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MATERIALS AND METHODS

III. MATERIALS AND METHODS

3.1 General considerations

The glassware used in this study were of neutral glass of Corning, Borosil

India Ltd., or Scott Durham (Germany) make. The culture media, buffers and other

biochemical reagents were prepared in MilliQ water (Millipore). The chemicals of

Analar, Excellar or molecular biology grade were used for the preparation of various

solutions and reagents. The culture media, buffers, reagents, enzymes, kits and other

requirements were obtained from M/s. HImedia, Mumbai; BD, BBL and DIFCO,

USA; Sisco Research Laboratories Pvt. Ltd., Mumbai; Bangalore Genei, Bangalore;

Chromous Biotech Pvt. Ltd., Bangalore; Sigma Aldrich, USA; Fermentas Life

Sciences, QIAGEN (Qiagen, Inc., Chatsworth, CA); E-Merck (India) Ltd., and

Machery-Nagel Gmbh & Co. Germany. Plasticware including syringes, micro

centrifuge tubes, micropipette tips, cryovials, Petri dishes and autoclave bags were

procured from M/s. Tarson Products Pvt. Ltd., Kolkata and Axygen Inc., USA.

3.1.1 Preparation of glassware

The glassware used in the study were prepared by soaking them in detergent

(Teepol) solution over night. The following day, they were washed thoroughly in running

tap water, followed by rinse in deionised / distilled water (DW). The oven dried

glasswares were packed and sterilized in hot air oven for one hr at 160 C as per Collee et

al. (1996).

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3.1.2 Preparation of plasticware

The new plasticware including micro centrifuge tubes and micropipette tips were

sterilized by autoclaving at 121oC for 15 min at 15 psi.

3.1.3 Media reagents and chemicals

The media and reagents were either obtained from HImedia, Mumbai or

prepared in the laboratory as per the standard procedures.

3.1.4 Media and reagents used

Streptococcus Selection broth, Blood agar, Brain Heart Infusion (BHI) broth

and agar, Tryptose phosphate broth, MacConkey agar, Mannitol salt agar, Nutrient

agar and broth, Pyrolidonyl reduction (PYR) agar,Toludine blue DNA agar, Glucose

phosphate peptone medium, Voges-Proskauer’s test medium, Modified Bile Esculin

Azide agar, Peptone water, Sodium hippurate, Ninhydrin and sugars like Glucose,

Sucrose, Mannitol, Arabinose, Ribose, Raffinose, Maltose and Trehalose, Hydrogen

peroxide, and Gram staining kit (M/s HImedia Laboratories Pvt. Ltd., Mumbai).

Media and reagents were prepared as recommended by the manufacturer and

sterilized by autoclaving.

3.1.4.1 Blood agar

Ready to use 5 per cent sheep blood agar plates were purchased from M/s JDM

Technologies, Bangalore.

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3.1.4.2 Nutrient-glycerol broth

Nutrient broth 85 mL (sterile)

Sterile glycerol 15 mL

Glycerol was sterilized in hot air oven at 160o C for one hr and added into nutrient

broth, mixed well and aliquoted into 1ml sterile tubes and stored at 4º C.

3.2 Biochemical test media and reagents

3.2.1 Phenol red peptone water carbohydrate media

Pancreatic digest of casein 1.0 g

Sodium chloride 0.5 g

Phenol red 1.8 mg

Carbohydrate 1.0 g

Distilled water 100 mL

The medium was prepared manually and distributed into sterile tubes and steamed

for 30 min. Two mL of the medium was supplemented with 4-5 drops of sterile serum

and was used for performing sugar fermentation reactions.

3.2.2 Ninhydrin reagent

Ninhydrin available as powder form was procured form M/s HImedia, Mumbai

and the reagent was prepared as mentioned below.

Ninhydrin powder 3.5 g

1:1 mixture of Acetone and Butanol 100 mL

During the test, the reagent was used at the rate of 0.2 mL per tube.

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3.2.3 Sodium Hippurate

Sodium hippurate, available as powder form was procured from M/s HImedia,

Mumbai and the reagent was prepared as mentioned below.

Sodium Hippurate 1 g

DW 100 mL

During the test, this reagent was used at the rate of 0.5 mL per tube.

3.3 Collection of milk samples

Milk samples were collected from four organized farms (Farm A, B, C and D) and

three unorganized sectors from various geographic locations in and around Bangalore.

Milk samples were collected in 10 mL sterile tubes following strict aseptic measures and

immediately transported to laboratory in refrigerated condition.

3.4 Screening for subclinical mastitis

A total of 246 milk samples were collected from different sources mentioned

below (Table 1).

Table 1. Farm wise details of milk samples collected

Sl. No. Farm code No. of milk samples1 Farm A 102 Farm B 173 Farm C 614 Farm D 275 Unorganized sector A 756 Unorganized sector B 307 Unorganized sector C 26

Total 246

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3.4.1 Measurement of Electrical Conductivity

Milk samples procured were subjected for EC test (Milk checker, Oriental

instruments limited, Tokyo, Japan). Five mL milk sample was used for this purpose. EC

of more than 6.5 milli Siemens (mS)/cm was taken as a criteria to declare the milk /

animal as subclinically mastitic /infected and such milk samples were subjected for

cultural examinations.

