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cara deteksi CMNV di udang
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Covert mortality disease (CMD)“bottom death”
Coastal Aquatic Animal Health Research Institute
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Causative:
RNA virus ◦ “ Convert Mortality Nodavirus (CMNV)”
◦ 32 nm particles
◦ 80% mortality
◦ 60-80 days post stocking
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Clinical signs:
◦ C l ti i f t i t d l◦ Coagulative necrosis of striated muscle
◦ Eosinophilic inclusions in epithelium of the tubules in hepatopancreas and lymphoid organ
◦ Mass karyopyknotic nuclei existed in muscle Mass karyopyknotic nuclei existed in muscle and lymphoid organ
◦ The tubular epithelium of hepatopancreas show significant atrophy
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Other Muscle necrosis:
I f ti i i (IMNV) Infectious myonecrosis virus (IMNV)
Penaeus vannamei nodavirus (PvNV)
Macrobrachium rosenbergii nodavirus (MrNV)
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PCR primerPCR primerPrimer: Products
CMNV6197F1 5’-AAA TAC GGC GAT GAC G-3’ First step 619 bp
CMNV6197R1 5’-ACG AAG TGC CCA CAG AC-3’
CMNV1657F2 5’-CAC AAC CGA GTC AAA CC-3’ Nested
165 bp CMNV1657R2 5’-GCG TAA ACA GCG AAG G-3’
EF1aS-F 5'-ATG-GTT-GTC-AAC-TTT-GCC-CC-3' Internal control 500 bp
EF1aS-R 5'-TTG-ACC-TCC-TTG-ATC-ACA-CC-3' EF1aS R 5 TTG ACC TCC TTG ATC ACA CC 3
การแปลผล: CMNV +ve : 619 bp first step / 165 bp nested
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PCR reaction:
PCR components: 1st step Volume (μl)/reaction
DEPC water 1.80
2X Reaction mix 5.00
20 μM CMNV619F1 (0.4 μM final conc.) 0.20
20 μM CMNV619R1 (0.4 μM final conc.) 0.20
M EF S F ( M fi l ) 2 5 μM EF1aS-F (0.1 μM final conc.) 0.20
5 μM EF1aS-R (0.1 μM final conc.) 0.20
Superscript III RT/Platinum Taq Mix 0.40
RNA Template 2.00
Total 10.00
เตรียม Nested master mix →เติมในหลอด 1st step ปริมาณ 15 μl
PCR components: Nested Volume (μl)/reaction PCR components: Nested Volume (μl)/reaction
DEPC water 6.50
2G Fast mastermix 7.50
20 μM CMNV6197F2 (0.4 μM final conc.) 0.50
20 μM CMNV6197R2 (0.4 μM final conc.) 0.50
1st step PCR product 10.00
Total 25.00
เตรียม Primer:
- เตรียม 20 μM Primer: จาก Stock primer 100 μM → 50 μL + 200 μL DEPC water = 20 μM → ดึงไปใช้ 0.2 μl/10 μl และ 0.5 μl/25 μl reaction = 0.4 μM final conc.
- เตรียม 5 μM Primer EF1aS-F/EF1aS-R: จากStock primer 100 μM →10 μL + 90 μL DEPC water = 10μM → 50 μL + 50 μL DEPC water = 5μM ดึงไปใช้ 0.2 μl/10 μl reaction (0.1μ M final conc.)
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PCR Profile:
CMNV 619 (1 hr 20 min.) Cycle No. Temperature Time
RT-PCR 1 50°C 30 min
94°C 4 min
Denature
15
94°C 30 s
Annealing 45°C 30 s
Extension 72°C 40 s
Final Extension 1 72°C 7 min
Cooling 15°C α
CMNV 165 Cycle No. Temperature Time
Initial denaturation 1 94°C 4 min
Denature
30
94°C 20 s
Annealing 50°C 20 s
Extension 72°C 20 s
Final Extension 1 72°C 7 min
Cooling 15°C α
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Juvenile shrimp : Gill
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