. Comm. 9(3): 349-356 (2016)

Recent advancements in molecular detection of

Vibrio

species in aquatic animals: A review

N.Z. Amalina

and M.Y. Ina-Salwany

1,2

Laboratory of Marine Biotechnology, Institute of Bioscience, Universiti Putra Malaysia, 43400 Serdang,

Selangor, Malaysia.

*Department of Aquaculture, Faculty of Agriculture, Universiti Putra Malaysia, 43400 Serdang,

Selangor, Malaysia

ABSTRACT

Vibriosis is one of a major problem in aquaculture sector causes by Vibrio species and gives bad impact on economy

and social development. In addition, number of the infected people with vibriosis is increasing every year through

consuming of contaminated  shes and other aquatic animals. To overcome this problem, researchers continuously

develop new methods for identi cation and detection of Vibrio species. Those methods can be categorized based on

the principle applied; conventional culture method, antigen-antibody based assay, nucleic acid ampli cation tech-

nology and lateral  ow dipstick. The conventional culture method is a basis in identi cation of Vibrio but it is time

consuming and requires skilled personnel. The nucleic acid ampli cation technology such as isothermal ampli cation

assay in a combination of lateral  ow dipstick is widely used since these assays offer rapid, easy to handle and high

sensitivity and speci city detection methods. This paper also reviews and describes the available application and

limitations of the studies involving Vibrio detection methods of aquaculture  eld. As a conclusion, the development

of new technologies is very important to improve the detection of Vibrio species as well as increasing the number of

food production to meet human demands.

KEY WORDS: ISOTHERMAL AMPLIFICATION, LAMP, LATERAL FLOW DIPSTICK, NUCLEIC ACID AMPLIFICATION, PCR,

VIBRIO

349

ARTICLE INFORMATION:

*Corresponding Author: salwany@upm.edu.my

Received 18

Aug, 2016

Accepted after revision 26

Sep, 2016

BBRC Print ISSN: 0974-6455

Online ISSN: 2321-4007

Thomson Reuters ISI ESC and Crossref Indexed Journal

NAAS Journal Score 2015: 3.48 Cosmos IF : 4.006

reserved.

Online Contents Available at: http//www.bbrc.in/

INTRODUCTION

Nowadays, aquaculture becomes an important food pro-

duction sector especially from  shes, molluscs, crusta-

ceans and other aquatic animals. According to FAO (2013),

128 million tons of  shes, molluscs and crustaceans were

provided for human food. The number of food produc-

tion will be increasing every year due to high demand for

food. In addition, aquaculture is a signi cant socio-eco-

nomic activity through employment, income generation,

350 MOLECULAR DETECTION FOR VIBRIOSIS BIOSCIENCE BIOTECHNOLOGY RESEARCH COMMUNICATIONS

Amalina and Ina-Salwany

domestic and international trade, and provide livelihoods

especially for rural communities (Edward et al., 2002).

Unfortunately, the diseases problem of aquaculture sec-

tor is increasing every years caused by bacteria, viruses,

fungi, parasites and other emerging pathogens (Adams

and Thompson, 2011). This problem also effects the eco-

nomic and social development worldwide.

Vibriosis is one of a major problem in aquaculture sector

and caused outbreaks of aquatic animal diseases. It leads to

signi cant mortality in aquatic animals, loss of food and

severe economic losses (Buller, 2014; Gauthier, 2015).

Besides, this bacterium can infect human through con-

sumption of raw or undercooked  sh and other aquatic

animals. Outbreaks of vibriosis have been reported world-

wide caused by Vibrio alginolyticus, V. parahaemolyticus,

V. harveyi, V. anguillarum, V. vulni cus and other Vibrio

species in various countries, including United State, Can-

ada, China, Taiwan, India and Japan (Chiou et al., 2000;

Alam et al., 2003; Deepanjali et al., 2005; Xie et al., 2005;

CDC, 2013). Over 80 000 cases and 100 deaths caused by

vibriosis were reported in United State every year (Scal-

lan et al., 2011). According to CDC, the number of cases in

U.S. was increasing 75% in 2013 compared to 2006 until

2008 with majority of cases caused by V. parahaemolyti-

cus and V. vulni cus (FoodNet, 2014).

Vibrio is a gram negative bacterium from family

Vibrionaceae, rod shape and undergoes facultative anaer-

obic respiration. Naturally Vibrio found in estuarine,

marine environments, normal  ora of organisms and also

can be pathogenic (Wang et al., 2009; Haldar et al., 2011;

Chatterjee and Haldar, 2012). Usually, there are no speci c

symptoms of infection of vibriosis observed in marine

life. The symptoms are similar with other bacteria dis-

eases starting with lethargy and loss of appetite. The skin

becomes discolored, red and boil-like sores may appear

on the skin surface. As a disease progress, erythema or

bloody blotches can be found around the mouth and  ns.

The gut and rectum can be bloody and  lled with  uid if

an early treatment not be given (Wang et al., 2009). Thus,

all these symptoms are not proof of a vibriosis and require

a further isolation and identi cation of bacteria.

Therefore, culture method has been developed for the

identi cation of bacteria species in aquatic animal and

marine environment. The bacteria are allowed for multi-

plying in culture media under controlled laboratory con-

ditions and further identi ed using gram staining and

biochemical tests. However, this method is tedious, time

consuming and requires skilled personnel to pick right

colonies of a plate. In addition, culture method unable

to differentiate species levels due to similar morphol-

ogy such as V. cholerae, V. alginolyticus and V.  uvialis

which appeared yellow color on agar plates (Oxoid, UK).

To overcome those limitations, the antigen-antibody

based assay was developed to test presence of speci c

antibody or antigen in aquatic samples. A speci c anti-

body is used for recognition of antigen from pathogenic

bacteria. Even though this assay is rapid and easy to

perform, but it is unable to differentiate Vibrio species

because they have same antigens (Chen et al., 1992).