3.4.2 Estimation of Somatic Cell Count

Somatic Cell Count was estimated using Nucleocounter (ChemoMetec, Denmark)

available at Project Directorate on Animal Disease Monitoring and Surveillance

(PD_ADMAS), Bangalore. Initially, 500 μL of milk sample was mixed with equal

quantity of the lysis buffer supplied by the manufacturer. The mixture was mixed gently

to lyse the cells. The lysed milk was then aspirated into the cassette and the cassette was

then inserted into the Nucleocounter and the SCC values were recorded. The SCC value

> 5,00,000 cells / mL of milk was taken as criteria to declare the milk / animal as

subclinically mastitic / infected and such milk samples were subjected for cultural

examinations.

3.5 Isolation of Streptococcus species, Staphylococcus and Escherichia coli from

milk

A total of 186 milk samples based on EC and SCC were subjected for

bacteriological examination. All the milk samples having SCC more than 5,00,000

cells / mL and EC more than 6.5 mS/cm were subjected for isolation.

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For isolation of Streptococci, 0.1 mL of milk sample was initially enriched in

Streptococcus Selection Broth, with 5-10 per cent CO2 tension for 6 hours (hr) at

37oC and then streaked onto Blood agar plates, incubated at 37oC for 48 hr. After

reading the haemolysis pattern and colony morphology, the colonies were again

streaked onto blood agar plates and incubated further at 37oC for 48 hr to obtain pure

culture. These pure cultures were then streaked onto BHI agar for further

identification procedures.

For isolation of Staphylococci, 0.1 mL of milk sample was initially enriched in

Brain heart infusion broth for 6 hrs at 37oC and then streaked onto Mannitol salt agar

and incubated at 370 C for 24 hr. After reading the colony morphology, the colonies

were further streaked onto BHI agar for further identification procedures.

For isolation of Escherichia coli, 0.1 mL of milk sample was initially enriched

in Tryptone phosphate broth for 18 hr. at 37oC and then streaked onto MacConkey

agar and incubated at 37oC for 24 hr. The lactose fermenting colonies were further

streaked onto EMB agar and incubated at 37oC for 24 hr. The metallic sheen colonies

were streaked onto BHI agar for further identification procedures.

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3.6 Identification of isolates

3.6.1 Streptococci

Pure cultures of isolates were subjected for catalase test. Further, catalase

negative cultures were streaked onto nutrient agar slants and preserved at 4oC. From

these slants, the pure cultures were subjected for various biochemical tests as per

standard procedures (Barrow and Feltham, 1993; FDA bacterialogical analytical manual,

2001; Collee et al., 2008).

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a) Haemolysis pattern: β haemolysis, a complete zone of clearance and α haemolysis, a

greenish discolouration around and beneath the colonies was recorded on 5 per cent

sheep blood agar plates.

b) Gram staining: Gram staining kit, which contained Crystal violet, Gram’s iodine,

Decolourizer and Safranin was procured from M/s HImedia, Mumbai. Staining of all the

culture isolates was carried out as per the kit.

3.6.1.1 Biochemical tests for Streptococcus

a) Catalase test: Evolution of gas bubbles after addition of 24 hr culture to 3 per cent

hydrogen peroxide indicated positive reaction. Absence of evolution of gas bubbles

indicated negative reaction.

b) Voges Proskauer test: Development of pink colour in two to five min and turning to

crimson red in 30 min after addition of one ml of 40 per cent Potassium hydroxide and

five mL of five per cent alpha-naphthol indicated a positive test. If the colour remained

yellow or failed to develop crimson red colour, then it was considered as negative.

c) Esculin Hydrolysis test: Modified Bile Esculin Azide agar plates were inoculated

with the culture and after incubation for 24 hr, development of black colour around the

colonies indicated positivity. When the colour remained unchanged, it was recorded

negative.

d) Hippurate Hydrolysis test: Development of purple colour within 30 min when

culture was inoculated to one per cent solution of Sodium Hippurate after adding a drop

of 1 per cent Ninhydrin reagent was considered as positive.

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e) Sugar fermentation test: Sugar fermentation media was inoculated with cultures and

incubated at 37ºC for 48 hr and observed for change in colour of the medium from red to

yellow which indicated that the organism was able to ferment the test sugar.

f) PYR test: Development of red colored colonies when the culture were grown on PYR

agar indicated positivity.

3.6.2 Staphylococci

Pure cultures of isolates were subjected for Gram staining and further by

catalase test. The catalase positive cultures were streaked onto nutrient agar slants and

preserved at 4oC. From these slants the pure cultures were subjected for various

biochemical tests as per standard procedures (Barrow and Feltham, 1993; FDA

bacterialogical analytical manual, 2001; Collee et al., 2008).

3.6.2.1 Biochemical tests for Staphylococcus

a) Catalase test: Evolution of gas bubbles after addition of 24 hr culture to 3 per cent

hydrogen peroxide indicated positive reaction. Absence of evolution of gas bubbles

indicated negative reaction.

b) Coagulase test: suspected culture of Staphylococcus in BHI broth (0.2-0.3mL) were

mixed with rabbit plasma (1:4 in BHI broth) and incubated at 37oC for 6 hr. Formation of

clot which will not fall on inversion of test tube was considered as positive for S. aureus.

c) Thermonuclease test: Wells of 6mm diameter were punctured in the Toludine blue

DNA agar. Twenty four hr suspected S. aureus cultures in BHI broth were boiled for 15

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min and cooled. Loaded 20 µL of these culture suspension onto the wells in the Toludine

blue DNA agar and incubated at 37oC for 24 hr. Presence of a pink halo around the well

with a dark blue peripheral ring was considered as positive for thermonuclease test.

d) Voges Proskauer test: carried out as per 3.6.1.1.b

e) Urease test: Cultures that turn the medium red or pink were considered positive and

those that turn it yellow as negative.

