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罗非鱼湖病毒微滴式逆转录数字PCR检测方法的建立

李敏 李永福 黄育浩 陈灼均 莫钻兰 钟群芳 李本旺 张险朋

李敏, 李永福, 黄育浩, 陈灼均, 莫钻兰, 钟群芳, 李本旺, 张险朋. 罗非鱼湖病毒微滴式逆转录数字PCR检测方法的建立[J]. 南方水产科学. doi: 10.12131/20220184
引用本文: 李敏, 李永福, 黄育浩, 陈灼均, 莫钻兰, 钟群芳, 李本旺, 张险朋. 罗非鱼湖病毒微滴式逆转录数字PCR检测方法的建立[J]. 南方水产科学. doi: 10.12131/20220184
LI Min, LI Yongfu, HUANG Yuhao, CHEN Zhuojun, MO Zuanlan, ZHONG Qunfang, LI Benwang, ZHANG Xianpeng. Establishment of reverse transcription droplet digital PCR assay for detection of Tilapia Lake Virus[J]. South China Fisheries Science. doi: 10.12131/20220184
Citation: LI Min, LI Yongfu, HUANG Yuhao, CHEN Zhuojun, MO Zuanlan, ZHONG Qunfang, LI Benwang, ZHANG Xianpeng. Establishment of reverse transcription droplet digital PCR assay for detection of Tilapia Lake Virus[J]. South China Fisheries Science. doi: 10.12131/20220184

罗非鱼湖病毒微滴式逆转录数字PCR检测方法的建立

doi: 10.12131/20220184
基金项目: 东莞市2021年省乡村振兴战略专项资金 (“大专项+任务清单”) 项目 (20211800400112)
详细信息
    作者简介:

    李敏:李 敏 (1982—),女,工程师,硕士,从事动物流行病学研究。E-mail: 362832680@qq.com

    通讯作者:

    张险朋 (1979—),男,高级兽医师,硕士,从事水生动物疫病防治研究。E-mail: xian.peng@163.com

  • 中图分类号: S 943

Establishment of reverse transcription droplet digital PCR assay for detection of Tilapia Lake Virus

  • 摘要: 建立一种敏感性高、特异性强、重复性好的罗非鱼湖病毒反转录微滴式数字PCR (RT-ddPCR) 检测方法,可为罗非鱼湖病毒的定量检测提供技术支持。参照NCBI中GenBank登陆的TiLV第3 段全基因序列,选择hypothetical protein gene 基因作为靶位基因设计合成了1 对引物和探针,以TiLV-cDNA为模板,摸索、优化反应方法,建立与实时荧光RT-PCR检测方法的线性关系,分析方法的敏感性、特异性、重复性,最后进行临床样品检测。结果显示,当引物、探针浓度分别为500、300 nmol·L−1且退火温度为54.2 ℃时,建立的TiLV RT-ddPCR扩增反应效率最高、阴阳性微滴分布界限最明显、平均拷贝数较高;敏感性强,检测低至2 拷贝·μL−1且在1~90 000 拷贝·μL−1范围内,与实时荧光RT-PCR检测的线性关系较好 (R2=0.995 8);检测变异系数低 (4.86%);与其他5 种常见的水生动物疫病病毒 (鲤浮肿病毒、锦鲤疱疹病毒、草鱼出血病毒、鲫造血器官坏死病毒、细胞肿大虹彩病毒) 阳性样品未发生交叉反应;在临床样品的检测中,48 份罗非鱼样品结果均为阴性,5份能力验证样品中3 份为阳性,与能力验证满意结果一致。该研究建立的罗非鱼湖病毒RT-ddPCR检测方法敏感性高、特异性强、重复性好,能够准确定量检测罗非鱼湖病毒,将为罗非鱼湖病毒相关研究提供有益参考。
  • 图  1  不同探针浓度时的扩增图

    Figure  1.  Fluorescence amplitude at different concentrations of probe

    图  2  不同引物浓度的ddPCR扩增图

    Figure  2.  Fluorescence amplitude with different primers concentrations

    图  3  退火温度的优化

    Figure  3.  Optimization of annealing temperature

    图  4  TiLV-cDNA的ddPCR与实时荧光PCR线性关系

    Figure  4.  Correlation between measured TiLV-cDNA numbers of TiLV-cDNA standard by ddPCR and real-time PCR

    图  5  TiLV的ddPCR特异性试验

    Figure  5.  Specificity for detection of ddPCR for TiLV

    图  6  ddPCR的组内重复性试验

    Figure  6.  Replicate of intra-assay using ddPCR

    图  7  TiLV RT-ddPCR 能力验证样品和部分临床样品检测结果

    Figure  7.  Proficiency testing samples and some clinical samples detection by TiLV RT-ddPCR

    图  8  2020 年TiLV 实时荧光RT-PCR 能力验证样品检测结果

    Figure  8.  Proficiency testing samples detection by TiLV real-time RT-PCR in 2020

