Decision No. 04/2004/QĐ-BTS On Issuing Industry Standards

Pursuant to …;

Regarding the Issuance of Industry Standards

 

MINISTER OF AQUATIC RESOURCES

Pursuant to Decree No. 43/2003/NĐ-CP dated May 2, 2003 of the Government stipulating the functions, tasks, powers, and organizational structure of the Ministry of Fisheries;

At the request of the Director of the Science and Technology Department,

 

DECISION:

Article 1. Issuing the following Industry Standard:

28 TCN 202: 2004 Procedure for Diagnosing White Spot Syndrome Virus Disease in Penaeid Shrimp Species Using Polymerase Chain Reaction Technique

Article 2. The above standard encourages application at disease testing and inspection facilities throughout the country and shall take effect fifteen days from the date of publication in the Official Gazette.

Article 3. The Head of the Ministry's Office; Heads of Departments, Directorates, and Inspectors General of the Ministry; Heads of subordinate units under the Ministry; Directors of Fisheries Departments and Departments of Agriculture and Rural Development responsible for fisheries management; disease testing and inspection facilities mentioned in Article 2 and related units are responsible for implementing this Decision.

                                                28 TCN202:2004:

DIAGNOSIS PROCEDURE FOR WHITE SPOT SYNDROME VIRUS DISEASE IN PENAEID SHRIMP SPECIES USING POLYMERASE CHAIN REACTION TECHNIQUE
                                            Polymerase Chain Reaction

1. SCOPE AND APPLICABILITY

1.1 This procedure specifies the sequence and content of methods for detecting the white spot syndrome virus (hereinafter referred to as WSSV) using the Polymerase Chain Reaction (hereinafter referred to as PCR) technique.

1.2 This procedure is used to diagnose WSSV disease in parent shrimp, shrimp broodstock, and commercial shrimp of Penaeid shrimp species using the PCR technique. It may also be used to diagnose WSSV disease in other crustaceans.

2.1 Lightner D.V. (Ed.) (1996). Handbook of Pathology and Diagnostic Procedures for Diseases of Penaeid Shrimp. World Aquaculture Society, Baton Rouge, USA.

2.2 Lo C.F., Ho C.H., Peng S.E., Chen C.H., Hsu H.C., Chiu Y.L., Chang C.F., Liu K.F., Su M.S., Wang C.H. & Kou G.H. (1996). White spot syndrome baculovirus (WSBV) detected in cultured and captured shrimps, crabs and other arthropods. Dis. Aquat. Org., 27, 215-225.

S., Wang C.

H. & Kou G.

H. (1996). White spot syndrome baculovirus (WSBV) detected in cultured and captured shrimps, crabs and other arthropods. Dis. Aquat. Org., 27, 215-225.

2.3 Lo C.F., Leu J.H., Ho C.H., Chen C.H., Peng S.E., Chen Y.T., Chou C.M., Yeh P.Y., Huang C.J., Chou H.Y., Wang C.H. & Kou G.H. (1996). Detection of baculovirus associated with white spot syndrome (WSBV) in penaeid shrimps using polymerase chain reaction. Dis. Aquat. Org., 25, 133-141.

2.4 Newton C.R. and Graham A. (1994). PCR. The Alden ss Ltd, Oxford, UK.

3.1 Polymerase chain reaction (PCR) technique is a method that uses a series of consecutive reactions to amplify the number of copies of a target DNA sequence through cycles consisting of three steps: denaturation of DNA, annealing with primers, and synthesis of new strands by DNA polymerase enzyme.

3.2 DNA polymerase is an enzyme that synthesizes a new DNA strand from a template strand and can participate in replication processes.

3.3 bp (base pair) is a pair of A-T or C-G bonds on a double-stranded DNA molecule and is the unit of measurement for the length of a DNA molecule.

3.4 Primer is a short DNA sequence that anneals to a template DNA strand carrying a free 3'-OH end, allowing DNA polymerase to initiate new strand synthesis.

3.5 Forward primer and reverse primer: understood according to the direction of reverse transcription.

3.6 Denaturation is the process of separating double-stranded DNA into single strands. The agent causing denaturation is usually heat.

3.7 Branchial filament is a structure composed of many branchial threads forming the gill of shrimp. Branchial threads consist of cells whose main function is respiration.

