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MATERIALS AND METHODS
3. MATERIALS AND METHODS
3.1 Sampling sites
The samples included adult shrimps and post larvae collected from both the east and
west coast of India. On the east coast, samples were collected from shrimp farms of Andhra
Pradesh located in Kakinada, Bhimavaram, Amalapuram and Palkol. On the west coast,
samples were collected from shrimp farms of Karnataka in Hejmadi, Kundapura and Kumta
regions. Samples of postlarvae were collected from the hatcheries in Kakinada (Andhra
Pradesh) and Kumta (Karnataka).
3.2 Sample collection
Moribund samples were collected from affected shrimp farms. The samples meant for
histopathology were fixed immediately in Davidson’s fixative as described by Bell and
Lightner (1988). Live moribund shrimps were injected with Davidson’s fixative and then the
shrimps were kept in fixative for 24-48 h depending on their size. After this period, these were
transferred to 50% alcohol and stored until further analysis. Samples for bacteriology and
Reverse Transcription-Polymerase Chain reaction (RT-PCR) were chilled immediately in ice,
transported to the laboratory and analyzed immediately or stored at -20 ºC or -80 ºC for later
analysis. Samples meant for RNA extraction were processed immediately or stored at -80 ºC.
Water samples were collected in sterile bottles to determine the physico-chemical parameters.
3.3 Bacteriology
Apparently healthy and moribund shrimps were dissected aseptically to separate
hepatopancreas and the hind gut for analysis. The organs were taken in a microfuge tube,
crushed using sterile inoculation loop and streaked onto trypticase soya agar with 1.0% NaCl
(TSAS) and thiosulphate citrate bile salt sucrose (TCBS) agar. The same procedure was
followed for the postlarval samples collected from hatcheries. TSAS and TCBS agar plates
were incubated for 24 and 36 h respectively at 30 °C for isolation of bacteria.
Trypticase Soy Agar (1% TSAS)
Tryptone 1.5%
Soy peptone 0.5%
Sodium chloride 1.0%
Agar 1.5%
pH 7.3± 0.2
Thiosulphate citrate bile salt sucrose (TCBS) Agar
Yeast Extract 0.5%
Peptone 1.0%
Sucrose 2.0%
Sodium thiosulphate 1.0%
Sodium citrate 1.0%
Ox bile 0.5%
Sodium chloride 1.0%
Ferric citrate 0.1%
Bromothymol blue 0.004%
Agar 1.5%
pH 8.6
3.3.1 Isolation and Identification of bacteria
Colonies exhibiting distinct colony morphology were isolated from TSAS and TCBS
plates, streaked on fresh plates to purify and stored on 1% TSAS slants for further
identification.
3.3.1.1 Gram Staining
Hucker’s modification of Gram staining procedure was followed. The stained smears
were observed under oil immersion objective to record the Gram reaction of each isolate.
3.3.2 Biochemical tests
A series of biochemical tests were performed to identify bacterial isolates following the
previously described identification scheme for Gram positive bacteria (Le Chevallier et al.,
1980) and the Gram negative bacteria (Bain and Shewan, 1968 and Le Chevallier et al., 1980).
The identification schemes are schematically represented in Fig.1A, 1B, 1C. For the
identification of Vibrio spp. the criteria proposed by Farmer and Hickman-Brenner (1992) was
followed.
3.3.2.1 Oxidation fermentation test
Hugh Leifson’s O/F medium was used to determine whether the organism was
fermentative, oxidative or inert in metabolism of glucose. About 3 ml each of media were
poured into a set of two tubes and autoclaved. Organisms were stabbed into the butt and one of
the tubes was overlaid with liquid paraffin. Fermentative or oxidative activity was recorded by
a change in the colour of the medium from purple to yellow because of the production of acid.
Inert organisms failed to produce acid and hence media colour was unchanged.
Hugh Leifson’s O/F medium
Peptone 0.2%
Yeast extract 0.5%
Sodium chloride 1.0%
Glucose 1.0%
Bromocresol purple 0.0015%
Agar 0.3-0.4%
pH 7.2±0.1
3.3.2.2 Oxidase test
Oxidase test was performed to determine whether the bacteria possess cytochrome
oxidase enzyme. The test was performed using pre-moistened filter paper stripes soaked with
1% oxidase reagent. Overnight cultures of bacteria were spotted on the oxidase paper with a
tooth pick. Development of dark purple colour within 10 sec indicated positive reaction.
Oxidase reagent
Oxidase reagent (N,N,N,N' tetramethyl
p-phenylenediaminedihydrochloride 1.0 g
Distilled water 100 ml
Whatman filter paper No.1 was cut into stripes of 2.5x1.0cms dipped in the reagent, dried and
stored in dark bottle at 4 ºC.
3.3.2.3 Sensitivity to O/129 compound
A lawn culture of the test organism was prepared on TSAS and O/129 disc placed at the
centre of the lawn. Development of a clear zone of inhibition around the disc was recorded as
sensitive and no clear zone of inhibition indicated resistance to the pteridine compound.
