MATERIALS AND METHODSshodhganga.inflibnet.ac.in/bitstream/10603/7011/7/07_chapter 3.pdf · A series of biochemical tests were performed to identify bacterial isolates following the

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%


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%


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%


Glucose 0.1%


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%


Sugars 1.0%


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%


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


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


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 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




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’


Hossain et

al., 2001a.

Table-3.4 Primers and PCR conditions for MBV



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’


II step

MBV1.4NF: 5’ TCC AAT CGC GTC TGC GAT ACT 3’

MBV1.4NR: 5’ CGC TAA TGG GGC ACA AGT CTC 3’


Belcher

and

Young,

1998.,

Otta et al.,

2003

Table-3.5 Primers and PCR conditions for HPV



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


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.

http://frodo.wi.mit.edu/

Table-3.7 Primer sequence and PCR protocol used for characterizing the new virus (LSNV).

Cycling Conditions Primer sequence


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


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

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