The molecular approaches based on synthesis of nucleic

acid were developed for producing more sensitive and

speci c assays such as polymerase chain reaction (PCR),

real-time PCR, multiplex PCR, reverse-transcriptase

PCR, touchdown PCR and so on. However, those assays

required expensive thermocycler machine to amplify the

nucleic acid based on three different temperatures.

Thus, a novel isothermal ampli cation assay was

developed in early 1990s as an alternative to the PCR

assay. The ampli cation of nucleic acid can be performed

in a single temperature (isothermal condition) which mim-

ics in vivo synthesis of DNA. The isothermal ampli cation

assay can be performed without the need for a thermocy-

cling apparatus. In addition, the isothermal ampli cation

assays also more sensitive and speci c, rapid and easy to

perform. However, the detection and visualization of iso-

thermal products still requires conventional agarose gel

electrophoresis system. This agarose gel electrophoresis

is laborious, requires special equipment, time consum-

ing and must be operated by skilled personnel. Thus, an

alternative method, a lateral  ow dipstick is developed for

the detection of isothermal products. Based on previous

study, the lateral  ow dipstick is rapid (within  ve to ten

mins), easy to perform, does not require special equip-

ment and can be visualized using naked eyes (Wain and

Hosoglu, 2008; Mugasa et al., 2009).

CONVENTIONAL CULTURE METHODS

Culture method is a gold standard for isolation of bac-

teria by using enrichment and selective medium. Basi-

cally, tryptic soy broth (TSB) is used to enrich amount of

Vibrio from samples. Vibrio can grow in different envi-

ronments due to its ability to live in saltwater, freshwa-

ter and living organisms. Vibrio grows well at pH 7 to 9,

from neutral to alkaline condition. It also able to grow

in high concentration of sodium chloride (NaCl) due to it

character halophilic. A sodium chloride (NaCl) is added

with the TSB when samples were collected from salt-

water. In laboratory, a halophilism test was performed

with different concentration of NaCl (0 to 10%) and V.

alginolyticus requires at least 3% NaCl and can tolerate

until 10% NaCl for growth (Gomathi et al., 2013).

Thiosulfate citrate bile salts-sucrose (TCBS) agar is a

common selective media used for isolation of Vibrio spe-

cies (Jones et al., 2012). In order to perform a bacteria

culture on the agar plate requires a lot of time to allow

optimum growth of bacteria colonies. For example, bac-

BIOSCIENCE BIOTECHNOLOGY RESEARCH COMMUNICATIONS MOLECULAR DETECTION FOR VIBRIOSIS 351

Amalina and Ina-Salwany

teria are incubated at 30

C for 16-24 hours when isolated

from environmental samples, while bacteria are incubated

at 35

C for 24 hours when isolated from other samples

(Oxoid, UK). Typical colonies of Vibrio can be varying in

terms of size and color of colonies (showed in Table 1).

The biochemical pro le of Vibrio species can be deter-

mined using API 20E diagnostic strip (bioMerieux, Durham,

NC). The API 20E test strip has 20 miniature biochemical

tests of different medium; Citrate (CIT), Voges-Proskauer

(VP), Urease (URE), hydrogen sul de (H

S), amino acid

decarboxylations (ADH), gelatin hydrolysis (GEL) and oth-

ers. Bacteria are mixed with two percents of NaCl as the

suspension solution, added to the API strip and incubated

at 37

C for 18-24 hours. After that, changes of the color

reaction were read and converted to a seven digit code

known as Analytical Pro le Index (API). The codes are

matched with the manufacturer’s database and give the

bacteria identi cation as a genus and species. According

to Jones (2012), API 20E test was able to identi ed 41.8%

of V. parahaemolyticus from oysters isolates and 54.6% of

V. parahaemolyticus from clinical isolates. The API codes

were misidenti ed several bacteria isolate such as V. vul-

ni cus, Aeromonas hydrophila, V.  uvialis, V.cholerae and

V. mimicus (Jones et al., 2012).

However, these conventional methods have disad-

vantages such as less speci c, tedious, require skilled

personnel and cannot differentiate between Vibrio spe-

cies, especially V. parahaemolyticus and V. alginolyticus

which are biochemically similar (Mustapha et al., 2013).

ANTIGEN-ANTIBODY BASED ASSAY

Vibrio species possesses three antigenic components;

heat-labile K-antigen (capsular polysaccharide), heat-

stable somatic O-antigen (lipopolysaccharide) and

H-antigen ( agellar). Those antigenic components of

Vibrio can be determined via agglutination tests where

the bacteria culture are reacted with speci c antiserum

to formed clumps or agglutinate particles. To determine

the O-antigens, the bacteria should be heated at boiling

temperature for one to two hours. This step is impor-

tant to removed K-antigen from Vibrio since K-antigen

masked the O-antigen.

The Mono-aqua test kit (BIONOR, Tamar Laboratory

Supplies Ltd., Israel) is a commercial agglutination tests

for rapid preliminaries screening of pathogens such as

V. anguillarum and V. salmonicida. The principle used is

a mono-disperse particles coated with speci c antibod-

ies will be reacted with  sh pathogenic bacteria (antigen)

and form a visible granular particle agglutination pattern.

This kit is rapid (can be performed in 30 minutes) and

inexpensive. However, the samples have to be cultured

overnight before testing and make this test tedious and

time consuming (Romalde et al., 1995). Also, Vibrio spe-

cies are serologically identical based on a study reported

that most of the Vibrio species share H-antigen at 52-kDa

protein including V. parahaemolyticus, V. alginolyticus,

V. anguillarum and V. ordalii (Chen et al., 1992).

An aqua-rapid and aqua-EIA test kits were devel-

oped as a modi cation of previous kit based on Enzyme

Immunoassay (EIA) principle. EIA is also known as

enzyme-linked immunosorbent assay (ELISA), combina-

tion of the antibody binding with certain enzyme for

the detection and quanti cation of antigen (pathogenic

bacteria) (Lequin, 2005). By using the EIA principle, both

kits can be used for direct identi cation of  sh patho-

gens without needs of culture. Thus, these kits are rapid,

easy to performed and suitable to be used at farm site.