3.6.3 Escherichia coli

Pure cultures of isolates were streaked onto nutrient agar slants and preserved

at 4oC. From these slants, the pure cultures were subjected for various biochemical

tests as per standard procedures (Barrow and Feltham, 1993; FDA bacteriological

analytical manual, 2001; Collee et al., 2008).

3.6.3.1 Biochemical tests for Escherichia coli

a) Indole test: Development of red colour in the alcohol layer after addition of Kovac’s

reagent to the 48 hr old culture medium was considered positive for indole production.

b) M.R test: Development of bright red color after the addition of five drops of methyl

red reagent to the 48 hr old inoculated medium was considered positive, whereas yellow

color was considered negative.

c) VP test: carried out as per 3.6.1.1.b

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d) Citrate test: Development of blue colour after 48 hr incubation of Simmon’s citrate

medium with the culture was considered positive.

3.7 Preservation of pure culture and revival of isolates

Pure cultures of Streptococcus, S. aureus and E. coli isolates were sub cultured

onto nutrient agar slants and preserved at 40C for further use. Also, a loopful of the

isolated organism was added to the sterile nutrient glycerol broth vials , mixed well,

labelled and preserved at -20ºC. The isolates were revived once in three months.

3.8 Molecular characterization of isolates by PCR

3.8.1 Reference isolates

The reference isolates of Streptococci were procured from Lead institution, NAIP

Subproject on Bovine mastitis: Unraveling molecular details of host-pathogen interaction

and development of molecular diagnostic methods, PD_ADMAS, Bangalore-24. For S.

aureus (Accession No. JN247783) and E. coli (Accession No. JF926686), the isolates

maintained at the Department of Veterinary Microbiology, KVAFSU, Veterinary

College, Bangalore were used in the study.

Sterptococcus agalactiae (AD1) Accession No. HM 355961

Streptococcus dysgalactiae (AD3) Accession No. HC 359248

Streptococcus uberis (AD2) Accession No HC 355971

Streptococcus uberis (AD6) Accession No HC 355972

3.8.2 Designing of the PCR primers

The gene sequences of interest were downloaded from the NCBI, GenBank.

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The downloaded sequences were then loaded into the EditSeq program of “Lasergene

DNA STAR” software.

The sequences were saved in the EditSeq format.

Thus saved sequences were then aligned by ClustalV method using MegAlign

program of the same software.

The alignment report contents were then split to fit them to a page.

The aligned sequences were then used for designing the primers manually.

Criteria for the selection of the primers

For the genus specific primers a region conserved through the genus was chosen.

For species specific primers variable region was chosen.

Other criteria such as GC content of 40-60 per cent and Tm value of 55-60,

nucleotide sequence length of 18-27 were also considered.

The primers were designed to detect Streptococcus and Staphylococcus at genus

level by targeting tuf gene, 16S rRNA gene based primers for S. agalactiae, S.

dysgalactiae, S. uberis, species specific Surface immunogenic protein (sip) gene based

primers for S. agalactiae, Plasminogen activator (pauA) gene based primers for S. uberis,

nuc and sodA gene based primers for S. aureus were designed and used for PCR studies.

For identification of E.coli by PCR, alanine racemase (alr) gene based primer

(Yokoigawa et al., 1999), multiplex primers designed at Lead centre, NAIP Subproject

on Bovine mastitis: Unraveling molecular details of host-pathogen interaction and

development of molecular diagnostic methods, PD_ADMAS, Bangalore-24 and TraT

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gene based primer designed at NAIP Subproject, Molecular Virology Laboratory,

Regional campus, IVRI, Bangalore-24 were used.

3.8.3 Extraction of genomic DNA

3.8.3.1 Extraction of genomic DNA

The genomic DNA was extracted as per the standard protocol, using the “QIAamp DNA

Mini and Blood mini kit” procured from Qiagen, Inc., Chatsworth, CA.

Materials

a. Microcentrifuge (Eppendorf, Germany)

b. Spectrophotometer

c. Micropipettes (Eppendorf, Germany)

d. Eppendorf tubes

List of materials provided in kit :

QIAamp Mini Spin Columns, Collection tubes (2 mL), Buffer ATL (Tissue lysis

buffer), Buffer AL (Lysis buffer), Buffer AW1 (Concentrate), Buffer AW2

(Concentrate), Buffer AE and Proteinase K.

Enzyme solution: (Composition/ mL)

Lysozyme : 20 mg/mL

Tris HCl : 20 mM (pH 8)

EDTA : 2 mM

Triton X : 1.2%

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3.8.3.2 Protocol for extraction of genomic DNA

One mL of 18 hr old pure bacterial culture was centrifuged at 7500 rpm for 5 minute

Bacterial pellet was suspended in 180 μL of enzyme solution and incubated for 30

min. at 37o C.

Twenty μL of proteinase K and 200 μL of buffer AL was added and mixed

thoroughly by vortexing.

Mixture was incubated at 56o C for 30 min. and 95oC for 15 min. in a water bath.

Mixture was centrifuged for a few seconds.

Two hundred μL of absolute ethanol (99 per cent) was added to the sample and mixed

by pulse-vortexing for 15 sec.

After mixing, the microcentrifuge tube was briefly centrifuged to remove the drops

from inside the lid.

