A TaqMan RT-qPCR Assay for Gyrovirus 3 Detection
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摘要:
目的 建立一种检测圆圈病毒3型 (Gyrovirus 3，GyV3) 的TaqMan 荧光定量PCR方法，为GyV3提供快速便捷的检测技术手段。 方法 根据NCBI数据库中GyV3 VP2基因保守区序列，设计一对特异性检测引物和一条标记FAM荧光基团的探针，经过条件优化，建立用于检测GyV3的TaqMan 实时荧光定量PCR快速检测方法，并进行特异性、敏感性、重复性以及模拟样品的检测试验。 结果 该方法灵敏性高，最低检测限度为1.585 copies·μL−1；特异性强，对其他常见禽病原不发生非特异性反应；重复性好，批内和批间变异系数均小于1%；对鸡肝脏组织DNA加入重组质粒制备的模拟样品同时进行TaqMan荧光定量PCR与SYBR Green Ⅰ实时荧光定量PCR检测，均扩增出与预期相符的扩增曲线。 结论 首次建立用于检测GyV3的TaqMan 实时荧光定量PCR快速检测方法，具有灵敏性高、特异性强、重复性好等优点，为GyV3的诊断和流行病学调查提供了技术支持。 Abstract:Objective An RT-qPCR method to accurately detect gyrovirus 3 (GyV3) was established. Methods A pair of specific primers and an FAM-labeled fluorophobe were designed according to the conserved region of GyV3 VP2 in Genbank database. Reaction conditions of a rapid TaqMan RT-qPCR assay were optimized, and specificity, sensitivity, repeatability, and simulation tests conducted to verify the applicability of the methodology. Results The assay exhibited a high sensitivity with a minimum detection limit of 1.585 copies·μL−1, a specificity free of cross-reactivity with other prevalent avian pathogens, and a reproducibility with less than 1% coefficients of variation on intra- and inter-batch determinations. On a simulated sample with an added recombinant plasmid to the chicken liver tissue DNA, the assay positively identified GyV3 with same result as did the SYBR Green Ⅰ PCR. Conclusion The newly developed TaqMan RT-qPCR assay showed high sensitivity, specificity, repeatability, and rapid turn-around time in detecting GyV3. It was considered appropriate for clinical diagnosis and epidemiological investigation on the virus. -
Key words:
- Gyrovirus 3 /
- RT-qPCR /
- detection assay /
- transmissible viral proventriculitis
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图 2 GyV3探针法荧光定量特异性
1～5分别为GyV3、IBDV、MDV、ALV、CIAV。
Figure 2. Specificity of TaqMan RT-qPCR assay
1–5 represent GyV3, IBDV, MDV, ALV, and CIAV, respectively.
图 3 GyV3探针法荧光定量方法的敏感性
1～9分别为质粒含量1.585×108～1.585×100 copies·μL−1。
Figure 3. Sensitivity of TaqMan RT-qPCR assay for GyV3
1–9 represent plasmid concentrations at 1.585×108–1.585×100 copies·μL−1, respectively.
图 4 GyV3 SYBR Green Ⅰ实时荧光定量PCR方法的敏感性
1～8分别为质粒含量1.585×108～1.585×101 copies·μL−1。
Figure 4. Sensitivity SYBR Green I PCR assay for GyV3
1–8 represent plasmid concentrations at 1.585×108–1.585×101 copies·μL−1, respectively.
图 5 GyV3探针法荧光定量PCR模拟样本检测
1～4分别为质粒含量1.585×103～1.585×106 copies·μL−1。
Figure 5. GyV3 TaqMan RT-qPCR detection on simulated sample
1–4 represent plasmid concentrations at 1.585×103–1.585×106 copies·μL−1, respectively.
表 1 引物和探针序列
Table 1. Sequences of primers and probe
引物名称
Primer name序列（5′-3′）
Sequences片段大小
Product size/bp备注
NoteGyV3-P FAM-CGACCTGGATGGTTCTGTAGTTCTTGG-BHQ1 — 探针 Probe GyV3-F CCGACTCCGACGAACTTATTG 113 探针法引物 Primers of TaqMan PCR GyV3-R AAACACGACTCTCCCTACCT GyV3-qPCR-F TGGATTACATCGCGAACAG 174 染料法引物 Primers of SYBR Green ⅠPCR GyV3-qPCR-R GCATATCGAAGTTTACGCC GyV3 VP2-F ATGTCATCCGGCGGTCTAG 720 普通PCR引物 Primers of conventional PCR GyV3 VP2-R TTACCAGGTAAGATCCCGGATG 
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表 2 荧光定量PCR重复性试验
Table 2. Repeatability and stability of qPCR on GyV3 detection
标准质粒浓度
Concentration of standard-plasmid/ (copies·μL−1)批内变异试验
Intra-assay variability批间变异试验
Inter-assay variability平均数±标准差
$ \overline{x} $±SD变异系数
CV/%平均数±标准差
$ \overline{x} $±SD变异系数
CV/%1.585×106 15.52±0.04 0.32 15.57±0.11 0.73 1.585×103 24.78±0.05 0.22 24.61±0.06 0.26 1.585×101 32.17±0.13 0.42 32.38±0.14 0.44
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