The aquaEIA test kit is using semiautomated system,

while aqua-rapid using manual system.

The dot immunoassay method is one of the antigen-

antibody based assay that has been used to detect V.

alginolyticus and A. hydrophila isolated from shrimps

and  shes. The bacteria culture is diluted before spot-

ted onto the nitrocellulose paper and allow reacting

with antiserum raised against V. alginolyticus and A.

hydrophila. Even though this method is simple, but it

is less speci c due to cross-reaction of V. alginolyticus

antiserum with V. parahaemolyticus, V. harveyi and V.

anguillarum (Mishra, 1998).

NUCLEIC ACID-BASED ASSAY

Molecular diagnostics involving nucleic acid ampli ca-

tion is the most common methods used for the detec-

tion and identi cation of infectious diseases. Polymer-

Table 1: Typical morphology of Vibrio colonies on TCBS agar

Organisms Size of colonies Color of colonies

Vibrio parahaemolyticus 3-5 mm diameter Blue colonies with green centers

Vibrio alginolyticus 3-5 mm diameter Yellow

Vibrio cholera 2-3 mm diameter Yellow and  at colonies

Vibrio vulni cus/ Vibrio mimicus 2-3 mm diameter Blue green

Vibrio harveyi 2-3 mm diameter Gray to bluish green colonies

Vibrio  uvialis 2-3 mm diameter Yellow

352 MOLECULAR DETECTION FOR VIBRIOSIS BIOSCIENCE BIOTECHNOLOGY RESEARCH COMMUNICATIONS

Amalina and Ina-Salwany

ase chain reaction (PCR) is a main technique used for

nucleic acid ampli cation. PCR provides a rapid, high

sensitivity and speci city compared to the conventional

culture methods for differentiation and identi cation of

species. It is also able to identify housekeeping genes

and different virulence genes among Vibrio species. In

addition, the PCR assay is able to simultaneous detec-

tion of multiple pathogens in a single reaction known

as multiplex PCR assay. The PCR assay is based on the

ampli cation of nucleic acids using speci c primer and

DNA polymerase at three different temperatures; dena-

turation, annealing and extension that repeated in a

number of cycles until certain amount of DNA obtained.

Thus, the PCR assay requires a thermal cycler to com-

plete the ampli cation process.

Researchers were performed extensive studies using

PCR assay for the identi cation and differentiation of

Vibrio infected  shes, molluscs, crustaceans and other

aquatic animals. Various genes were used in design-

ing primers for speci c ampli cation of target gene of

Vibrio. A 16S rRNA gene is highly conserved among

bacteria species based on hyper variable region that

contains similar species-speci c sequence which is use-

ful for bacteria identi cation. In 2006, Bramhachari and

Duey developed a PCR for detection of V. harveyi using

16S rRNA. Kim and Jeong (2001) was developing a PCR

targeting the same 16S rRNA gene to identify patho-

genic V. vulni cus in marine environments. Similarly,

Yong in 2006 targeted gene for identi cation of bacteria

from aquaculture systems.

A toxR gene is a virulence gene encodes for regula-

tory gene of toxin operon. A PCR for detection of V. har-

veyi targeting toxR gene was successfully developed with

100% speci city (Conejero and Hedreyda, 2003). In 2016,

the toxR gene is found to be more speci c compared to the

gyrB gene in identi cation of V. parahaemolyticus in  sh

and coastal environment in Jordan (Alaboudi et al., 2016).

GyrB gene is encodes for the B subunit protein of DNA

gyrase (topoisomerase type II). Previous study was found

the degree of homology of toxR gene between V. para-

haemolyticus and V. cholerae was 52% (Kim et al., 1999).

Other virulent genes used for identi cation of Vibrio

species were thermostable direct hemolysin (tdh), thermo-

labile hemolysin (tlh) and thermostable direct hemolysin-

related hemolysin (trh) (Jones, 2012). Mohammed and

Jerjees (2015) proved that the 12% and four percents of

V. parahaemolyticus isolated from fresh shrimp have the

tdh and trh genes, respectively. Other study revealed the

tdh and trh genes were 98% homology between V. algi-

nolyticus and V. parahaemolyticus isolated from shrimp

(Gargouti et al., 2015). In 2016, Li and his friends found

a unique gene for V. parahaemolyticus, bla

CARB-17

gene

which has the lowest degree of similarity (78% homology)

with V. alginolyticus compared with other virulence genes

such as tlh (85%), atpA (97%) and toxR (86%) genes. The

PCR assay targeting for bla

CARB-17

gene was produced

100% sensitivity and speci city (Li et al., 2016).

A multiplex PCR (mPCR) assay was used for simul-

taneous detection of more than one Vibrio species in a

single test. Xie (2005) was developing the mPCR assay

for detection of seven virulence genes targeted for V.

parahaemolyticus and V. alginolyticus from shrimp,  sh

and seawater in Guangdong Coast, China (tlh, trh, tdh,

toxR, toxRS, ctxA and VPI). This study found that the V.

alginolyticus, V. parahaemolyticus and V. cholerae pos-

sess homology using toxR, tlh and VPI genes. In 2011,

Izumiya was developed a highly sensitive and speci c

mPCR assay for the detection of V. cholerae, V. para-

haemolyticus and V. vulni cus from seawater sample

using dnaJ gene. Similarly, Xu in 2014 was targeted

dnaJ gene for detection of  ve Vibrio species; V. para-

haemolyticus, V. vulni cus, V. alginolyticus, V. cholerae

and V. mimicus from seafood samples.