The mixture from the above step (including the precipitate) was carefully applied to

the QIAamp mini spin column along with 2 mL collection tube without wetting the

rim. The cap was closed and centrifuged at 8000 rpm for 1 min. Spin column was

placed in a new clean 2 mL collection tube and collection tube containing the filtrate

was discarded.

Five hundred μL of buffer AW1 was carefully added without wetting the rim of the

spin column.

The cap was closed and centrifuged at 8000 rpm for 1 min. and the collection tube

was discarded.

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The spin column was carefully opened and add 500 μL of buffer AW2 was added

without wetting the rim of the spin column.

The cap was closed and centrifuged at 13,000 rpm for 5 min.

QIAamp mini spin column was placed in a clean 1.5 mL microcentrifuge tube and

carefully 200 μL buffer AE or distilled water was added and incubated at room

temperature for 1 min. Finally the DNA was eluted at 8000 rpm for 2 min.

3.8.4 Extraction of DNA from milk samples

The DNA from milk samples were extracted by following the procedure of

Cremonesi et al. (2006) with modifications.

Materials: Microcentrifuge (Eppendorf, Germany), Spectrophotometer,

Micropipettes (Eppendorf, Germany), Eppendorf tubes, EDTA, Tris-HCL, DL-

dithiothreitol, Guanidine thiocyanate, Silica, Absolute ethanol, Isopropanol, NaCl, Triton

X-100, milliQ water, Heat block.

Lysis buffer:

3M-guanidine thiocyanate

10 mM Tris-HCl (pH 6.8)

20 mM EDTA

10mg/mL DL-dithiothreitol

40 mg/mL Triton X-100

Binding solution:

40 mg/mL Silica suspended in Lysis buffer

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Washing solution:

25% absolute ethanol

25% isopropanol

100 mM NaCl


Elution buffer:


1mM EDTA

3.8.4.1 Procedure for extraction of DNA from milk sample

500 µL of milk sample was diluted with 500 µL of sterilized normal saline solution

and centrifuged at 5000 rpm for 15 min.

Discarded the supernatant and repeated the above step once.

Resuspended the pellet in 50 µL saline solution.

300 µL of lysis buffer and 200 µL of binding solution were added to the resuspended

pellet. Mixed and incubated for 5 min. at RT.

Centrifuged at 8000 rpm for 5 min. and discarded the supernatant.

200 µL of lysis buffer was added to the pellet, mixed well and centrifuged at 8000

rpm for 3 min. and the supernatant was discarded.

The above step was repeated once.

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200 µL of washing solution was added, mixed well and centrifuged at 8000 rpm for 3

min.

The supernatant was discarded and the procedure was repeated once.

200 µL of absolute ethanol was added to the pellet, mixed well and centrifuged at

8000 rpm for 3 min. and the supernatant was discarded.

The pellet was vacuum dried in heat block for 10 min.

50 µL of elution buffer was added to the dried pellet, resuspended and incubated for

15 min. at 650C in a heat block.

Centrifuged the suspension at 8000 rpm for 3 min. and transferred the supernatant in

a clean tube.

3.9 Determination of purity and yield of the DNA samples

The purity and concentration of the extracted genomic DNA was estimated by UV

spectrophotometry. Twenty microliter (20 µL) of DNA sample was dissolved in 980 µL

of sterile DW. The diluted DNA was transferred into 1mL microcuvette and checked the

optical density (OD) at 260 nm and 280 nm in a UV spectrophotometer. Sterile DW was

used as blank.

The ratio of 260/280 OD was calculated. A ratio of 1.7 to 1.9 was considered

pure. Further, the purity of the DNA sample was further checked by electrophoresis on

0.8 per cent of the agarose gel.

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3.10 DNA confirmation by Agarose Gel Electrophoresis

Equipments

a. Weighing balance (Shimazdu Philippines Manufacturing Inc., Japan)

b. Horizontal electrophoresis apparatus with power pack (Bangalore Genie, India)

c. Microwave oven (M/s. BPL Pvt. Ltd.)

d. Gel documentation unit (Biorad, USA)

Reagents

a. Agarose (M/s. Sigma Aldrich, USA)

b. Tris Borate EDTA buffer (10X, pH 8.2)

Tris base 108.0 g

Boric acid 55.0 g

EDTA disodium salt 8.3 g

Double distilled water up to 1000.0 mL

The stock solution was sterilized by autoclaving and made to 0.5X before use.

c. Gel loading dye (6X)

Bromophenol blue 0.25% (w/v)

Xylene cyanol 0.25% (w/v)

Sucrose 40% (w/v) in distilled water

The dye was procured from M/s. Bangalore Genei, Bangalore and stored at 4oC.

d. Ethidium bromide (10 mg / mL)

Ethidium bromide (Bangalore Genie) 100 mg

Double distilled water 10 mL

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The suspension was stirred to ensure that the dye had dissolved. The container was

then wrapped in aluminium foil.

3.10.1 Procedure

Required quantity of agarose was weighed and dissolved in proportionate

volume of 1X Tris Borate EDTA buffer (TBE) and melted in a microwave oven until a

clear uniform suspension was obtained. (About 0.2 g of analytical grade agarose was

dissolved in 20 mL of 1X TBE buffer by heating in microwave oven to obtain uniform

solution). Prior to casting the gel, the molten agarose was allowed to cool to about 50°C,

after which ethidium bromide was added to a final concentration of 0.5 g/mL and mixed

thoroughly. Gel was casted on an appropriate gelcasting tray fitted with acrylic comb and

left for setting. The acrylic comb was carefully removed after the gel was set. The tray

with gel was then submerged in an electrophoresis tank containing 1X TBE buffer.