O/129 reagent (150mg)
O/129 (2-4, diamino 6-7 diisopropyl pteridine) 15 mg
Acetone 1 ml
O/129 compound was dissolved in acetone and 100 discs of 6.5 mm diameter each
(presterilised at 140 ºC for 1h) were soaked in the solution, dried and stored at 4 ºC. Each disc
contained 150 μg of the pteridine compound.
Scheme for identification of bacteria from shrimp samples
Gram positive organisms
Cocci* Rods Branching filaments Catalase + Spores No spores Oxidase - Not acid fast colonies O/F glucose rubbery, firmly + - attached to agar medium Bacillus Cell Actinomycetes +/+ +/- or -/- morphology Staphylococcus Micrococcus Rod-Coccus Snapping division Transformation Corynebacterium Fig. 1a. Flow chart for identification of microorganisms
* Excludes catalase negative streptococci Source : Lechevallier et.al.(1980)
Gram negative organisms
O/F Glucose -/- or +/- O/F Glucose +/+ Non fermentative bacteria fig 1c Cocci or Coccobacilli Rods Oxidase (or sensitivity to penicillin) Motility + Moraxella Acinetobacter Water soluble Indole Blue green pigments + Pseudomonas No acid or gas No acid or gas Moraxella like From sugars pigments not Alcaligenes soluble O/F Glucose Flavobacterium Oxidative Alkaline No action Green fluorescent No diffusible No diffusible No diffusible diffusible pigment pigment pigment pigment Pseudomonas Pseudomonas Pseudomonas Pseudomonas Group I Group II Group III Group IV Fig1b. Flow chart for identification of Microorganisms. Source : Modified from Bain and Shewan, 1968 and Le chavellier et al., 1980.
Gram negative organisms O/F Glucose +/+
(Fermentative) Oxidase test
+ - Sensitivity to 0/129 Enterobacteriaceae (C) + - - Vibrio Aeromonas Plesiomonas Escherichia Klebsiella Enterobacter Salmonella Hafnia Serratia Proteus
*Arginine - + + Catalase + + + + + + + Lysine + - + Oxidase - - - - - - - Ornithine + - + Motility + - + + + + + Indole + - - - - D D M.R. + - - + - D D V.P. - - + - D + - Citrate - + + + + + D Urease - + D - - - +
D. Some strains positive and some negative Fig. 1c Flow chart for identification of Microorganisms Source: *Carpenter (1966) (C) Cruickshank et.al.,1975
Table-3.1 BIOCHEMICAL CHARACTERISTICS OF VIBRIO SPECIES WITH REPORTED ABILITY TO CAUSE HUMAN INFECTIONS Growth in
tryptone broth with
Acid from
Species
Oxi
dase
0/
129
sens
itive
O/F
TSI
Nitr
ate
-> n
itrite
A
rgin
ine
dihy
drol
ase
Lysi
ne d
ecar
boxy
lase
O
rnith
ine
0% N
aCl
3% N
aCl
8% N
aCl
11%
NaC
l Sw
arm
ing
on B
A
Indo
le
Vog
es –
Pro
skau
er
ON
PG
Gas
from
– D
-Glu
cose
Glu
cose
Su
cros
e
Lact
ose
Ara
bino
se
Cel
lobi
ose
Salic
in
Mal
tose
Man
nito
l
Man
nose
V. alginolyticus + S F A/A + - + V - + + + + (+) + - - + + - - + - + + + V. carchariae + S F A/A + - + + - + + - + + - - - + + - - + - + + + V. cholerae + S F A/A + - + + + + - - - + + + - + + - - - - + + (+) V. cincinnatiensis + S F A/A + - + - - + + - - - - + - + + - + + + + + + V. damsela + S F A/A + + + - - + V - - - + - V + (-) - - + - + - + V. fluvialis + S F A/A + + + - + + (+) - - (+) - - - + + - + (-) (+) + + (+) V. furnissii + S F A/A + + + - + + + - - - - - + + + - + - - + + + V. hollisae + S F A/A + - + - - + + - - + - - - + - - + - - - - + V. metschnikovii - S F A/A - V - - - + + - - - + - - + + (-) - - (-) + (+) + V. mimicus + S F A/A + - + + + + - - - + - + - + - - - - - + (+) - V. parahaemolyticus + S F A/A + - + + - + + - (+) + - - - + - - + - - + (+) + V. vulnificus + S F A/A + - + + - + - - - + - + - + (-) (+) - + - + (+) +
Symbols: + 90-100% positive; (+) 75-90% positive; V 25-75% positive; (-) 10-25% positive; - 0-10% positive.