In addition, the 16S rRNA gene was included in the

mPCR assay as a Vibrio species control to avoid any doubt

regarding target of V. parahaemolyticus, V. mimicus, V.

vulni cus and V. cholerae from shrimp and crab samples

(Amin and Salem, 2012). The 16S rRNA gene also used

as an internal ampli cation control (IAC) in the mPCR

assay targeting V. alginolyticus, V. parahaemolyticus, V.

vulni cus and V. cholerae (Wei et al., 2014). The inter-

nal ampli cation control is a mandatory for diagnostic

using PCR assay. IAC is used for validation of negative

result whether it is truly negative or due to malfunction

of thermal cycler, incorrect PCR mixture or presence of

inhibitory substances in the sample (Hoorfar et al., 2003).

Based on previous studies, the PCR assay is highly

sensitive and speci c, able to simultaneous detection of

many pathogens in a single tube and successful detect

Vibrio species. However, this assay requires expensive

equipment, time consuming and requires trained per-

sonnel. Based on the WHO guidelines, a develop diag-

nostic device must be affordable, sensitive, speci c, user

friendly, rapid and robust, equipment free and deliverable

to end used (ASSURED) (Mabey et al., 2004). Thus, an

alternative platform known as isothermal ampli cation

assay was developed in order to ful ll this guideline.

ISOTHERMAL AMPLIFICATION ASSAY

Isothermal ampli cation techniques have been devel-

oped since early 1990s as an alternative to the poly-

merase chain reaction (PCR) assay. This technique is

used to amplify DNA, RNA, cells and proteins. The iso-

thermal ampli cation assay can be performed at one

temperature rather than the PCR assay which required

three different temperatures (Notomi et al., 2000; Chow

BIOSCIENCE BIOTECHNOLOGY RESEARCH COMMUNICATIONS MOLECULAR DETECTION FOR VIBRIOSIS 353

Amalina and Ina-Salwany

et al., 2008; Fang et al., 2010). Therefore, the isother-

mal ampli cation can be performed using simple heat-

ing devices (e.g., water bath or heating block) without

the need of thermocycling apparatus. In addition, the

isothermal product can be directly visualized by naked

eyes on the form of turbidity or  uorescence, eliminat-

ing the need for electrophoresis system as used in the

PCR assay. Thus, the isothermal ampli cation are rapid,

user friendly, high sensitivity and speci city and can be

performed without expensive equipment.

Nowadays, several types of isothermal ampli ca-

tion technique were developed for the detection of

pathogenic bacteria such as loop-mediated ampli ca-

tion (LAMP), nucleic acid sequence-based ampli ca-

tion (NASBA), strand displacement ampli cation (SDA),

helicase dependent ampli cation (HDA), rolling circle

ampli cation (RCA) and transcription mediated ampli-

 cation (TMA) assays (Compton, 1991; Walker et al.,

1992; Fire and Xu, 1995; Notomi et al., 2000; Vincent et

al., 2004). Each isothermal assay had their own mecha-

nisms to promote a next cycle of DNA synthesis. Differ

with the PCR assay that used heat denaturation of double

stranded DNA to initiate next cycles of DNA synthesis;

the isothermal assays use various accessory proteins to

open the double stranded DNA (Gill and Ghaemi 2008).

Loop-mediated ampli cation (LAMP) assay is the

most popular isothermal ampli cation technique used in

aquaculture  eld. LAMP assay was developed by Notomi

et al. (2000) for the identi cation of Hepatitis B virus.

This assay was designed based on four to six primers to

recognize six or eight distinct sequences on the template

DNA. The nucleic acid is extended by the Bst polymerase

to synthesis new DNA strands. The entire LAMP reac-

tion can be performed in simple heating devices at 60 to

C for less than one hour. The ampli ed products will

be mixture of stem-loop DNAs with various length and

cauli ower-like structures with multiple loops (Notomi

et al., 2000; Mori and Notomi, 2009). The products can

be identi ed by using agarose gel electrophoresis (pro-

duced multiple bands of different sizes from ~300bp),

restriction enzyme digestion (such as BamHI, HindIII

and others to produce two different bands) and South-

ern blot hybridization. Also, the LAMP products can be

observed by naked eyes through usage of magnesium

pyrophosphate, calcein, SYBR Green or other  uores-

cence dye (Prompamorn et al., 2011).

LAMP assay has been developed for the detection of

Vibrio species such as V. alginolyticus, V. harveyi and

V. parahaemolyticus from aquatic animals and environ-

ment (Cai et al., 2010, Di et al., 2015). The LAMP assay

was developed for the detection of V. alginolyticus in

mariculture  sh using gyrB gene and it is ten-fold more

sensitive than PCR assay (Cai et al., 2010). In addition,

several studies revealed that the LAMP assay is supe-

rior to the PCR assay and conventional culture method

for the detection of V. parahaemolyticus from environ-

mental samples (Di et al., 2015; Kongrueng et al., 2015;

Malcolm et al., 2015). A universal LAMP primer from

conserved region of 16S rRNA was designed as a Vibrio

species control and result showed the LAMP assay 100

times more sensitive than PCR assay with 100% speci c-

ity (Xu et al., 2012).

In addition, a multiplex LAMP (mLAMP) assay is

developed for simultaneous detection of two or more

Vibrio species by amplifying their DNA in a single tube.

Two or more sets of each of the four LAMP primers with

total eight or more primers are used in the same reac-

tion mixture (Biswas et al., 2014). A study on a multiplex

LAMP was done for the detection of V. harveyi, V. anguil-

larum and V. alginolyticus from Japanese  ounder, shark,

seabass and shrimp (Yu et al., 2013). Three sets of four

species-speci c primers were designed and performed in

a single reaction. This assay showed 10

to 10

times more

sensitive than the traditional PCR assay. However, in the

detection of mLAMP products, the agarose gel electro-

phoreis was used followed with sequencing or further

con rmation of species among these three Vibrios. This

detection methods are tedious, time consuming, expen-

sive and requires electrophoresis equipments.