The DNA to be analyzed was mixed with appropriate volume of 6X DNA loading

buffer and charged into wells alongside 100 bp DNA molecular weight marker.

Electrophoresis was carried out at five V/cm until the tracking dye (Bromophenol Blue)

had just passed out of the gel. Following the electrophoresis, DNA bands were visualized

and the images were captured by using Gel Doc XR (Bio Rad, USA).

3.11 Polymerase chain reaction

Reagents

a. 10X Taq buffer

b. dNTPs (Deoxy nucleoside triphosphate mix)

70

c. Taq DNA polymerase (5U / µL)

d. Nuclease Free Water (NFW)

e. Primer ( synthesized by Bioserve, Hyderabad)

f. Template DNA extracted from reference isolates (positive control) and test isolates,

g. 50 bp / 100 bp DNA ladder (Bangalore Genie)

3.11.1 Amplification of Streptococcus genus specific sequence

Genus specific primers were designed targeting the tuf gene of streptococci as

detailed in the section 3.8.2 and synthesized commercially from M/s. Bioserve

Biotechnologies (India) Pvt. Ltd, Hyderabad. Primers are enlisted with their base sequences

below in Table 2. These primers were reconstituted in Nuclease free water (NFW) to obtain

the required concentration and aliquot was stored at -20oC.

Table 2. Nucleotide sequences of Streptococcus genus specific (tuf gene) primers

Name of theprimer Nucleotide Sequence 5’─ 3’ Product

length (bp)

S tuf-F 5’- CAA CTT GAC GAA GGT CCT GCA -3’110

S tuf-R 5’- TGG GTT GAT TGA ACC TGG TTT A-3’

3.11.1.1 Protocol for Partial amplification of nucleotide sequence of tuf gene by

genus specific primers

The test was initially standardized using known positive and negative controls.

The amplification reactions were carried out in 0.2mL micro centrifuge tubes using a

programmable thermal cycler (M/s Eppendorf, Germany).

71

Each 25 µL of the PCR mixture comprised of 2.5 µL of 10X PCR Taq Buffer A,

1 µL (100 µM) of each dNTPs, 0.5 µL (10 pmol) of each S- tuf F and S- tuf R primers

and and 0.2 µL (1U) of Taq DNA polymerase and 3 µL of Streptococcal template DNA.

Filtered quartz water was added to make a final volume of 25 µL.

PCR Cycling conditions:

Cycling conditions

Initialdenaturation Denaturation Annealing Extension Final extension

940C

5 min.

940C for 30 sec. 500C for 30 sec. 720C for 30 sec.720C for 10 min.

Repeated for 30 cycles

After completion of PCR reaction, 25 µL amplified products along with 100 bp

DNA ladders, added with 6X gel loading dye, and were subjected to electrophoresis in

1.8 per cent agarose gel following the procedure described earlier. The images were

captured using gel documentation system (Gel Doc XR, M/s, BioRad., U.S.A).

3.11.1.2 Amplification of Streptococcus species specific sequence

In this study, a total of three different sets of species specific primers for S.

agalactiae (Saga), S. dysgalactiae (Sdys) and S. uberis (Sub) were designed by targeting

16S rRNA gene as detailed in the section 3.8.2 and synthesized commercially at M/s.

Bioserve Biotechnologies (India) Pvt. Ltd, Hyderabad. Primers are enlisted with their

base sequences as follows in Table 3. Primers were reconstituted in NFW as per the

requirement and stored at - 20OC.

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Table 3. Nucleotide sequences of Streptococcus species specific (16 S rRNA) primers


length (bp)

Saga F 5’- GCC TCA TAG CGG GG ATA AC- 3’329

Saga R 5’-ACG TTC TTC TCT AAC AAC AGA- 3’

Sdys F 5’- GGA GTG GAA AAT CCA CCA T -3’549

Sdys R 5’- CGG TCA GGA GGA TGT CAA GAC- 3’

Sub F 5’- GTA CCC TAT TTA AAA GGG GCA AAT- 3’854

Sub R 5’- C TCC GAT GTA CCG AAG TAA AGC TCT- 3’

3.11.1.2.1 Protocol for Partial amplification of Streptococcus species specific (16S

rRNA) sequence

The amplification reactions were carried out in 0.2mL micro centrifuge tubes

using a programmable thermal cycler (M/s Eppendorf, Germany).

a. Each 25 µL of the PCR mixture comprised of 2.5 µL of 10X PCR Taq Buffer A, 1

µL (100 µM) of each dNTPs, 0.5 µL (10 pmol) of each SagaF/ SdysF/ SubF and

SagaR/ SdysR/ SubR primers and, and 0.2 µL (1U) of Taq DNA polymerase and 3

µL of Streptococcal DNA. Filtered quartz water was added to make a final volume of

25 µL.

b. PCR conditions:

Primers(forward

and reverse)

Cycling conditionsInitial

denaturation Denaturation Annealing Extension Finalextension

Saga F/RSdys F/RSub F/R

94oC, 5min. 94oC, 30sec. 50oC,30sec.

72oC,30sec.

72oC10min.


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After completion of PCR reaction, 25 µL amplified products along with 100bp

DNA ladders, added with 6X gel loading dye were subjected to electrophoresis in 1.8 per

cent agarose gel as described. The images were captured using gel documentation system

(Gel Doc XR, M/s, BioRad., U.S.A).