Table-3.2 BIOCHEMICAL CHARACTERS OF VIBRIO SPECIES WHICH ARE GENERALLY NOT INVOLVED IN HUMAN INFECTIONS
Growth in tryptone broth with
Acid from
Species O
xida
se
0/12
9 se
nsiti
ve
O/F
Nitr
ate
-> n
itrite
Indo
le
Vog
es –
Pro
skau
er
Arg
inin
e di
hydr
olas
e
Lysi
ne d
ecar
boxy
lase
Orn
ithin
e de
carb
oxyl
ase
0% N
aCl
3% N
aCl
8% N
aCl
11%
NaC
l
Gas
from
– D
-Glu
cose
Glu
cose
Sucr
ose
Lact
ose
Ara
bino
se
Cel
lobi
ose
Salic
in
Mal
tose
Man
nito
l
Man
nose
V. aestuarianus + S F + + - + V - - + V - - + + (+) - + - + + + V. anguillarum + S F + (+) + (+) - - - + - - - + + - (+) (+) - + + + V. campbellii + S F + (+) - - + (-) - + V - - + - - - + - + V + V. costicola + S F V - (+) V - - - + + V - + V - - (-) - (+) V (+) V. diazotrophicus + S F + + - - - - - + + - - + + V + + + + + (+) V. fischeri + S F (+) - - - V - - + - - - + - (-) - + (+) + V + V. gazogenes - S F - - V - - - - + + + + + + (+) + + + + + + V. harveyi + S F + + - - + (+) - + (+) - - + + - - + - + + + V. logei + S F + - - - + V - + - - - + (-) (-) - + - + (+) + V. mediterranei + S F + + - V V - - + V - - + + - - + - + + + V. marinus + S F + - - - - - - + - - - + - - - - - + - - V. natriegens + S F + - - - - - - + + - - + + - + (+) + + + (+) V. nereis + S F + V - (+) - - - + + - - + + - - - - + - + V. nigripulchritudo + S F + + - - - - - + - - - + - - - + - + - V V. ordalii + S F + - - - - - - + - - - + + - - - - + - - V. orientalis + S F + + - - + - - + + - - + + - - + - + + + V. pelagicus biogroup1 + S F + - - - - - - + + - - + + (-) - (-) - + + (+) V. pelagicus biogroup2 + S F + + - - - - - + - - - + V - - - - + + + V. proteolyticus + S F + + - + + - - + + + - + - - - + - + + + V. salmonicida + S F + - - - - - - + - - - + - - - - - - + + V. splendidus biogroup1 + S F + + - (-) - - - + V - - + (+) - - + - + + + V. splendidus biogroup2 + S F + + - - - - - + V - - + - - - + - + + - V. tubiashii + S F + + - - (+) - - + V - - + + - - (+) - + + + V. vulnificus biogroup2 + S F + - - - - + - + - - - + - V - + - + - V
Symbols: + 90-100% positive, (+) 75-90% positive, V 25-75% positive , (-) 10-25% positive, - 0-10% positive
3.3.2.4 Amino acid decarboxylase test
Ability of the bacteria to decarboxylate aminoacids lysine, ornithine and arginine
was tested by inoculating test cultures into media containing aminoacids in separate tubes
and to a tube with only basal medium which served as control. After overlaying with
liquid paraffin, all the tubes were incubated at 30 ºC. When the colour of media turned to
yellow and returned to purple, the reaction was taken as positive, whereas the control
tube without aminoacid remained yellow.
Basal medium
Peptone 0.5%
Yeast extract 0.3%
Sodium chloride 1.0%
Glucose 0.1%
Bromocresol purple 0.0016%
pH 7.2 ± 0.2
The basal medium was divided into four parts and aminoacids L-Lysine
monohydrochloride, L-ornithine monohydrochloride and L-arginine monohydrochloride
were added individually to each quarter at a concentration of 0.5, 0.5 and 0.4%
respectively. The last quarter served as control. The media was distributed into tubes, in
4ml volumes and sterilized at 110 ºC for 15 min.
Liquid paraffin
Liquid paraffin was sterilized at 180 ºC for 2 h in a hot air oven.
3.3.2.5 Sugar fermentation test
Different sugars viz glucose, sucrose, lactose, arabinose, cellobiose, salicin,
maltose and mannitol were used as the substrates to observe the ability of the bacterial
cultures to break down the same. Cultures were inoculated into presterilized individual
sugar media in tubes and incubated. Colour change from purple to yellow indicated
fermentation of the sugar.
Sugar fermentation medium
Peptone 0.2%
Sodium chloride 1.0%
Sugars 1.0%
Bromocresol purple 0.0016%
pH 7.1 ± 0.2
The ingredients were dissolved in distilled water and then dispensed in 4 ml volume into
test tubes containing Durham’s tubes and sterilized by autoclaving at 110 ºC for 15 min.
3.3.2.6 Indole test
The cultures were grown in tryptophan broth for 24 h and then a few drops of
Kovac’s reagent was added. Formation of a pink indole ring floating on the surface of
culture was recorded as a positive reaction.
Tryptophan broth
Tryptophan 1.0%
Sodiumchloride 1.0%
pH 7.1 ± 0.2
This was distributed in 5 ml volumes into tubes and autoclaved at 121 ºC for 15 min.
Kovac’s reagent
p-Dimethyl aminobenzaldehyde (DMAB) 5.0 g
Amyl alcohol 75 ml
Hydrochloric acid 25 ml
DMAB was dissolved in amyl alcohol and then the concentrated hydrochloric acid was
added slowly. The solution was stored and used to perform the test whenever required.
3.3.2.7 Methyl red test
The ability of the bacteria to break down glucose to produce stable acid end
products was tested by growing the cultures in glucose phosphate peptone broth for 48-72
h and adding few drops of methyl red indicator. A red colouration indicates positive
reaction.