Even though LAMP assay offers a rapid, high sen-

sitivity and speci city and user friendly, but it has a

complicated primer design especially for new users. As

we know, LAMP assay requires more than one set of

primers that target six or eight regions within a target

DNA (Notomi et al., 2000). Sometimes, the termination

step is required to stop the LAMP reaction by heating at

C for two to ten minutes (Biswas et al., 2014). Instead

of the LAMP assay, the simplest reaction HDA assay can

be used since it requires one set of primers and requires

reaction buffer and enzyme mix similar to the PCR assay

(Vincent et al., 2004). Moreover, this assay can be per-

formed at 60 to 65

C for less than one hour. However in

aquaculture  eld, there is no publication found regard-

ing the detection of Vibrio species using HDA assay.

LATERAL FLOW DIPSTICK

Nowadays, many publications in aquaculture  eld

focused on the detection of Vibrio species by LAMP assay

combined with lateral  ow dipstick (LFD). Lateral  ow

dipstick is used as an alternative detection method to the

agarose gel electrophoresis, turbidity,  uorescence and

restriction enzyme digestion. LFD detection method is a

rapid, user-friendly, and easy to perform, highly sensitive

and speci c and stable at room temperature (Posthuma-

Trumpie et al., 2009). Also, it is able to detect multiple

pathogenic bacteria simultaneously in one strip. Thus, a

354 MOLECULAR DETECTION FOR VIBRIOSIS BIOSCIENCE BIOTECHNOLOGY RESEARCH COMMUNICATIONS

Amalina and Ina-Salwany

multiplex LAMP can be developed and detected via lat-

eral  ow dipstick. LFD is known as oligochromatography

lateral  ow or immunochromatographic test (Brandonisio

et al., 2002). It is based on the chromatography technique

where the reagents were immobilized on the nitrocel-

lulose strip and reactivated using sample/ampli cation

product for the detection of amplicon.

LAMP assay combined with LFD was developed for

the detection of V. alginolyticus targeting rpoX gene

(Plaon et al., 2015). The biotin-labeled LAMP product

was ampli ed at 60

C for one hour and hybridized with

a  uorescein isothiocyanate (FITC)-labeled probe and

result was visualized within  ve minutes. Result showed

that the LAMP-LFD was ten times less sensitive in detec-

tion of V. alginolyticus with 100% sensitivity and spec-

i city (Plaon et al., 2015). In 2010, Prompamorn was

developed the LAMP-LFD for the detection of V. para-

haemolyticus from shrimp samples. The LAMP ampli -

cation was performed at 65

C for 90 mins using primers

labeled with biotin. The biotinylated LAMP amplicons

were then hybridized with a FITC-labeled probe and

detected via LFD for  ve mins. Result showed the detec-

tion limit of the LAMP-LFD was 1.8 X 10

CFU g

-1

which

is ten times more sensitive than PCR assay.

Surasilp (2011) was developed LAMP combined with

LFD for the detection of V. vulni cus targeting RNA

polymerase subunit sigma factor S (rpoS) gene. The

LAMP-LFD was able to detect 1.5 X 10

CFU ml

-1

of V.

vulni cus from pure culture and also has 100% sensitiv-

ity and speci city. Other study showed the LAMP-LFD

is successfully developed for the detection of V. harveyi

from black tiger shrimp, green mussel and bloody clam

(Thongkao et al., 2013). The ampli cation was performed

for 60 mins to produce biotinylated-LAMP products and

hybridize with FITC-labeled DNA probe and detected via

LFD for  ve to ten mins. Result showed that the LAMP-

LFD is ten times more sensitive than PCR assay in the

detection of V. harveyi from shrimp samples.

The combination of LAMP assay and lateral  ow dip-

stick for the detection of vibriosis offers several advantages

such as rapid (30 min to one hour), highly sensitive and

speci c, equipment free and deliverable to end user. Thus,

a multiplex LAMP combine with LFD can be a potential

diagnostic test for simultaneously detection of Vibrio spe-

cies from  sh, mollusks and other aquatic animals.

CONCLUSION

In conclusion, the isothermal ampli cation assay espe-

cially LAMP is capable for the detection of Vibrio spe-

cies. The simplicity of lateral  ow dipstick and LAMP

assay offer great potentials for the development of

nucleic acid diagnostic devices that could be used to

detect vibriosis at point-of-care or in the  eld.

ACKNOWLEDGEMENT

This work (LAMP development) was supported by the

grants provided by Ministry of Higher Institution via

Higher Institution Centre of Excellence (HICoE) under

Vote No: 6369100.

REFERENCES

Adams, A. and Thompson, K.D. (2011) Development of diag-

nostics for aquaculture: challenges and opportunities. Aqua-

culture Research. Vol. 42 Pages 93-102.

Alaboudi, A.R., Ababneh, M., Osaili, T.M. and Shloul, K.A.

(2016) Detection, Identi cation, and Prevalence of Pathogenic

Vibrio parahaemolyticus in Fish and Coastal Environment in

Jordan. Journal of Food Science.Vol. 81 No (1) Pages 130-134.

Alam, M.J., Miyoshi, S. and Shinoda, S. (2003) Studies on

pathogenic Vibrio parahaemolyticus during a warm weather

season in the Seto Inland Sea, Japan. Environmental Microbi-

ology.Vol. 5 Pages 706-710.

Amin, R.A. and Salem, A.M (2012) Speci c Detection of Patho-

genic Vibrio species inShell sh by Using Multiplex Polymerase

Chain Reaction. Global Veterinaria. Vol. 8 No (5) Pages 525-

531.

Biswas, G. & Sakai, M. (2014) Loop-mediated Isothermal

Ampli cation (LAMP) Assays for Detection and Identi ca-

tion of aquaculture pathogens: Current State and Perspectives.

Applied Microbiology and Biotechnology. Vol 98 Page 2881.

Bramhachari, P.V. and Dubey, S.K. (2006) Rapid and speci c

detection of luminous and non-luminous Vibrio harveyi iso-

lates by PCR ampli cation. Current Science. Vol. 90 Pages

1105-1108.