3.11.1.3 Amplification of Surface immunogenic protein (sip) for S. agalactiae;

Plasminogen activator gene (pauA) for S. uberis

In this study, a total of two different sets of Virulence gene specific primers

{Surface immunogenic protein (sip) for S. agalactiae (Strep sip-I); Plasminogen activator

gene (pauA) for S. uberis (Strep pau-I)}, were also designed as detailed in the section

3.8.2 and synthesized commercially from M/s. Bioserve Biotechnologies (India) Pvt. Ltd,

Hyderabad, The details of the primers and their base sequences are given below in

Table 4. Thus obtained primers were reconstituted in NFW as per the requirement and

stored at -20OC.

Table 4. Nucleotide sequences of Streptococcus agalactiae sip and

Streptococcus uberis pauA gene specific primers

Name of theprimer Sequence 5’─ 3’

Productlength(bp)

Strep sip I-F

Strep sip I-R

5’-ACTATTGACATCGACAATGGCAGC-3’266

5’-GTTACTGTCAGTGTTGTCTCAGGA- 3’

Strep pau I-F

Strep pau I-R

5’-TGCTACTCAACCATCAAAGGTTGC-3’439

5’-TAGCAGTCTCAGTAGGATGAGTGA- 3’

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3.11.1.3.1 Protocol for Partial amplification of S. agalactiae sip gene and S. uberis

pauA gene sequence

The amplification reactions were carried out in 0.2mL micro centrifuge tubes using

a programmable thermalcycler (M/s Eppendorf, Germany).

a. Each 25 µL of the PCR mixture comprised of 2.5 µL of 10X PCR Taq Buffer A, 1

µL (100 µM) of each dNTPs, 0.5 µL (10 pmol) of each Strep sip I-F / Strep pau I-F

and Strep sip I-R / Strep pau I-R primers and and 0.16 µL (0.8U) of Taq, DNA

polymerase and 3 µL of Streptococcal DNA. Filtered quartz water was added to

make a final volume of 25 µL.

b. PCR conditions:

Primers(forward

and reverse)

Cycling conditionsInitial

denaturation Denaturation Annealing Extension Finalextension

Strep sip I-F/R 94oC, 5min.

94oC, 30sec. 52oC,30sec.

72oC,30sec. 72oC,

10min.Repeated for 30 cycles

Strep pau I-F/R 94oC, 5min.

94oC, 30sec. 54oC,30sec.

72oC,30sec. 72oC,


c. After completion of PCR reaction, 25 µL amplified products along with 100 bp DNA

ladders, added with 6X gel loading dye, were subjected to electrophoresis in 1.8 per

cent agarose gel. The images were captured using gel documentation system (Gel

Doc XR, M/s, BioRad., U.S.A).

3.11.2 Amplification of Staphylococcus genus specific sequence

Genus specific primers to detect Staphylococcus at genus level were designed

targeting the tuf gene of staphylococci as detailed in the section 3.8.2 and synthesized

75

commercially from M/s. Bioserve Biotechnologies (India) Pvt. Ltd, Hyderabad. Primers are

enlisted with their base sequences below in Table 5. These primers were reconstituted in

NFW to obtain the required concentration and aliquot was stored at -20oC.

Table 5. Nucleotide sequences of Staphylococcus genus specific (tuf gene) primers

Name of theprimer Nucleotide Sequence 5’─ 3’ Product length

(bp)Staph tuf-F 5’-GAA GAA TTA TTA GAA TTA GT-3’

235Staph tuf-R 5’ GTG ATT GAG AAT ACG TCC TCA AC-3’

3.11.2.1 Protocol for Partial amplification of nucleotide sequence of tuf gene by

Staphylococcus genus specific primers

a. The amplification reactions were carried out in 0.2mL micro centrifuge tubes using a


Each 25 µL of the PCR mixture comprised of 2.5 µL of 10X PCR Taq Buffer A, 1

µL (100 µM) of each dNTPs, , 0.5 µL (10 pmol) of each S tuf F and S tuf R primers

and and 0.2 µL (1U) of Taq, DNA polymerase and 3 µL of Staphylococcal DNA.

Filtered quartz water was added to make a final volume of 25 µL.

b. PCR conditions:

Cycling conditions

Initialdenaturation Denaturation Annealing Extension Final

extension

94oC, 5min.94oC, 30sec. 50oC, 30 sec. 72oC, 30sec.

72oC,10min.Repeated for 30 cycles

c. After completion of PCR reaction, 25 µL amplified products along with 100 bp DNA

ladders, added with 6X gel loading dye, and were subjected to electrophoresis in 1.8

76

per cent agarose gel. The images were captured using gel documentation system (Gel


3.11.2.2 Amplification of nuc gene for identification of S. aureus

In this study, primers were designed targeting nuc gene for the detection of S.

aureus (SAU-nuc-I) as detailed in the section 3.8.2 and synthesized commercially from

M/s. Bioserve Biotechnologies (India) Pvt. Ltd, Hyderabad, The details of the primers

and their base sequences are given below in Table 6. Thus obtained primers were

reconstituted in NFW as per the requirement and stored at -20OC.