Glucose phosphate peptone broth
Peptone 0.5%
Dipotassium hydrogen phosphate (K2HPO4) 0.5%
Sodium chloride 1.0%
Glucose 0.5%
pH 7.6
The media after preparation was dispersed into tubes in 5 ml volumes and sterilized by
autoclaving at 110 ºC for 15 min.
Methyl red reagent
Methyl red 0.1 g
Alcohol (95%) 300 ml
Methyl red was dissolved in ethyl alcohol and the volume made upto 500 ml, filtered and
stored.
3.3.2.8 Voges-Proskauer (VP) test
Production of neutral end products like acetyl methyl or dimethyl carbinol was
tested by growing the bacteria for more than 48 h in glucose phosphate peptone broth and
adding an alkaline α-Naphthol solution. Development of port wine colour was recorded
as positive reaction.
VP-A solution
Potassium hydroxide 40 g
Distilled water 100 ml
VP-B solution
α -Naphthol 5.0 g
Ethyl alcohol (95%) 100 ml
0.2 ml of VP-A and 0.6 ml of VP-B solution were added to about 5 ml of the culture and
the tubes held for 5 min to 2 h to obtain the reaction colour.
3.3.2.9 Citrate utilization test
Ability of bacteria to use citrate as the sole source of carbon for metabolism was
tested by growing the cultures in Simmon’s citrate agar slants. Change of colour from
green to prussian blue colour and growth of bacteria was recorded as a positive reaction.
Simmon’s citrate agar
Sodium chloride 1.0%
Magnesium sulphate 0.02%
Ammonium dihydrogen phosphate 0.1%
Potassium dihydrogen phosphate 0.1%
Sodium citrate 0.2%
Bromothymol blue 0.008%
Agar 2.0%
pH 6.8
The medium was boiled to dissolve, distributed into tubes in 3 ml volumes and sterilized
at 121 ºC for 15 min. Slants were prepared after it was autoclaved.
3.3.2.10 Urease test
Cultures were grown on urea slants to see their ability to break down urea. The
reaction was recorded as positive if the slants changed colour from yellow to pink after
incubation with the culture.
Basal medium
Peptone 0.1%
Dipotassium hydrogen phosphate 0.2%
Glucose 0.1%
Phenol red 0.0012%
Agar 1.5%
pH 6.9
Autoclave the basal medium at 110 ºC for 15 min. To 90 ml of this molten medium 10ml
of 20% filter sterilized urea solution was added, distributed in 4 ml volume in to sterile
tubes and slants prepared.
3.3.2.11 Salt tolerant test
This test was carried out to see the ability of bacteria to grow in varying
concentration of salt. Cultures were inoculated to tryptone broth containing 0%, 3%, 8%,
and 11% sodium chloride and incubated at 37 ºC for 18 to 24 h.
Tryptone broth
Tryptone 1.0%
Yeast extract 0.3%
pH 7.2
The ingredients were dissolved in distilled water. To this basal medium sodium chloride
was added to give final concentration as mentioned above.
3.3.2.12 Triple sugar iron agar (TSI) test
TSI test was performed by stabbing the culture in the butt of TSI tube and
streaking the slant. Tubes were incubated at 30 ºC for 24 h and observed for characteristic
reaction. Cultures showing an acid butt/ acid slant or acid butt/ alkaline slant with /
without H2S production were further proceeded with.
Triple sugar iron agar (TSIA)
Peptone 1.0%
Tryptone 1.0%
Yeast extract 0.3%
Beef extract 0.3%
Lactose 1.0%
Saccharose 1.0%
Dextrose 0.1%
Ferrous sulphate 0.02%
Sodium chloride 1.0%
Sodium thiosulphate 0.03%
Phenol red 0.0024%
Agar 1.2%
pH 7.4 ± 0.2
After melting the medium was dispersed in 5-7 ml volume tubes and after autoclaving
was allowed to set at an appropriate angle to get a butt of about 1inch and a slant.
3.3.2.13 Catalase test
On dipping young cultures of bacteria in 3 % v/v hydrogen peroxide, release of
oxygen as effervescence was observed and recorded as positive reaction.
3.4 Histopathology
The histopathological analysis of shrimps was carried out according to the method
of Bell and Lightner (1988). The fresh specimen was fixed in the Davidson’s AFA
(Humason, 1972) fixative by injecting the fixative to the live moribund shrimps and
cutting the head part in a cross section to allow the easy and rapid penetration of fixative.
After 24 h, the shrimps were transferred to 50% ethyl alcohol (EtOH).
Shrimps were removed from 50% ethyl alcohol (EtOH) and placed on a cutting
board. Using a scalpel, the shrimps (larger than 3cm) were bisected transversely. The
distal 80% of head appendages were removed. Then the cephalothorax was bisected
longitudinally and some shrimps cross sectionally. Then the required tissue blocks were
processed in the following ascending grades of alcohol for dehydration.
70% EtOH Two separate 1h baths
80% EtOH Two separate 1h baths
95% EtOH Two separate 1h baths
100% EtOH Two separate 1h baths
Xylene (E-Merck) Two separate 1h baths
Paraffin Wax (E-Merck) Two to four 1h baths.
The wax infiltration step was carried out on a Leica EG1120 hot plate (Leica,
Germany) and tissues successfully embedded were placed in molten wax filled
embedding moulds and transferred to a Leica EG1130 Cryoplate (Leica Germany), to
form blocks.