4 Equipment, tools, primers, and chemicals

4.1 Equipment, tools

4.1.1 Sterile sample grinding apparatus.

4.1.2 Sample mixer.

4.1.3 Magnetic stirrer.

4.1.4 Distillation apparatus.

4.1.5 Centrifuge (speed not less than 13,000 rpm).

4.1.6 Thermal cycler.

4.1.7 Electrophoresis system including power supply and horizontal electrophoresis tank.

4.1.8 Result reading table with UV light, wavelength 302 nm.

4.1.9 Photography equipment to record results.

4.1.10 Refrigerator at -20°C or lower.

4.1.11 Refrigerator at 4°C.

4.1.12 Sterile workbench.

4.1.13 Tool drying cabinet.

4.1.14 Microwave oven.

4.1.15 Analytical balance (accuracy to 0.001 g).

4.1.16 Various micropipettes: 1-5, 5-10, 20-100, and 100-1000 μl.

4.1.17 Filter-tipped pipette.

4.2 Primers, chemicals

4.2.1 Primers Specific primers for the white spot syndrome virus (WSSV) used in two-step PCR technique include forward primers (146F1, 146F2) and reverse primers (146R1, 146R2) designed from the DNA SalI 146 sequence. The specific primer sequences are as follows: 146F1: 5' ACT ACT AAC TTC AGC CTA TCT AG 3' (forward primer); 146R1: 5' TAA TGC GGG TGT AAT GTT CTT ACG A 3' (reverse primer); 146F2: 5' GTA ACT GCC CCT TCC ATC TCC A 3' (forward primer); 146R2: 5' TAC GGC AGC TGC TGC ACC TTG T 3' (reverse primer). The concentration of primers used in analysis is diluted to 25 mM in TBE buffer solution. TBE buffer consists of the following components: 10 mM Tris-HCl (pH = 8.0) and 1 mM EDTA.

4.2.2 Chemicals

4.2.2.1 DNA extraction solution: sodium dodecyl sulfate - NaOH (SDS - NaOH) NaOH 0.5 N (20 X): dissolve 2 g NaOH in 100 ml sterile distilled water. SDS 0.25% (20 X): dissolve 0.25 g SDS in 100 ml sterile distilled water (do not autoclave sterilize).

4.2.2.2 NaOH 0.025 N - SDS 0.0125% solution: 5 ml SDS 0.25%, 5 ml sterile distilled water, make up to 100 ml. Store the solution at 4°C.

4.2.2.3 Master mix: the amount of PCR master mix for 50 ml amplification reaction includes: Taq DNA Polymerase: 1.25 units Tris-HCl (pH = 8.8 at 25°C): 75 mM (NH4)2SO4: 20 mM MgCl2: 1.5 mM dATP, dCTP, dGTP, and dTTP: 0.2 mM each

4.2.2.4 DNA ladder fX174/HaeIII

4. 2.2.5 TBE Buffer (Tris-Borate-EDTA) EDTA (ethylene diamine tetra acetic acid) 0.5 M: dissolve 93.05 g EDTA in 350 ml water, then adjust the solution to pH = 8 with NaOH. Add distilled water to make up to 500 ml. Sterilize by autoclaving and store at room temperature. TBE 1X: Tris: 108 g Boric Acid: 55 g EDTA 0.5 M: 40 ml Prepare a 10X concentrated solution by dissolving 108 g Tris and 55 g boric acid in approximately 600 ml water. Then add 40 ml EDTA 0.5 M and adjust the volume to 1 liter with water. Store at room temperature. Dilute with sterile distilled water to 1X when using.

4. 2.2.6 1% Agarose in TBE Solution

4. 2.2.7 Sample Loading Solution (6X) containing: bromophenol blue 0.25%, glycerol 40%

4. 2.2.8 Ethidium Bromide Solution (10 mg/ml) 5 Sample Preparation 5.1 Sample Quantity

5. 1.1 For postlarvae shrimp samples, the amount of sample for each reaction is 100 individuals taken whole.

5. 1.2 For parent shrimp samples, the amount of sample for each reaction is 100 mg taken from the pleopod, eyestalk, or swimming legs.