Table 6. Nucleotide sequences of S. aureus specific (nuc gene) primers


length (bp)

SAU-nuc-I F 5’ GTG CTG GCA TAT GTA TGG CAA TTG T-3’181

SAU-nuc-I R 5’ TAC GCC GTT ATC TGT TTG TGA TGC- 3’

3.11.2.2.1 Protocol for partial amplification of nucleotide sequence of nuc gene by

S. aureus specific primers



b. Each 25 µL of the PCR mixture comprised of 2.5 µL of 10X PCR Taq Buffer A, 1

µL (100 µM) of each dNTPs, 0.5 µL (10 pmol) of each SAU nuc F and SAU nuc R

primers and, and 0.2 µL (1U) of Taq, DNA polymerase and 3µL of Staphylococcus

aureus DNA. Filtered quartz water was added to make a final volume of 25 µL.

77

c. PCR conditions:

Primers(forward

and reverse)

Cycling conditions


extension

SAU-nuc-IF/R

94oC, 5min. 94oC, 30sec 54oC,30 sec.

72oC,30 sec 72oC,


d. After completion of PCR reaction, 25 µL amplified products along with 100 bp DNA




3.11.2.3 Amplification of sodA gene for identification of S. aureus

In this study, primers were designed targeting sodA gene for the detection of S.

aureus (SAU-sodA-I) as detailed in the section 3.8.2 and synthesized commercially from

M/s. Bioserve Biotechnologies (India) Pvt. Ltd, Hyderabad, The details of the primers

and their base sequences are given below in Table 7. Thus obtained primers were

reconstituted in NFW as per the requirement and stored at -20OC.

Table 7. Nucleotide sequences of S. aureus specific (sodA gene) primers


length (bp)

SAU-sodA-I F 5’ AGA TCA AAG AAC AAT GGA GTT TCA TC-3’159

SAU-sodA-I R 5’ ACG GAC TGA CAT CCT CAT CGC TTC- 3’

78

3.11.2.3.1 Protocol for Partial amplification of nucleotide sequence of sodA gene by

S. aureus specific primers




µL (100 µM) of each dNTPs, 0.5 µL (10 pmol) of each SAU sodA-F and SAU sodA-

R primers and 0.2 µL (1U) of Taq, DNA polymerase and 3µL of Staphylococcus

aureus DNA. Filtered quartz water was added to make a final volume of 25 µL.

c. PCR conditions:

Primers(forward

and reverse)

Cycling conditions


extension

SAU-sodA-IF/R

94oC,5 min.

94oC,30 sec.

54oC,30sec.

72oC,30sec. 72oC,






3.11.3 Amplification of alr gene for Escherichia coli

In the present study, to identify E. coli, alr gene based primer published by

Yokoigawa et al. (1999) was used. The details of the primers and their base sequences

are given in Table 8.

79

Table 8. Nucleotide sequence of E. coli specific alr gene primers

Name ofthe primer Nucleotide Sequence 5’─ 3’ Product length

(bp)

EC-alr-F 5’-CTG GAA GAG GCT AGC CTG GAC GAG-3’366

EC-alr-R 5’-AAA ATC GCC ACC GGT GGA GCG ATC-3’

3.11.3.1 Protocol for Partial amplification of nucleotide sequence of alr gene by

E. coli specific primers




µL (100 µM) of each dNTPs, , 0.5 µL (10 pmol) of each EC-alr -F and EC-alr -R

primers, 0.2 µL (1U) of Taq, DNA polymerase and 3 µL of E. coli DNA and filtered

quartz water was added to make a final volume of 25 µL.

c. PCR conditions:

Primers(forward

andreverse)

Cycling conditions


extension

EC-alrF/R

94oC, 5min. 94oC, 30sec. 57oC, 30sec. 72oC, 30sec. 72oC10min.Repeated for 30 cycles

3.11.3.2 Amplification of TraT gene for Escherichia coli

In this study, primers designed at NAIP Subproject, Molecular Virology

Laboratory, Regional campus, IVRI, Bangalore-24, targeting TraT gene was used for the

80

detection of E. coli. The details of the primers and their base sequences are given in

Table 9.

Table 9. Nucleotide sequences of E. coli specific TraT gene primers

Name of theprimer Nucleotide Sequence 5’─ 3’ Product length

(bp)

EC traT-F 5’-TGG TGC CGC GTT AGG TGC TG - 3’ 313

EC traT -R 5’- GCC CTG ACG AAG AGC GGC AA - 3’

3.11.3.2.1 Partial amplification of nucleotide sequence of TraT gene by E. coli

specific primers

a. The amplification reactions were carried out in 0.2 mL micro centrifuge tubes using a


b. Each 25 µL of the PCR mixture comprised 2.5 µL of 10X PCR Taq Buffer A, 1 µL

(100 µM) of each dNTPs, 0.5 µL (10 pmol) of each EC traT-F and EC traT -R

primers 0.2 µL (1U) of Taq, DNA polymerase and 3 µL of E. coli DNA. Filtered

quartz water was added to make a final volume of 25 µL.

c. PCR conditions:

Primers(forward

and reverse)

Cycling conditions


extension

EC-traT FEC-traT R

94oC, 5min. 94oC, 30sec. 55oC, 30sec. 72oC, 30sec 72oC,10min.Repeated for 30 cycles



81



3.11.3.3 Amplification of multiplex PCR for identification of E. coli

Primers designed at Lead centre, NAIP Subproject on Bovine mastitis:

Unraveling molecular details of host-pathogen interaction and development of molecular

diagnostic methods. PD_ADMAS, for identification of E. coli and differentiation from

Shigella spp. and other Enterobacteriaceae were used. It was based on specific

amplification of five house keeping genes specific for E. coli. The details of the primers

and their base sequences are given below.