The blocks were sectioned in a Leica RM 2125 microtome (Leica Germany) using
disposable knives. The blocks were first face trimmed till the desired tissue was exposed
after which they were cut into sections of 5μm thickness. The ribbons were floated on
water maintained at 50 ºC in a water bath to remove the wrinkles. Good sections were
selected and transferred to egg white coated slides. After brief air drying, the slides were
kept on 55 ºC hot plate for at least 4 h to coagulate the egg white to attach the sections to
the slides.
The slides were stained by placing them in couplin jars, following the sequence
solutions and time mentioned below.
Xylene 5 min
Xylene 5 min
100% EtOH 10 dips
100% EtOH 10 dips
95% EtOH 10 dips
95% EtOH 10 dips
80% EtOH 10 dips
80% EtOH 10 dips
50% EtOH 10 dips
Distilled water 5 min
Harris hematoxylin (E-Merck) 15 min
Running tap water 5 min
Acid Alcohol 3-5 dips
Tap water Brief rinsing
Ammonia water 3-5 dips
Running tap water 30 min
Eosin (Qualigens) 1-3 sec
95% EtOH 10 dips
95% EtOH 10 dips
100% EtOH 10 dips
100% EtOH 10 dips
Xylene 10 dips
Xylene 10 dips
Xylene 10 dips
Xylene 10 dips
The sections were then mounted in a DPX mountant (Qualigens, Mumbai) with
appropriate cover slips and left for drying overnight at room temperature.
3.5 Detection of viruses by polymerase chain reaction (PCR)
3.5.1 Procedure for extraction of DNA
Approximately 100 mg of individual organs like hepatopancreas, gills and gut
were removed aseptically from the shrimps, transferred to a sterile disposable plastic
pouch and homogenized. Eight hundred μl of digestion buffer (10mM Tris-HCl, pH 8.0,
0.1M EDTA, pH 8.0, 0.5% Triton X 100, 6M Guanidine hydrochloride, 0.1M Sodium
acetate) was added and the homogenate was incubated at room temperature (28º C) for 30
min. Following incubation, the homogenate was transferred to a 1.5 ml microfuge tube
and centrifuged at 6000x g for 10 min. The supernatant was transferred to a fresh tube,
equal volume of chloroform added vortexed and then centrifuged at 6000x g for 10 min.
The aqueous phase containing DNA was transferred to a fresh tube, precipitated by
adding 2-3 volumes of 100% ethanol, and centrifuged at 14000x g for 20 min. The pellet
was washed twice with 1 volume of 95% ethanol and then vacuum dried. Finally pellet
was dissolved in 100 μl of 1x TE buffer or 100 μl of distilled water. The extracted DNA
was stored at -20 ºC till further use.
3.5.2 Primers and PCR protocol for WSSV
PCR was carried out in 30 μl reaction mixture containing 2.0 μl of template DNA,
1X assay buffer (10mM Tris-HCl, pH 9.0, 1.5mM MgCl2, 50mM KCl, 0.01% Gelatin),
100 μM of each of the four dNTP’s, 10 pico moles of forward and reverse primers and
0.9 U of Taq DNA polymerase (Bangalore GENEI, Bangalore). Primer sequence and
cycling conditions were given in Table-3.3.
3.5.3 Primers and PCR protocol for MBV
DNA extracted from hepatopancreas served as template for PCR detection of
monodon baculovirus (MBV). The PCR was carried out in 30ul reaction mixture
containing 2.0 μl template DNA, 1X assay buffer (10mM Tris-HCl, pH 9.0, 1.5mM
MgCl2, 50mM KCl, 0.01% Gelatin), 100 μM of each of four dNTP’s, 10 pico moles of
forward and reverse primers and 0.9 U of Taq DNA polymerase (Bangalore GENEI,
Bangalore). Primer sequence and cycling conditions were given in Table-3.4.
3.5.4 Primers and PCR protocol for HPV
PCR was carried out in 30ul reaction mixture containing 2.0 μl template DNA,
1X assay buffer (10mM Tris-HCl, pH 9.0, 1.5mM MgCl2, 50mM KCl, 0.01% Gelatin),
100 μM of each of four dNTP’s , 10 pmol of forward and reverse primers and 0.9 U of
Taq DNA polymerase (Bangalore GENEI, Bangalore). Primer sequence and cycling
conditions were given in Table-3.5.
3.5.5 PCR detection of NHP
PCR was carried out in 30 μl reaction mixture containing 2.0 μl template DNA,
1X assay buffer (10mM Tris-HCl , pH 9.0, 1.5mM MgCl2, 50mM KCl, 0.01% Gelatin ),
100 μM of each of four dNTPs, 10 pmol of forward and reverse primers and 0.9 U of
Taq DNA polymerase (Bangalore GENEI, Bangalore). The reactants are heated at 94ºC
for 5min and the amplification profile consists of 35 cycles of 30 s at 94ºC, 30 s at 58ºC
and 1 min at 72ºC. The final extension of flush ends was performed at 72ºC for 5 min.
The forward primer and reverse primer-1 amplify a 441bp fragment. The forward primer
and the reverse primer-2 amplify a 660bp fragment (Loy et al., 1996).
