Poseidon

NESTED PCR FOR DETECTION OF WSSV IN SHRIMP

1. KIT COMPONENTS

Reagents for rapid DNA extraction: stored at room temperature.

DNA extraction buffer - 1 Bottle 75 ml
Reagents for PCR reaction: stored at -20 o C.
Ready-to-use master mix in PCR tube 110 tubes 7.5 ml / tube
WSSV first Primers 1 tube 450 ml / tube
WSSV nested primers 1 tube 450 ml / tube
Marker (100bp) 1 tube 15 ml / tube
Negative Control 1 tube 15 ml / tube
Positive Control Template 1 tube 15 ml / tube
Other Accessories
Grinding sticks 55 nos
Centrifuge tubes 55 nos
Gloves 50 nos

2. EQUIPMENT AND MATERIALS REQUIRED

Thermal cycler, Electrophoresis equipment and reagents, UV transluminator, Pipettes, Table top centrifuge, Absolute alcohol

3. DNA EXTRACTION

DNA purification is achieved using the DNA extraction buffer. Sample of 50 mg from the individual shrimp tissue or hemolymph or 20 PL are homogenized in 600 ml of DNA extraction buffer. Homogenized tissue / PL is allowed to dissolve in DNA extraction buffer for 10 min at room temperature. The sample is centrifuged at 5000 rpm for 5 min and take 200 ml of supernatant in another tube. The DNA is precipitated by addition of 2 volume of 100% ethanol (EtOH) and sample is mixed well. Centrifuge the samples at 10,000 rpm for 10 min to pellet the DNA. Slowly pour off the supernatent by taking care that the DNA pellet remains at the bottom of the tube. Dissolved the DNA pellet in 100 ml of DD H 2 O by mixing gently with a micro pipette. This is served as template for PCR.

4. POLYMERASE CHAIN REACTION

FIRST PCR

Take one PCR tube containing PCR master mix. Add 6.5 µl of first primer mix and one µl DNA template to PCR tube. Mix gently, spin down briefly. Place into a PCR machine as quick as possible and run with following program

Pre-denaturation temperature 95°C 5 min
30 cycle of Post Extension temperature 72°C for 10min
HOLD 4°C

NESTED PCR

Take one PCR tube containing PCR master mix. Add the 6.5 µl of nested primer mix and one µl of first PCR product to PCR tube. Mix gently, spin down briefly. Place into a hot PCR machine as quick as possible and run with following PCR progaram.

Pre-denaturation temperature 95°C for 5 min
30 cycle of Post Extension temperature 72°C 10min
HOLD 4°C
Analyze samples for a PCR product by 1 -1. 2% agarose gel electrophoresis. For fractionation of 155bp use 1.2% agarose in TBE buffer.

5. AGAROSE GEL ANALYSIS OF PCR PRODUCTS

To prepare the gel, using 1X TBE and agarose by melting in a microwave oven, mixing, then cooling to 50-55°C. Add ethidium bromide (Etbr) (1m of 1%) to agarose gel solution before pouring in gel casting platform. Set up the gel by carefully pouring agarose solution into the sealed gel-casting platform, and inserting the gel comb. The gel should cover only about 1/3 the height of the comb teeth, use a pipette tip to move large bubbles or solid debris to the sides or end of the tray, while the gel is still liquid. After the gel has hardened, remove the seal from the gel casting platform and withdraw the gel comb. Place into an electrophoresis tank and fill with 0.5 X TBE buffer (or 1X TAE buffer) so the buffer just covers the gel. After cycling is complete, spin tubes briefly in microcentrifuge then load in designated wells in gel. Load 3 µl of molecular weight marker (100 bp DNA ladder) into outer lane.

Attach the leads so that the DNA migrates to the anode or positive lead. Turn off the power supply when the loading dye has migrated a distance judged sufficient for separation of the DNA fragments. View the gel on a UV transluminator. Interpret the results.

6. DIAGNOSIS

In the nested PCR, the positive control lane should contain three bands of 720 bp, 310 bp and 210 bp and negative control lane should not contain any band. Test samples containing three bands of 720 bp, 310 bp and 210 bp should be considered as high positive for WSSV whereas test samples with a single band of 210 bp should be considered as positive. The test samples that have no band in the lane should be considered as a negative for WSSV.

Lane M : 100 bp Molecular weight marker
Lane P : WSSV high positive with 3 bands of 720 bp, 310 bp and 210 bp.
Lane N : WSSV negative control without band.
Lane 1 to 5 : WSSV positive samples in different dilution