5. 1.3 For commercial shrimp samples, the amount of sample needed for each reaction is 100 mg taken from the pleopod. Commercial shrimp samples should be collected in two cases:

a. If there are diseased shrimp in the pond, collect approximately 10-20 individuals showing the most obvious signs of disease.

b. If the shrimp pond is developing normally or shows no signs of disease, randomly collect approximately 20-30 individuals.

5. 2 Requirements for Samples for Analysis Samples must be collected while the shrimp are alive and immediately preserved in 95% ethanol. The volume of ethanol used for preservation must not be less than three times the volume of the sample to be analyzed. After fixation, the samples are stored at a temperature of about 25-30°C for one week. If longer storage is required, the ethanol must be replaced.

6 Methodology

6. 1 Sample Processing

6. 1.1 The sample is ground in a sterile sample grinder (4.1.1) with 900 ml DNA extraction buffer (4.2.2.1). The ground solution is transferred to a 1.5 ml Eppendorf tube (4.1.17) and denatured by boiling in a water bath for 5-10 minutes, then rapidly cooled by placing it in ice water.

6. 1.2 Centrifuge the ground solution for 5 minutes using a centrifuge (4.1.5) at 13,000 rpm. Then, collect the supernatant for the PCR reaction. If the sample is not analyzed immediately, it must be stored at -20°C for up to one week.

6. 2 PCR Amplification Reaction The reaction includes two rounds of specific amplification of a WSSV gene segment based on specific primer pairs (4.2.1). Use primer pair 146F1, 146R1 for the first amplification to produce a product of 1441 bp and primer pair 146F2, 146R2 for the second amplification to produce a product of 941 bp.

6. 2.1 First Amplification Step The components of a 50 μl PCR reaction for the first step are as follows:

6. 2.1.1 For test samples: draw 46 μl PCR master mix into a 0.2 ml Eppendorf tube, then add 1 μl each of primers 146F1 and 146R1 at 25 mM concentration (4.2.1) and 2 μl of DNA extract from the sample to be analyzed obtained in Article 6.1.2.

6. 2.1.2 Positive control sample: replace the DNA extract from the sample to be analyzed with DNA extract from a known white spot syndrome virus (WSSV) infected sample.

6. 2.1.3 Negative control sample: replace the DNA extract from the sample to be analyzed with sterile distilled water.

6. 2.2 Second Amplification Step Draw 46 μl PCR master mix into a 0.2 ml Eppendorf tube, then add 1 μl each of primers 146F2 and 146R2 at 25 mM concentration (4.2.1) and 2 μl of the first amplification product. The PCR amplification reactions for both steps are performed on a thermal cycler (4.1.6) and set with the same reaction program. The amplification reaction program: at 94°C for 4 minutes (1 cycle); at 94°C for 1 minute; at 55°C for 1 minute; at 72°C for 2 minutes (39 cycles); at 72°C for 2 minutes (1 cycle); at 4°C until analysis. The second amplification product is immediately electrophoresed on a 1% agarose gel (4.2.2.6) containing ethidium bromide (4.2.2.8).

6. 3 Electrophoresis

6. 3.1 Preparing the Agarose Gel Mix 1% agarose in 1X TBE buffer (4.2.2.5). After completely melting the agarose, cool it to about 60°C, then add 5 ml of ethidium bromide solution (4.2.2.8) and 100 ml of 1% agarose (4.2.2.6). Pour the agarose mixture into a mold with combs to form wells. The electrophoresis gel should have a thickness of about 3-4 mm. After preparation, immerse the gel in 1X TBE buffer.

6. 3.2 Electrophoresis Chamber The electrophoresis chamber contains 1X TBE buffer (4.2.2.5).

6. 3.3 Electrophoresis Mix 10 ml of amplification product (6.2) with 2 ml of loading dye (4.2.2.7) and load it into the wells. Electrophorese for 30 minutes at 100 V and 50 mA.

7 Reading Results The results are read on a UV reader (wavelength 302 nm). The specific amplification products of WSSV in the first step are 1441 bp and in the second step are 941 bp. Based on the DNA ladder, the sample is determined to be positive or negative for WSSV according to the specific amplification products of the first and second amplification steps.

8 Safety Regulations During the operation process, the following regulations must be strictly followed:

8. 1 Ethidium bromide stain is a carcinogenic substance that can cause cancer in humans and animals. Therefore, when using this chemical, gloves and protective goggles must be worn. Used solutions must pass through activated charcoal before disposal.