Below is the sequence of oligonucleotide primers used for amplifying variable regions of
the 16s rRNA sequence:
Forward : 5’ ACG TTG GAG GTT CGT CCT TCA G 3’
Reverse -1 : 5’ TCA CCC CCT TGC TTC TCA TTG T 3’
Reverse -2 : 5’ CCA GTC ATC ACC TTT TCT GTG GTC 3’
3.6 Detection of RNA viruses of shrimp and freshwater prawn by reverse
transcription PCR (RT-PCR)
3.6.1 Extraction of RNA
All the glasswares and plasticwares were prepared for RNA work as described by
Sambrook et al (1989).
Reagents used for RNA extraction
Trizol LS reagent (Invitrogen, USA)
Chloroform
Isopropyl alcohol
75% Ethanol (in DEPC treated water)
RNase free water
Procedure
RNA extracted using TRIzol LS reagent (Invitrogen, USA)
Steps
1. 50-100 mg of fixed hepatopancreas/PL (shrimp) or muscle/pleopods/eggs
(freshwater prawn) tissue was aseptically transferred to an RNase free, disposable
plastic pouch.
2. The tissue was homogenized in 0.75 ml of Trizol LS reagent and the homogenate
was transferred to RNase free 1.5 ml microfuge tube and the volume adjusted to
1ml with diethylpyrocarbonate (DEPC) treated water.
Table-3.3 Primers and PCR conditions for WSSV
Cycling conditions Primers
Denaturation Annealing Extension
No. of
cycles
Product
size Reference
I Step
IK-1: 5’ TGG CAT GAC AAC GGC AGG AG 3’
IK-2 : 5’GGC TTC TGA GAT GAG GAC GG 3’
94ºC for 30s 55ºC for 30s 72ºC for 30s 30 486bp
II step
IK-3: 5’ TGT CAT CGC CAG CAC GTG TGC 3’
IK-4: 5’ AGA GGT CGT CAG AGC CTA GTC 3’
94ºC for 30s 55ºC for 30s 72ºC for 30s 30 310bp
Hossain et
al., 2001a.
Table-3.4 Primers and PCR conditions for MBV
Cycling conditions Primers
Denaturation Annealing Extension
No. of
cycles
Product
size Reference
I Step
MBV1.4F: 5’ CGA TTC CAT ATC GGC CGA ATA 3’
MBV1.4R: 5’ TTG GCA TGC ACT CCC TGA GAT 3’
94ºC for 30s 65ºC for 30s 72ºC for 30s 40 533bp
II step
MBV1.4NF: 5’ TCC AAT CGC GTC TGC GAT ACT 3’
MBV1.4NR: 5’ CGC TAA TGG GGC ACA AGT CTC 3’
94ºC for 30s 60ºC for 30s 72ºC for 30s 35 361bp
Belcher
and
Young,
1998.,
Otta et al.,
2003
Table-3.5 Primers and PCR conditions for HPV
Cycling conditions Primers
Denaturation Annealing Extension
No. of
cycles
Product
size Reference
I Step
HPV441F: 5’ GCA TTA CAA GAG CCA AGC AG 3’
HPV441R: 5’ ACA CTC AGC CTC TAC CTT GT 3’
94ºC for 30s
55ºC for 30s
72ºC for 30s
30
441bp
II step
HPVnF: 5’ATA GAA CGC ATA GAA AAC GCT 3’
HPVnR: 5’ CAG CGA TTC ATT CCA GCG CCA CC 3’
94ºC for 30s
55ºC for 30s
72ºC for 30s
30
310bp
Phromjai
et al.,
2001.,
Manjanaik
et al.,
2005;
Umesha et
al., 2006
3. The homogenate was then incubated for 5min at room temperature (28º ± 1ºC) to
permit the Complete dissociation of nucleoprotein Complexes.
4. 200 μl of Chloroform was then added, mixed by vigorous shaking by hand for 15
sec and incubated for 15 min at room temperature (28º ± 1ºC).
5. Sample was centrifuged at 12000x g for 15min at 4ºC.
6. The upper colourless aqueous phase containing RNA was transferred to a fresh
microfuge tube and 0.5 ml of isopropyl alcohol was added, incubated for 10min at
room temperature (28º ± 1ºC ) to precipitate the RNA.
7. The RNA precipitate was sedimented by centrifugation at 12000x g for 10 min at
4ºC.
8. The supernatant was removed and the RNA pellet was washed with 1 ml of 75%
ethanol. Sample was mixed by vortexing and centrifuged at 7500x g for 5 min at
4ºC.
9. The supernatant was removed and the pellet briefly dried under vacuum,
dissolved in DEPC treated water and stored at -80ºC.
3.6.2 Determination of purity and integrity of RNA preparation (Farrell, 1998)
The integrity of the RNA isolated was determined by electrophoresis through a
standard, non-denaturing 1% agarose gel.
Steps
1. 1% agarose gel was prepared in 1 X TAE buffe with ethidium bromide (Sigma
Chemicals Co. USA) added to a final concentration of 0.5 μg/ml.
2. Five μl of RNA preparation was diluted in 10 ml diethylpyrocarbonate (DEPC,
Calbiochem) treated water. To this 2.5 μl of formaldehyde (37% stock solution )
and 0.5 μl formamide (Himedia, Mumbai) were added.