Name of theGene targetted Nucleotide Sequence 5’─ 3’ Product

length (bp)

lacYCTA CCG GTG AAC AGG GTA CG

289GTC GCT GAA AAA CGC ACT TC

lacZATG AAA GCT GGC TAC AGG AAG G

517CTC CAC AGT TTC GGG TTT TC

uidACGC CGA TGC AGA TAT ATT CG

603GCT GTG ACG CAC AGT TCA TAG

cydACG CTA TAC GCC AAA CGT G

398ACA ATT CTG CCA CCA GGA AC

phoAGGT AAC GTT TCT ACC GCA GAG TTG

468CAG GGT TGG TAC ACT GTC ATT ACG

3.11.3.3.1 Protocol for Partial amplification of nucleotide sequence of multiplex

PCR for E. coli



82

b. Each 25 µL of the PCR mixture comprised of 2.5 µL of 10X PCR Taq Buffer A, 1 µL

(100 µM) of each dNTPs, 0.2 µL (1U) of Taq DNA polymerase, 0.5 µL (0.15 µM

lacY, 0.5 µM lacZ, 0.4 µM uidA, 0.4 µM cyd and 0.6 µM phoA ) of each primers and

3 µL of E. coli DNA template. Filtered quartz water was added to make a final

volume of 25 µL.

c. PCR conditions:

Primers(forward and

reverse)

Cycling conditions


extension

lacYlacZuidAcyd

phoA

94oC, 5min.

94oC, 30 sec. 60oC, 30 sec. 72oC, 45 sec.

72oC5 min.Repeated for 30 cycles





3.12 Sensitivity and detection limit of PCR

a. Determination of Colony count: colony count for each reference strains was carried

out by spread plate method. For Streptococcus, BHI agar plates were used, while for

S. aureus MSA plates and E. coli, MCA plates were used.

b. Serial tenfold dilutions of 24 hr culture of reference strains were done in PBS.

83

c. 25 µL of diluted culture was used for inoculating the plates. Plates in duplicate were

used for each dilution.

d. Incubated at 370C for 24 hr.

e. Highest dilution of the plates where 50 to 300 colonies could be counted was taken

for calculating the count.

f. Final colony count was calculated

g. Colony forming unit (cfu) from 1cfu to 107cfu was prepared in normal saline and

spiked onto sterile milk and normal saline.

h. DNA was extracted from the spiked milk and normal saline following the procedure

adopted for milk DNA extraction..

i. PCR was carried out for each reference strain from 1cfu to 107cfu/mL

Detection limit:

a. DNA concentration of the reference strains was estimated

b. Serial tenfold dilutions of each reference strain DNA was carried out in NFW.

c. PCR was carried out using each diluted DNA as template.

3.13 Standardization of two tube multiplex PCR

Two tube multiplex for detection of five mastitis pathogens was standardized.

One tube containing specific primers sip and pauA to detect S. agalactiae and S. uberis,

other tube containing three specific primers 16S rRNA dys, alr and nuc to detect S.

dysgalactiae, E. coli and S. aureus respectively was used.

84

a. Each 25 µL of the PCR mixture in one tube comprised of 2.5 µL of 10X PCR Taq

Buffer A, 1 µL (100µM) of each dNTPs, 0.2 µL (1U) of Taq, DNA polymerase,

0.25 µL(5 pm) each of Strep sip-F and Strep sip-R and 0.5 µL each of Strep pau-F

and Strep pau-R primers, 3 µL each of S .agalactiae and S. uberis DNA template.

Filtered quartz water was added to make a final volume of 25 µL

b. Each 25 µL of the PCR mixture in other tube comprised of 2.5 µL of 10X PCR Taq

Buffer A, 1 µL (100µM) of each dNTPs, 0.2 µL (1U) of Taq, DNA polymerase,

0.375 µL(7.5pm) each of alr-F and alr-R primers; 0.5 µL(10pm) each of nuc-F and

nuc-R primers; 0.25 µL(5pm) each of 16 S dys-F and 16 S dys-R primers and 3 µL

each of E. coli, S. aureus and S. dysgalactiae DNA templates. Filtered quartz water

was added to make a final volume of 25 µL.

c. m-PCR conditions

Primers(forward and

reverse)

Cycling conditions

Initialdenaturatio

nDenaturation Annealing Extension Final

extention

Strep sip-F/R,Strep pauA-F/R 94oC

5 min.

94oC, 30sec. 57oC, 30sec. 72oC, 30sec.72oC

10 min.

EC-alr-F/R,16 S dys-F/R,nuc-F/R





Doc XR, M/s, BioRad., U.S.A).The two tube multiplex PCR was standardized using

85

reference strains and the same was used directly to screen the milk DNA samples to

detect these five major mastitis pathogens simultaneously.

3.13.1 Screening of milk samples for subclinical mastitis pathogens

DNA from the processed milk samples were extracted and subjected for two tube

m-PCR. The detected isolates were compared with the isolates obtained from these

samples

3.13.2 Screening of Bulk milk samples for subclinical mastitis pathogens

Bulk milk samples were procured from chilling centres of Bangalore Milk Union

covering Bangalore district and DNA was extracted from these milk samples and

subjected for m-PCR for simultaneous detection of S. agalactiae, S, dysgalactiae,

S. uberis, S. aureus and E. coli. Following are the details of bulk milk collection at

DCMS level.

Sl. No Chilling Centre No. of Bulk Milk Samples (DCMS)

1 A 39

2 B 36

3 C 36

4 D 36

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