Brandonisio, O., Fumarola, L., Maggi, P., Cavaliere, R., Spinelli,

R., Pastore, G. (2002) Evaluation of a Rapid Immunochromato-

graphic Test for Serodiagnosis of Visceral Leishmaniasis. Euro-

pean Journal of Clinical Microbiology & Infectious Diseases.

Vol 21 Pages 461–464.

Buller, N.B. (2014) Bacteria and Fungi from Fish and other

Aquatic Animals, 2

edition: A Practical Identi cation Man-

ual. CABI Publishing, UK.Pages 214-217.

Cai, S.H., Lu, Y.S., Wu, Z.H., Jian, J.C., Wang, B. and Huang,

Y.C. (2010) Loop-mediated isothermal ampli cation method for

rapid detection of Vibrio alginolyticus, the causative agent of

vibriosis in mariculture  sh. The Society for Applied Micro-

biology, Letters in Applied Microbiology.Vol. 50 Pages 480–

485.

Canajero, M.J.U. and Hedreyda, C.T. (2003) Isolation of partial

toxR gene of Vibrio harveyi and design of toxR-targeted PCR

primers for species detection. Journal of Applied Microbiology.

Vol. 95 Pages 602–611.

Centers for Disease Control and Prevention (CDC) (2013)

Increase in Vibrio parahaemolyticus illnesses associated with

consumption of shell sh from several Atlantic coast harvest

areas, United States, 2013. Accessed on 11 Mei 2016 at http://

www.cdc.gov/vibrio/investigations/

BIOSCIENCE BIOTECHNOLOGY RESEARCH COMMUNICATIONS MOLECULAR DETECTION FOR VIBRIOSIS 355

Amalina and Ina-Salwany

Chatterjee, S. and Haldar, S. (2012) Vibrio Related Diseases in

Aquaculture and Development of Rapid and Accurate Identi -

cation Methods. Journal of Marine Science: Research & Devel-

opment. Vol.1 Pages 1-17.

Chen, D., Hanna, P.J., Altmann, K., Smith, A., Moon, P. and

Hammond, L.S. (1992) Development of Monoclonal Antibodies

That Identify Vibrio Species Commonly Isolated from Infec-

tions of Humans, Fish, and Shell sh. Applied and Environ-

mental Microbiology. Vol. 58 No (11) Pages 3694-3700.

Chiou, C.S., Hsu, S.Y., Chiu, S.J., Wang, T.K. and Chao, C.S.

(2000) Vibrio parahaemolyticus serovar O3:K6 as cause of

unusually high incidence of food-borne disease outbreaks in

Taiwan from 1996 to 1999. Journal of Clinical Microbiology.

Vol. 38 Pages 4621-4625.

Chow, W. H. A., McCloskey, C., Tong, Y., Hu, L.,You, Q., Kelly,

C. P., Kong, H., Tang, Y.-W. and Tang, W. (2008). Application

of Isothermal Helicase-Dependent Ampli cation with a Dis-

posable Detection Device in a Simple Sensitive Stool Test for

Toxigenic Clostridium dif cile. The Journal of Molecular Diag-

nostics.Vol. 10 No (5) Pages 452-458.

Compton, J. (1991). Nucleic acid sequence-based ampli cation.

Nature.Vol.350 No (6313) Pages 91-92.

Deepanjali, A., Kumar, H.S., Karunasagar, I. and Karunasagar,

I. (2005) Seasonal variation in abundance of total and patho-

genic Vibrio parahaemolyticus bacteria in oysters along the

southwest coastal of India. Applied and Environmental Micro-

biology. Vol. 71 Pages 3575-3580.

Di, H., Ye, L., Neogi, S.B., Meng, H., Yan, H., Yamasaki, S. and

Shi, L. (2015) Development and Evaluation of a Loop-Mediated

Isothermal Ampli cation Assay Combined with Enrichment

Culture for Rapid Detection of Very Low Numbers of Vibrio

parahaemolyticus in Seafood Samples.Biological and Pharma-

ceutical Bulletin.Vol. 38 Pages 82–87.

Edwards, P., Little, D.C. and Demaine, H., (2002) Rural aquacul-

ture. CABI publisihing, UK. Page 358.

Fang, X., Liu, Y., Kong, J. and Jiang, X. (2010) Loop-mediated

isothermal ampli cation integrated on micro uidic chips for

point-of-care quantitative detection of pathogens. Analytical

Chemistry.Vol.82 No (7) Pages 3002-3006.

Fire, A. and Xu, S.Q. (1995) Rolling replication of short DNA

circles. Proceedings of the National Academy of Sciences.

Vol.92 No (10) Pages 4641-4645.

Food and Agriculture Organization of the United Nations

(FAO) (2013) FAO Statistical Yearbook 2013 World Food and

Agriculture. Pages 146-149.

Foodborne Diseases Active Surveillance Network (FoodNet):

FoodNet Surveillance Report for 2014 (Final Report). Atlanta,

Georgia: U.S. Department of Health and Human Services,

CDC. 2016. http://www.cdc.gov/foodnet/reports/annual-

reports-2014.html. Accessed April 21, 2016.

Gargouti, A.S., Ab-Rashid, M. N.K., Ghazali, M.F., Mitsuaki,

N., Haresh, K.K. and Radu, S. (2015) Detection of tdh and trh

Toxic Genes in Vibrio Alginolyticus Strain from Mantis Shrimp

(Oratosquilla Oratoria). Journal of Nutrition & Food Sciences.

Vol.5 No (5) Pages 1-4.

Gauthier, T. (2015) Bacterial zoonoses of  shes: A review and

appraisal of evidence for linkages between  sh and human

infections. The Veterinary Journal. Vol. 203 Pages 27–35.