3. The solution was heated to 65ºC for 5 min and pulse centrifuged to collect the
sample at the bottom of the tube.
4. The solution was then mixed with 6X loading buffer (0.25% Bromophenol blue,
40% w/v sucrose in water) and loaded into the gel.
5. The samples were electrophoresed at 80-120 volts and the bands were visualized
under UV light.
3.6.3 Reverse Transcription Polymerase Chain Reaction (RT-PCR)
To detect the presence of Laem-singh virus, an RNA virus, reverse transcription
polymerase chain reaction was carried out using the primers LLV(F) and LLV(R) as
described previously by Sritunyalucksana et al (2006).
3.6.4 Synthesis of complementary DNA (cDNA) from RNA
Extracted RNA (100 ng) was incubated at 42ºC for 1 h to make cDNA in a 10 μl
reaction mixture containing 5x first strand buffer (250 mM Tris-HCl, pH 8.3, 375mM
KCl, 15mM MgCl2), 10 mM Dithiotritol (DTT), 100 U of superscript II Reverse
Transcriptase (RT) (Invitrogen life technologies), 20 U of human placental RNase
inhibitor (Bangalore GENEI, Bangalore), 200 mM each dATP, dCTP, dGTP and dTTP,
0.5 μl of formamide (Himedia, Mumbai), 35 pmol each of LLV(F) and LLV(R) primer
mix.
Following cDNA synthesis, the mixture was incubated at 99ºC for 5min to
inactivate the reverse transcriptase and then the product was cooled to 5º C.
3.6.5 Polymerase Chain Reaction (PCR)
PCR was carried out in 30 μl reaction mixture containing 2.0 μl of reverse
transcription product (cDNA), 1X assay buffer (10mM Tris-HCl, pH 9.0, 1.5mM MgCl2,
50mM KCl, 0.01% Gelatin ), 100 μM of each of four dNTP’s, 10 pmol of forward and
reverse primers LLV (F) and LLV (R) and 0.9 U of Taq DNA polymerase (Bangalore
GENEI, Bangalore).
The cycling conditions consisted of: one cycle at 94ºC for 5 min, 35 cycles at
94ºC for 1 min, 60ºC for 1 min, 72ºC for 1 min and a final incubation at 72ºC for 10 min.
The primers LLV-F and LLV-R amplify 200bp fragment (Table-3.6).
New set of primers (LSN-F and LSN-R) (Table-3.6) were developed internal to
the 200bp fragment and these primers after amplification yielded a product of 154bp.
Except annealing temperature of 58ºC for 1min, the PCR conditions were same as that for
previous primers.
Table-3.6 Primer sequences used for detection of LSNV
Virus Primer
name Primer sequence (5’- 3’) Reference
LSNV LLV-F TTG CCT TCT CCC GAG TGG TC LLV-R CCG GCT GAG GTA GCT GCT TG
Sritunyalucksana
et al., 2006
LSN-F GCG CAA GAG TTC TCA GGC TT LSN-R ATC ACC GCA GGC TAA TAT AG
This study.
3.6.6 Electrophoresis
All the PCR and RT-PCR products were analysed in agarose gel at appropriate
percentage (w/v). 20 μl of the amplicons were mixed with 4μl of 6X loading buffer and
loaded into the wells of agarose gel containing 0.5 μg/ml ethidium bromide. A 100bp
ladder (Bangalore GENEI, Bangalore) was used as a molecular weight marker. The
electrophoresis was carried out at 80-120 volts and the bands were visualized under UV
light.
TAE buffer
Stock solution (50X)
Per liter composition
Tris base 242 g
Glacial acetic acid 57.1 ml
0.5M EDTA (pH 8.0) 100 ml
Working solution (1X)
0.04M Tris-acetate
0.001M EDTA
Stored at room temperature (28º ± 1ºC)
6X Loading buffer
0.25% Bromophenol blue
40% w/v Sucrose in water
stored at 4ºC.
3.7 Cloning of PCR products
3.7.1 Purification of PCR product for cloning
LSNV PCR products were purified using QIAquick PCR Purification Kit
(QIAGEN). In this procedure PCR fragments ranging from 100bp to 10kb can be purified
from primers, nucleotides, polymerases and salts.
Steps
1. 5 μl of buffer PB was added to 1 volume of the PCR sample and mixed.
2. QIAquick spin column was placed in 2 ml collection tube.
3. The sample is applied to the QIAquick column to bind DNA (PCR Product) and
centrifuged at 13000 rpm (17900 x g) for 30-60s.
4. The flow through was discarded. To wash the DNA, 0.75 ml buffer PE was added
to the QIAquick column and then centrifuged at 13000 rpm (17900 x g) for 30-
60s.
5. The flowthrough was discarded and the QIAquick column was centrifuged for
additional 1 min at 13000 rpm (17900 x g).
6. QIAquick column was placed in a 1.5 ml microcentrifuge tube.
7. To elute DNA (PCR Product), 50 μl of buffer EB (10mM Tris-Cl, pH 8.5) or
water was added to the center of the QIAquick membrane and centrifuged the
column for 1 min and then centrifuged at 13000 rpm (17900 x g) for 1 min. The
elute collected in the microcentrifuge tube was the pure PCR Product which can
be used for cloning or directly sent for sequencing.
