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    This document summarizes research on a virus causing covert mortality disease (CMD), also known as "bottom death", in coastal aquatic animals. The causative agent is a 32nm RNA virus called Convert Mortality Nodavirus (CMNV). Clinical signs include coagulative necrosis of striated muscle, eosinophilic inclusions in epithelial tissues, and karyopyknotic nuclei in muscle and lymphoid organs. PCR was used to detect the virus using specific primers. The PCR profile and reaction components are described to detect both the full 619bp sequence and a nested 165bp fragment. Images show histopathological findings in infected juvenile shrimp gill tissue.

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    cara deteksi CMNV di udang

    Original Title

    Cm Nv Diagnosis

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    This document summarizes research on a virus causing covert mortality disease (CMD), also known as "bottom death", in coastal aquatic animals. The causative agent is a 32nm RNA virus called Convert Mortality Nodavirus (CMNV). Clinical signs include coagulative necrosis of striated muscle, eosinophilic inclusions in epithelial tissues, and karyopyknotic nuclei in muscle and lymphoid organs. PCR was used to detect the virus using specific primers. The PCR profile and reaction components are described to detect both the full 619bp sequence and a nested 165bp fragment. Images show histopathological findings in infected juvenile shrimp gill tissue.

    Copyright:

    © All Rights Reserved
    Download as PDF, TXT or read online from Scribd
    Flag for inappropriate content
    Download as pdf or txt
    40 views13 pages

    CM NV Diagnosis

    Uploaded by

    mbah37
    This document summarizes research on a virus causing covert mortality disease (CMD), also known as "bottom death", in coastal aquatic animals. The causative agent is a 32nm RNA virus called Convert Mortality Nodavirus (CMNV). Clinical signs include coagulative necrosis of striated muscle, eosinophilic inclusions in epithelial tissues, and karyopyknotic nuclei in muscle and lymphoid organs. PCR was used to detect the virus using specific primers. The PCR profile and reaction components are described to detect both the full 619bp sequence and a nested 165bp fragment. Images show histopathological findings in infected juvenile shrimp gill tissue.

    Copyright:

    © All Rights Reserved
    Download as PDF, TXT or read online from Scribd
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    of 13

    03/11/57

    Covert mortality disease (CMD)


    bottom death
    Coastal Aquatic Animal Health Research Institute

    03/11/57

    Causative:
    y RNA

    virus

    Convert Mortality Nodavirus (CMNV)

    32 nm particles
    80% mortality
    60-80 days post stocking

    03/11/57

    Clinical signs:
    Coagulative
    C
    l ti necrosis
    i off striated
    t i t d muscle
    l
    Eosinophilic inclusions in epithelium of the
    tubules in hepatopancreas and lymphoid organ
    Mass karyopyknotic nuclei existed in muscle
    and lymphoid organ
    The tubular epithelium of hepatopancreas show
    significant atrophy

    03/11/57

    Other Muscle necrosis:


    y

    I f ti
    Infectious
    myonecrosis
    i virus
    i (IMNV)

    Penaeus vannamei nodavirus (PvNV)

    Macrobrachium rosenbergii nodavirus (MrNV)

    03/11/57

    PCR primer
    Primer:
    CMNV6197F1
    CMNV6197R1
    CMNV1657F2
    CMNV1657R2
    EF1aS-F
    EF1aS-RR
    EF1aS

    Products
    5-AAA TAC GGC GAT GAC G-3
    5-ACG AAG TGC CCA CAG AC-3
    5-CAC AAC CGA GTC AAA CC-3
    5-GCG TAA ACA GCG AAG G-3
    5'-ATG-GTT-GTC-AAC-TTT-GCC-CC-3'
    55'-TTG-ACC-TCC-TTG-ATC-ACA-CC-3'
    TTG ACC TCC TTG ATC ACA CC 3

    :
    CMNV +ve

    First step
    Nested
    Internal control

    619 bp
    165 bp
    500 bp

    : 619 bp first step / 165 bp nested

    03/11/57

    PCR reaction:
    PCR components: 1st step
    DEPC water
    2X Reaction mix
    20 M CMNV619F1 (0.4 M final conc.)
    20 M CMNV619R1 (0.4 M final conc.)
    5 M
    M EF1aS-F
    EF S F (0.1
    ( M
    M final
    fi l conc.))
    5 M EF1aS-R (0.1 M final conc.)
    Superscript III RT/Platinum Taq Mix
    RNA Template
    Total
    Nested master mix 1st step 15 l
    PCR components: Nested
    DEPC water
    2G Fast mastermix
    20 M CMNV6197F2 (0.4 M final conc.)
    20 M CMNV6197R2 (0.4 M final conc.)
    1st step PCR product
    Total

    Volume (l)/reaction
    1.80
    5.00
    0.20
    0.20
    0.20
    2
    0.20
    0.40
    2.00
    10.00
    Volume (l)/reaction
    6.50
    7.50
    0.50
    0.50
    10.00
    25.00

    Primer:
    - 20 M Primer: Stock primer 100 M 50 L + 200 L DEPC water = 20 M
    0.2 l/10 l 0.5 l/25 l reaction = 0.4 M final conc.
    - 5 M Primer EF1aS-F/EF1aS-R: Stock primer 100 M 10 L + 90 L DEPC water =
    10M 50 L + 50 L DEPC water = 5M 0.2 l/10 l reaction (0.1 M final conc.)

    03/11/57

    PCR Profile:
    CMNV 619 (1 hr 20 min.)
    RT-PCR
    Denature
    Annealing
    Extension
    Final Extension

    Cycle No.
    1

    15
    1

    Cooling
    CMNV 165
    Initial denaturation
    Denature
    Annealing
    Extension
    Final Extension
    Cooling

    Cycle No.
    1
    30
    1

    Temperature
    50C
    94C
    94C
    45C
    72C
    72C
    15C

    Time
    30 min
    4 min
    30 s
    30 s
    40 s

    Temperature
    94C
    94C
    50C
    72C
    72C
    15C

    Time
    4 min
    20 s
    20 s
    20 s
    7 min

    7 min

    03/11/57

    Juvenile shrimp : Gill

    03/11/57

    03/11/57

    10

    03/11/57

    11

    03/11/57

    12

    03/11/57

    13

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