    表  1  RT-ddPCR和实时荧光RT-PCR检测罗非鱼湖病毒的引物、探针

    Table  1.   Primer and probe of RT-ddPCR and real-time RT-PCR for TiLV detection

    引物、探针  
    Primer, probe  
    引物序列
    Primer sequences (5'–3')
    上游引物
    Upstream primer
    TTCGAGTGCTCAAAGTTCCT
    下游引物
    Downstream primer
    CGTGCGTACTCGTTCAGTATA
    探针
    Probe
    FAM-TCAAGACCACACTCCTCA
    CCRCAG-BHQ1
    下载: 导出CSV

    表  2  逆转录反应体系和反应程序

    Table  2.   Reaction mixture and protocol of reverse transcription

    反应体系
    Reaction mixture
    用量
    Volume/μL
    反应程序
    Protocol
    5×FastKing-RT SuperMix 4 42 ℃去除基因组及反转录15 min;95 ℃酶灭活
    3 min
    Total RNA 2
    RNase-Free ddH2O 14
    下载: 导出CSV

    表  3  TiLV-cDNA ddPCR反应体系和反应程序

    Table  3.   Reaction mixture and protocol of ddPCR for TiLV-cDNA detection

    反应体系
    Reaction mixture
    用量
    Volume/mL
    终浓度
    Final concentration
    反应程序 Protocol
    温度
    Temperature/℃
    时间
    Time
    循环次数
    Cycle
    2×ddPCR SupermixTM for Probes 10 95 10 min 1
    引物 Primer 0.36 900 nmol·L−1 94 30 s 40
    探针 Probe 0.1 250 nmol·L−1 60 60 s 1
    cDNA模板 cDNA template 1.0 98 10 min 1
    dd-H2O 8.54 4
    下载: 导出CSV

    表  4  TiLV-cDNA 实时荧光PCR反应体系和反应程序

    Table  4.   Reaction mixture and protocol of real-time PCR for TiLV-cDNA

    反应体系
    Reaction mixture
    用量
    Volume/μL
    反应程序 Protocol
    温度
    Temperature/℃
    时间
    Time
    循环次数
    Cycle
    5×PCR buffer 5 95 3 min 1
    dNTPs 2
    Taq DNA聚合酶 Polymerase 0.6 95 15 s 45
    引物/探针 Primer/Probe 0.1/0.08
    cDNA模板 cDNA template 2 55 (收集荧光) 30 s
    dd-H2O 15.12
    下载: 导出CSV

    表  5  不同探针浓度的ddPCR检测的cDNA分子数

    Table  5.   Normalized detected target DNA by ddPCR at different probe concentrations

    探针浓度
    Probe concentration/
    (nmol·L−1)
    cDNA分子数
    Normalized detected target DNA/
    (拷贝·μL−1)
    15016 400±1 624.84
    20016 040±1 020.89
    25015 060±1 175.79
    30016 080±1 561.57
    下载: 导出CSV

    表  6  不同引物浓度的ddPCR检测的cDNA分子数

    Table  6.   Normalized detected target cDNA by ddPCR at different primer concentrations

    引物浓度
    Primer concentration/
    (nmol·L−1)
    cDNA分子数
    Normalized detected target DNA/
    (拷贝·μL−1)
    20015 580±1 165.28
    30016 540±2 645.52
    40016 500±900.57
    50017 360±336.39
    60016 800±331.46
    70016 440±507.02
    80016 760±131.99
    90017 500±861.06
    1 00016 720±244.59
    1 10015 620±1 082.75
    1 20015 760±1 268.10
    下载: 导出CSV

    表  7  不同退火温度ddPCR检测的cDNA分子数

    Table  7.   Normalized detected target cDNA by ddPCR at different annealing temperatures

    退火温度
    Annealing temperature/ ℃
    cDNA分子数
    Normalized detected target DNA/
    (拷贝·μL−1)
    61.016 280±1 420.83
    60.215 140±2 237.92
    58.815 780±4 166.57
    56.715 980±1 908.79
    54.216 360±2 022.83
    52.116 020±2 432.50
    50.816 260±1 830.22
    50.015 740±1 942.53
    下载: 导出CSV