Media and reagents for cloning
Ampicillin stock solution (50 mg/ml)
Ampicillin (HiMedia, Mumbai) 50 mg
Sterile distilled water 1 ml
The solution was filtered using 0.22 μ syringe filter (Millipore, USA) and stored in the
refrigerator at 4 °C.
Luria Bertani Medium (HiMedia, Mumbai)
Casein enzymic hydrolysate 10.0 g
Yeast extract 5.0 g
Sodium chloride 10.0 g
Agar agar 20.0 g
Distilled water 1000 ml
pH 7.5± 0.2
The medium was autoclaved at 121°C for 15 min at 15 lb pressure and cooled to
50°C. Antibiotic solution was added to a desired concentration and poured into pre-
sterilised plates. The antibiotic containing LB agar plates were stored in refrigerator for
not more than a week.
X-Gal (MBI Fermentas, USA)
X-Gal (5-bromo-4-chloro-3-indolyl-β-
D-galactopyranoside) 40 mg
Dimethylformamide 1 ml
The solution was stored at –20°C in brown bottles
Reagents supplied
i) Vector: pCR 2.1- TOPO. It is a linearised vector with a single overhanging 3’
deoxy thymidine (T) residue. This will allow insertion of Taq polymerase
amplified PCR fragments with single deoxyadenosine (A) at 3’ ends efficiently.
ii) Competent cells: TOP10, One shot chemically competent cells
iii) SOC medium
Procedure
Cloning of the LSNV PCR products was performed using TOPO TA cloning kit
(Invitrogen, USA). Cloning was performed using the reagents and competent cells
supplied along with the kit. Briefly, a cloning reaction was set up with 4 μl of pure PCR
product, 1 μl of salt solution and 1 μl of pCR 2.1 TOPO vector. This mixture was
incubated for 30 minutes at 22°C. After incubation, 2 μl of cloning reaction was added to
a vial of one shot chemically competent cells (E. coli), mixed gently and incubated in ice
for 30 minutes. The cells were then given a heat shock for 30 seconds at 42°C. A 250 μl
of SOC medium was added to the heat shocked cells and incubated for 1 h at 37°C with
vigourous shaking (200 rpm) (Orbitek, India). Different volumes (10-50 μl) of
transformation mixture were then spread plated on LB agar containing 50 μg/ml
ampicillin and X-Gal and incubated for 24-48 h at 37°C. Selection of colonies was based
on blue white phenotype. White and light blue colonies were picked from the plates and
transferred onto fresh LB-ampicillin plates and grown overnight. The clones were stored
in the refrigerator at 4°C until further analysis.
3.7.2 Amplification of other genes of Laem Singh virus
In an attempt to amplify other genes of Laem-Singh Virus, the sequence of the
coat protein of closely related virus (Mushroom bacilliform virus) (Accession number:
AAA53090) was selected from the GenBank and three sets of primers were developed
using “Primer3” software (http://frodo.wi.mit.edu/). The primer sequences and PCR
protocols are given in Table-3.7.
Detection of Macrobrachium rosenbergii noda virus (MrNV) and Extra small virus
(XSV) by RT-PCR
One pair of primer specific to MrNV –RNA1 was designed from sequence data of
the MrNV genome (GenBank Accession No. AY222839). The primer sequences and
PCR protocols are given in Table-3.8.
Table-3.7 Primer sequence and PCR protocol used for characterizing the new virus (LSNV).
Cycling Conditions Primer sequence
Denaturation Annealing Extension
No. of
cycles
Product
size Reference
LSF1: 5’ CTA ATC GAG CAA CCC AGA GC 3’
LSR1: 5’ TCG AAT GCC ATC AAA CTC AA 3’ 94ºC for 1min 45ºC for 1min 60ºC for 1min 35 500bp This Study.
LSF2: 5’ CTA ATC GAG CAA CCC AGA GC 3’
LSR2: 5’ CTA GGA ACA GTC GGC GTA GC 3’ 94ºC for 1min 45ºC for 1min 60ºC for 1min 35 300bp This Study.
LSF3: 5’ CGC AGC ATC TCC TAC GAA AC 3’
LSR3: 5’ GGA ACC ACT CGA CAA CCA TT 3’ 94ºC for 1min 45ºC for 1min 60ºC for 1min 35 284bp This Study.
Table-3.8 Primer sequence and PCR protocol used for detecting MrNV and XSV.
Cycling Conditions Primer sequence
Denaturation Annealing Extension
No. of
cycles
Product
size Reference
MrNV-F: 5’ TGC TTG GTG TCA AGA GAA CG 3’
MrNV-R: 5’ GCT GGG CAA CTA ACC AAC AT 3’ 94ºC for 40 s 55ºC for 40s 68ºC for 1min 30 108bp This Study.
XSV-F: 5’ GGA GAA CCA TGA GAT CAC G 3’
XSV-R: 5’ CTG CTC ATT ACT GTT CGG AGT C 3’ 94ºC for 40 s 55ºC for 40 s 68ºC for 1min 30 500bp Sri Widada
et al., 2004