Establishment of reverse transcription droplet digital PCR assay for detection of Tilapia Lake Virus
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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，检测限低至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 (Carp edema virus, CEV)、锦鲤疱疹病毒 (Koi herpesvirus, KHV)、草鱼出血病毒 (Grass carp reovirus, GCRV)、鲫造血器官坏死病毒 (Cyprinid herpesvirus 2, CyHV-2)、细胞肿大虹彩病毒 (Red sea bream iridovirus, RSIV)] 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。
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关键词:
- 罗非鱼湖病毒 /
- 微滴式逆转录数字PCR /
- 线性关系 /
- 特异性 /
- 变异系数
Abstract: To establish an assay of reverse transcription droplet digital PCR (RT-ddPCR) for Tilapia Lake Virus (TiLV), we designed a pair of specific primers and probe based on the conserved region of TiLV segment 3 and evaluated the specificity, sensitivity and repeatability of this method. The structured standard curve was evaluated by using TiLV-cDNA as a template. Finally, the samples were tested. When the concentrations of primers and probes were 500 and 300 nmol·L−1 and the annealing temperature was 54.2 ℃, the established TiLV RT-ddPCR amplification reaction efficiency was the highest, the distribution boundary of the positive and negative droplets was the most obvious, and the average copy number was higher. The RT-ddPCR of TiLV had a lower limit of detection with 2 copies·μL−1 and showed a good linear relationship between 1–90 000 copies·μL−1 (Correlation coefficient R2=0.995 8). There was no amplification reaction to other viruses in aquatic animals. The CV of ddPCR for TiLV-cDNA was 4.86%. There was no cross reaction with the positive samples of other five common aquatic animal disease viruses [Carp edema virus (CEV), Koi herpesvirus (KHV), Grass carp reovirus (GCV), Cyprinid herpesvirus 2 (CyHV-2), Red sea bream iridovirus (RSIV)]. Among the 53 detected samples, 48 were negative, three of five proficiency testing samples were positive, consistent with satisfactory previous proficiency testing results. -
图 2 不同引物浓度的ddPCR扩增图
Figure 2. Fluorescence amplitude with different primers concentrations
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 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 (5'—3')Primer sequence (5'–3') 上游引物Upstream primer TTCGAGTGCTCAAAGTTCCT 下游引物 Downstream primer CGTGCGTACTCGTTCAGTATA 探针 Probe FAM-TCAAGACCACACTCCTCACCRCAG-BHQ1 
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表 2 TiLV-cDNA ddPCR反应体系和反应程序
Table 2. 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 1× 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 双蒸水 ddH2O 8.18 — 4 ∞ —
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表 3 不同探针浓度的ddPCR检测的cDNA分子数
Table 3. 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) 150 16 400±1 624.84 200 16 040±1 020.89 250 15 060±1 175.79 300 16 080±1 561.57
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表 4 不同引物浓度的ddPCR检测的cDNA分子数
Table 4. 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) 200 15 580±1 165.28 300 16 540±2 645.52 400 16 500±900.57 500 17 360±336.39 600 16 800±331.46 700 16 440±507.02 800 16 760±131.99 900 17 500±861.06 1 000 16 720±244.59 1 100 15 620±1 082.75 1 200 15 760±1 268.10
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表 5 不同退火温度ddPCR检测的cDNA分子数
Table 5. 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) 61.0 16 280±1 420.83 60.2 15 140±2 237.92 58.8 15 780±4 166.57 56.7 15 980±1 908.79 54.2 16 360±2 022.83 52.1 16 020±2 432.50 50.8 16 260±1 830.22 50.0 15 740±1 942.53
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表 6 TiLV-cDNA ddPCR和实时荧光PCR方法敏感性试验
Table 6. 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分子数的平均值±标准差$\overline { X}\pm { \rm {SD}}$ cDNA分子数的变异系数CV/% Ct值Ct value 平均值±标准差$\overline { X}\pm { \rm {SD}}$ 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.
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表 7 4倍稀释的TiLV-cDNA ddPCR组内重复性试验
Table 7. 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-1 18 200 TiLV-2 18 300 TiLV-3 18 200 TiLV-4 19 220 TiLV-5 18 180 TiLV-6 20 880 TiLV-7 18 620 TiLV-8 18 840 TiLV-9 18 600 TiLV-10 17 380 TiLV-11 17 380 TiLV-12 17 880 TiLV-13 17 400 TiLV-14 19 220 TiLV-15 18 340 平均数±标准差 $\overline { X}\pm { \rm {SD}}$ 18 442.67±896.15 变异系数 CV/% 4.86
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表 8 TiLV (能力验证样品) RT-ddPCR与实时荧光RT-PCR检测结果
Table 8. 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 sample cDNA分子数Normalized detected target DNA/(拷贝·μL−1) Ct值*Ct value 10 17 760 19.90 95 1 920 21.58 122 0 Undet 171 518 24.85 243 0 Undet 注：*. 实时荧光RT-PCR方法 (SC/T 7223—2020)。 Note: *. Real-time RT-PCR (SC/T 7223—2020).
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