    表  8  TiLV-cDNA ddPCR和实时荧光PCR方法敏感性试验

    Table  8.   Sensitivity test of ddPCR and real-time PCR for TiLV-cDNA

    微滴式数字 PCR ddPCR实时荧光PCR real-time PCR
    样品/稀释倍数
    Sample/Dilution
    ratio
    总微滴数*
    Number of
    droplets
    阳性微滴数*
    Positive
    droplets
    cDNA分子数
    Normalized detected
    target DNA/
    (拷贝·μL−1)
    cDNA分子的
    平均数±标准差
    X±SD
    cDNA分子数的
    变异系数
    CV/%
    Ct值
    Ct Value
    平均值±标准差
    Mean±SD
    变异系数
    CV/%
    cDNA 15 547 15 023 79 800 82 800±4 233.20 5.11 20.12 20.41±0.37 1.83
    18 355 17 778 81 400 20.83
    17 782 17 357 87 800 20.27
    cDNA/2 17 010 13 679 38 360 40 880±3 070.53 7.51 21.75 21.60±0.17 0.80
    17 982 15 251 44 340 21.41
    17 358 14 207 40 140 21.63
    cDNA/4 18 420 11 442 22 840 23 220±340.78 1.47 23.13 22.81±0.45 1.97
    17 543 11 058 23 420 23.01
    18 527 11 687 23 440 22.3
    cDNA/8 19 145 6 374 9 520 10 360±3 731.40 36.02 24.05 23.78±0.28 1.16
    19 992 7 041 4 220 23.79
    18 496 7 108 1 1420 23.5
    cDNA/16 19 583 3 906 5 240 5 002±274.95 5.50 24.94 24.86±0.40 1.61
    19 997 3 870 5 060 24.43
    19 031 3 845 4 700 25.22
    cDNA/32 18 624 2 007 2 740 2 736±170.10 6.22 26.32 26.39±0.22 0.85
    18 828 1 946 2 560 26.64
    18 722 2 176 2 900 26.21
    cDNA/64 18 708 757 972 1 092±120.01 10.99 26.79 27.15±0.33 1.20
    18 944 858 1 090 27.25
    18 618 935 1 212 27.42
    cDNA/128 17 010 525 738 738±11.02 1.49 28.41 28.24±0.32 1.14
    17 723 556 750 28.44
    18 609 567 728 27.87
    cDNA/256 18 922 320 402 374±27.15 7.26 29.73 29.78±0.24 0.80
    17 785 277 370 30.04
    16 723 245 348 29.57
    cDNA/512 17 195 134 184 162±19.70 12.16 30.81 30.67±0.19 0.63
    13 343 88 156 30.75
    16 586 103 146 30.45
    cDNA/1 024 18 273 55 70 66±5.29 8.01 32.11 32.10±0.01 0.03
    17 070 49 68 32.1
    17 303 44 60 32.09
    cDNA/2 048 17 316 23 32 29.8±8.84 29.66 32.74 32.86±0.15 0.47
    18 791 30 38 32.8
    18 324 16 20.6 33.03
    cDNA/4 096 18 590 15 19 18.4±0.42 2.26 33.39 33.68±0.46 1.36
    16 562 13 18.4 34.21
    16 821 13 18.2 33.44
    cDNA/8 192 17 892 4 5.2 10.2±5.10 50.00 35.99 35.44±0.61 1.73
    16 910 11 15.4 35.55
    16 276 7 10.2 34.78
    cDNA/16 384 17 413 4 5.4 6.2±0.69 11.17 38.95 37.01±1.70 4.59
    17 684 5 6.6 36.27
    17 941 5 6.6 35.8
    cDNA/32 768 17 761 4 5.2 3.6±1.45 40.18 38.55 37.95±0.73 1.92
    18 207 2 2.6 38.16
    17 341 2 2.8 37.14
    cDNA/65 536 17 395 1 1.4 2±0.60 30.00
    18 230 2 2.6
    16 999 1 2
    cDNA/131 072 18 359 0 0
    18 619 0 0
    14 472 0 0
    cDNA/262 144 15 121 0 0
    14 538 0 0
    13 526 0 0
    cDNA/524 288 15 733 0 0
    14 537 0 0
    15 672 0 0
    注:*. 20 μL反应体系。 Note: *. 20 μL reaction mixture.
    下载: 导出CSV

    表  9  4倍稀释的TiLV-cDNA ddPCR组内重复性试验

    Table  9.   Replicate of intra-assay for 4-fold dilution of TiLV-cDNA ddPCR

    样品编号
    Sample No.
    cDNA分子数
    Normalized detected target DNA/
    (拷贝·μL−1)
    TiLV-118 200
    TiLV-218 300
    TiLV-318 200
    TiLV-419 220
    TiLV-518 180
    TiLV-620 880
    TiLV-718 620
    TiLV-818 840
    TiLV-918 600
    TiLV-1017 380
    TiLV-1117 380
    TiLV-1217 880
    TiLV-1317 400
    TiLV-1419 220
    TiLV-1518 340
    平均数±标准差 X±SD18 442.67±896.15
    变异系数 CV/%4.86
    下载: 导出CSV

    表  10  TiLV(能力验证样品) RT-ddPCR与实时荧光RT-PCR检测结果

    Table  10.   Results of TiLV RT-ddPCR and real-time RT-PCR for proficiency testing samples

    能力验证样品编号
    No. of proficiency testing
    cDNA分子数
    Normalized detected target DNA/
    (拷贝·μL−1)
    Ct值*
    Ct value
    1017 76019.90
    951 92021.58
    1220Undet
    17151824.85
    2430Undet
    注:*. 实时荧光RT-PCR方法 (SC/T 7223—2020)。 Note: *. Real-time RT-PCR (SC/T 7223–2020).
    下载: 导出CSV
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  • 文章访问数:  47
  • 被引次数: 0
出版历程
  • 收稿日期:  2022-06-30
  • 修回日期:  2022-07-24
  • 录用日期:  2022-08-09
  • 网络出版日期:  2022-09-05

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