Gill, P. and Ghaemi, A. (2008) Nucleic Acid Isothermal Ampli-

 cation Technologies – AReview. Nucleosides, Nucleotides and

Nucleic Acids.Vol.27 No (3) Pages 224 – 243.

Gomathi, R.S., Vinothkumar, R. and Arunagiri, K. (2013) Isola-

tion and identi cation Vibrios from marine seafood samples.

International Journal of Current MicrobiologyandAppliedSci-

ences. Vol.2 No (2) Pages36-43.

Haldar, S., Chatterjee, S., Sugimoto, N., Das, S., Chowdhury,

N., Hinenoya A.,Asakura M.,Yamasaki S. (2011) Identi cation

of Vibrio campbelii isolated from diseased farm-shrimps from

South India and establishment of its pathogenic potential in an

Artemia model. Microbiology.Vol. 157 Pages 79-188.

He, L. and Xu, H.S. (2011) Development of a multiplex loop-

mediated isothermal ampli cation for direct detection of Myco-

bacterium tuberculosis complex, M.avium and M.intracellulare

in sputum samples. Journal of Clinical Microbiology. Vol. 41

No (6) Pages 2616-2622.

Hoorfar, J., Cook, N., Malorny, B., Wegner, M., Medici, D.D.,

Abdulmawjood, A. and Fach, P. (2003) Making internal ampli-

 cation control mandatory for diagnostic PCR. Journal of

Clinical Microbiology. Vol.41 No (12) Pages 5835.

Izumiya, H., Matsumoto, K., Yahiro, S., Lee, J., Morita, M.,

Yamamoto, S., Arakawa, E. and Ohnishi, M. (2011) Multiplex

PCR assay for identi cation of three major pathogenic Vibrio

spp., Vibrio cholerae, Vibrio parahaemolyticus, and Vibrio vul-

ni cus. Molecular and Cellular Probes. Vol.25 Pages 174-176.

Jones, J.L., Hara-Kudo, Y., Krantz, J.A., Benner Jr, R.A.,

Smith, A.B., Dambaugh, T.R., Bowers, J.C. and DePaola, A.

(2012) Comparison of molecular detection methods for Vibrio

pahaemolyticus and Vibrio vulni cus. Food Microbiology.

Vol.30 Pages 105-111.

Kim, M.S. and Jeong, H.D. (2001) Development of 16S rRNA

tageted PCR methods for the detection and differentiation

of Vibrio vulni cus in marine environments. Aquaculture.

Vol.193 Pages 199-211.

Kim, Y.B., Okuda, J., Matsumoto,C., Takahashi, N., Hashimoto,

S. and Nishibuchi, M. (1999) Identi cation of Vibrio para-

haemolyticus strains at the species level by PCR targeted to

the toxR gene. Journal of Clinical Microbiology. Vol. 37 No (4)

Pages 1173-1177.

Kongrueng, J., Tansila, N., Mitraparp-arthorn, P., Nishibuchi,

M., Vora, G.J. and Vuddhakul, V. (2015) LAMP assay to detect

Vibrio parahaemolyitcus causing acute hepatopancreatic

necrosis disease in shrimp. Aquaculture International.Vol. 23

Pages 1179-1188.

Lequin, R.M. (2005) Enzyme Immunoassay (EIA)/Enzyme-

linked Immunosorbent Assay (ELISA). Clinical Chemistry.Vol.

51 No (12) Pages 2415-2418.

Li, R., Chious, J., Chan, E.C. and Chen, S. (2016) A novel PCR-

based approach for accurate identi cation of Vibrio parahaemo-

lyticus. Frontiers in Microbiology. Vol.44 No (7) Pages 1-5.

356 MOLECULAR DETECTION FOR VIBRIOSIS BIOSCIENCE BIOTECHNOLOGY RESEARCH COMMUNICATIONS

Amalina and Ina-Salwany

Mabey, D., Peeling, R.W., Ustianowski, A., Perkins, M.D. and

Free, R. (2004) Diagnostics for the developing world. Tropical

Infectious Diseases. Vol.2 Pages 1-10.

Malcolm, T.T.H., Cheah, Y.K., Radzi, C.W.J.W.M., Kasim, F.A.,

Kantilal, H.K., John, T.Y.H., Martinez-Urtaza, J., Nakaguchi, Y.,

Nishibuchi, M. and Son, R. (2015) Detection and quanti cation

of pathogenic Vibrio parahaemolyticus in shell sh by using

multiplex PCR and loop-medated isothermal ampli cation

assay. Food Control.Vol. 47 Pages 664-671.

Mishra, S.S. (1998) Use of dot immunoassay for rapid detec-

tion of pathogenic bacteria Vibrio alginolticus and Aeromonas

hydrophila from shrimps and  shes. Indian Journal of Geo-

Marine Sciences.Vol.27 No (2) Pages 222-226.

Mohammed, A.M. and Jerjees, S.G. (2015) Detection of tdh and

trh genes from Vibrio parahaemolyticus isolated from gastro-

enteritis cases and shrimp samples. International Journal of

Advanced Research. Vol. 3 No (2) Pages 288-294.

Mori, Y. and Notomi, T. (2009). Loop-mediated isothermal

ampli cation (LAMP): a rapid, accurate, and cost-effective

diagnostic method for infectious diseases. Journal of Infection

and Chemotherapy.Vol.15 No (2) Pages 62-69.

Mugasa, C. M., Laurent, T., Schoone, G. J., Kager, P. A., Lubega,

G. W., and Schallig,H.D.F.H. (2009). Nucleic Acid Sequence-

Based Ampli cation with Oligochromatography for Detection

of Trypanosoma brucei in Clinical Samples. Journal of Clinical

Microbiology.Vol.47 No (3) Pages 630-635.

Mustapha, S., Mustapha, E.M. and Nozha, C. (2013) Vibrio

alginolyticus: An emerging pathogen of foodborne diseases.

International Journal of Science and Technology.Vol.2 No (4)

Pages 302-309.

Notomi, T., Okayama, H., Masubuchi, H., Yonekawa, T., Wata-

nabe, K., Amino, N. and Hase, T. (2000). Loop-mediated iso-

thermal ampli cation of DNA. Nucleic Acids Research.Vol.28

No (12) Pages E63.

Plaon, S., Longyant, S., Sithigorngul, P. & Chaivisuthangkura,

P. (2015) Rapid and Sensitive Detection of Vibrio alginolyticus

by Loop Mediated Isothermal Ampli cation Combined with a

Lateral Flow Dipstick Targeted to the rpoX Gene. Journal of

Aquatic Animal Health. Vol 27 No (3) Pages 156-163.

Posthuma-Trumpie, G.A., Korf, J. & van Amerongen, A. (2009)

Lateral  ow (immuno) assay: Its Strengths, Weaknesses, Oppor-

tunities and Threats. Analytical and Bioanalytical Chemistry.

Vol. 393 No (2) Pages 569-582.

Prompamorn, P., Sithigorngul, P., Rukpratanporn, S., Longant,

S., Sridulyakul, P. and Chaivisuthangkura, P. (2011) The devel-

opment of loop-mediated isothermal ampli cation combined

with lateral  ow dipstick for detection of Vibrio parahaemo-

lyticus.Letters in Applied Microbiology. Vol. 52 Pages 344-351.

Romalde, J.L., Magarinos, B., Fouz, b., Bandin, I., Nunez, S.

and Toranzo, A.E. (1995) Evaluation of BIONOR Mono-kits for

rapid detection of bacterial  sh pathogens. Diseases of Aquatic

Organisms. Vol.21 Pages 25-34.

Scallan, E., Hoekstra, R.M., Angulo, F.J., Tauxe R.V.,Widdow-

son M.A.,Roy S.L.,Jones J.L.,Grif n P.M. (2011) Foodborne

illness acquired in the United States- major pathogens. Emerg-

ingInfectious Disease. Vol.17 No (1) Pages 7-15.

Surasilpa, T., Siwaporn, L., Rukpratanpornb, S., Sridulyakula,

P., Sithigorngula, P., Chaivisuthangkura, P. (2011) Rapid and

sensitive detection of Vibrio vulni cus by loop-mediated iso-

thermal ampli cation combined with lateral  ow dipstick tar-

geted to rpoS gene. Molecular and Cellular Probes Vol. 25 No

(4) Pages 158–163.

Thongkao, K., Siwaporn L., Silprasit K., Sithigorngul, P. &

Chaivisuthangkura, P. (2013) Rapid and Sensitive Detection

of Vibrio harveyi by Loop-mediated Isothermal Ampli cation

Combined with Lateral Flow Dipstick Targeted to vhhP2 Gene.

Aquaculture Research. Pages 1–10

Vincent, M., Xu, Y. and Kong, H. (2004). Helicase-dependent

isothermal DNA ampli cation. EMBO reports. Vol.5 No (8)

Pages 795-800.

Wain, J., and Hosoglu, S. (2008). Review Article: The labora-

tory diagnosis of enteric fever.Journal of Infection inDevelop-

ing Countries.Vol.2 No (6) Pages 421-425.

Walker, G. T., Little, M. C., Nadeau, J. G. and Shank, D. D.

(1992). Isothermal in vitro ampli cation of DNA by a restric-

tion enzyme/ DNA polymerase system. Proceedings of the

National Academy of Sciences.Vol.89 No (1) Pages 392-396.

Wang, X., Yuan, Y., Wang, H., Zhou, Q., Zhu, J., Chang, Y., Cui,

Y. and Qiu, X. (2009) Phylogenetic analysis of four Vibrio strains

of pathogenic bacteria based on hemolysin genes and 16S rRNA

genes. Book of Abstract; 3rd International Conference on Bioin-

formatics and Biomedical Engineering. Pages 1-4.

Wei, S., Zhao, H., Xian, Y., Hussain, M.A. and Wu, X. (2014)

Multiplex PCR assays for the detecton of Vibrio alginolyticus,

Vibrio parahaemolyticus, Vibrio vulni cus and Vibrio cholerae

with an internal ampli cation control.Diagnostic Microbiol-

ogy and Infectious Disease.Vol. 79 Pages 115-118.

Xie Z.Y., Hu C.Q., Chen, C., Zhang, L.P. and Ren C.H. (2005)

Investigation of seven Vibrio virulence genes among Vibrio

alginolyticus and Vibrio Parahaemolyticus strains from the

coastal mariculture systems in Guangdong, China. Letters in

Applied Microbiology.Vol. 41Pages 202-207.

Xu, H., He, L., Lv, S., Gong, Q. and Li, S. (2012) Establishment

of universal loop-mediated isothermal ampli cation method

(LAMP) for rapid detection of pathogenic Vibrio spp. In aquatic

organisms. African Journal of Microbiology Research. Vol.6

No (14) Pages 3447-3454.

Xu, Y., Cui, L., Liu, Z., Li, D., Li, S. and Zhao, M. (2014) Simult-

neous detection of  ve pathogenic Vibrio species in seafood by

a multiplex polymerase chain reaction coupled with high per-

formane liquid chromatography assay. Food Control. Vol.53

Pages 109-116.

Yong, L., Guanpin, Y., Hualei, W., Jixiang, C., Xianming, S.,

Guiwei, Z., Qiwei, W. and Xiuqin, S. (2006) Design of Vibrio

16S rRNA gene speci c primers and their application in the

analysis of seawater Vibrio community. Journal of Ocean Uni-

versity of China.Vol.5 No (2) Pages 157-164.

Yu, L. P., Hu, Y. H., Zhang, X. H, Sun, B. G. (2013) Development

of a Triplex Loop-Mediated Isothermal Ampli cation Method

for Rapid On-Site Detection of Three Vibrio Species Associ-

ated With Fish Diseases. Aquaculture Vol. 414–415 Pages 267–

273.