甘蔗条点病毒荧光定量PCR检测方法的建立及应用

doi:10.13560/j.cnki.biotech.bull.1985.2024-0129

摘要/Abstract

摘要：

【目的】 建立一种快速、灵敏、特异的检测甘蔗条点病毒（sugarcane striate virus，SStrV）的SYBR Green I 荧光定量PCR方法。【方法】 从SStrV基因组保守区域序列设计特异性扩增引物，构建含有SStrV基因组序列的重组质粒pMD19-T-SStrV-qN作为阳性质粒标准品，以其为模板建立SStrV荧光定量PCR检测方法，并对该方法的灵敏性、特异性、稳定性进行了测试，随后用该方法对甘蔗不同组织部位中SStrV载量进行检测。【结果】 将含有SStrV基因组序列的重组质粒按10倍比稀释成标准品，将其作为模板进行荧光定量PCR，获得标准曲线y = -3.337 x + 38.197，相关系数r²=0.999，说明Cq值与标准品浓度拷贝数的对数呈线性关系；建立的荧光定量PCR最低可以检测到13拷贝重组质粒/μL，是普通PCR灵敏度的100倍。该方法能特异的检测SStrV，特异性高，组内和组间的变异系数在0.13% - 0.94%之间，表明该方法重复性良好。SStrV的载量在甘蔗的不同组织部位中差异较大，+4叶中SStrV的载量最高，与其他组织部位达到了显著差异。【结论】 建立了能灵敏特异检测SStrV的SYBR Green I 荧光定量PCR方法，明确了+4叶是甘蔗中SStrV检测的最佳采样部位。

关键词: 甘蔗条点病毒, 荧光定量PCR, 病毒载量, 检测

Abstract:

【Objective】 It is to establish a rapid, sensitive and specific SYBR Green I quantitative PCR method for the detection of sugarcane striate virus（SStrV）.【Method】 A specific amplification primer was designed from the conserved sequence of the SStrV genome sequence, the recombinant plasmid pMD19-T-SStrV-qN containing SStrV gene was constructed as a positive plasmid standard. Using it as template, the SStrV fluorescence quantitative PCR assay was established. And the sensitivity, specificity, stability, and subsequently of this method were tested, then the SStrV loads in different tissue sites of sugarcane were detected.【Result】 The recombinant plasmid containing SStrV genome sequence was diluted into a standard at a 10-fold ratio, and they were used as a template for real-time PCR, the standard curve y = -3.337 x + 38.197 was obtained, and the correlation coefficient r² = 0.999 was obtained, indicating that the Cq value was linearly related to the logarithm of the copy number of the standard concentration. With this established real-time PCR, the least detection limit was 13 copies of the recombinant plasmid/μL, which was 100 times more sensitive than ordinary PCR. The method specifically detected SStrV with high specificity, and the coefficient of variation within and between groups was n 0.13%-0.94%, indicating that the method was of good repeatability. The load of SStrV was significantly different among different tissue sites of the sugarcane, and the load of SStrV was the highest in +4 leaves, which was significantly different from other tissue sites.【Conclusion】 The SYBR Green I quantitative PCR method is established to provide an efficient quantitative detection method for the diagnosis of SStrV, and it is determined that +4 leaves are the best sampling site for the detection of SStrV in sugarcane.

Key words: sugarcane striate virus, real-time PCR, viral load, detection

王超敏, 何美丹, 王文治, 袁潜华, 张树珍, 沈林波. 甘蔗条点病毒荧光定量PCR检测方法的建立及应用[J]. 生物技术通报, 2024, 40(6): 126-133.

WANG Chao-min, HE Mei-dan, WANG Wen-zhi, YUAN Qian-hua, ZHANG Shu-zhen, SHEN Lin-bo. Establishment and Application of Real-time PCR for Sugarcane Striate Virus[J]. Biotechnology Bulletin, 2024, 40(6): 126-133.

图/表 6

图1 SStrV 片段PCR扩增 M：Marker DS2000; 1-2:扩增的SStrV目的片段；3：阴性对照

Fig. 1 PCR amplification of SStrV fragments M：Marker DS2000; 1-2: amplified SStrV target fragment; 3: negative control

图2 荧光定量PCR标准曲线（A）、扩增曲线（B）以及熔解曲线（C） A为标准曲线，分别由拷贝数为1.31 × 108 copies /μL SStrV质粒DNA标准品经10倍梯度稀释而构建；B为扩增曲线，从左到右分别是由拷贝数为1.31 × 108 copies /μL SStrV质粒DNA标准品经10倍梯度稀释而构建；C为熔解曲线，其峰值对应的温度大于80℃

Fig. 2 PCR standard curve（A）, amplification curve（B）and melting curve（C） A is standard curve, built with a 10-fold serial dilution of SStrV plasmid DNA of 1.31 ×108 copies /μL constructed, respectively. B is amplification curve, from left to right were developed with a 10-fold serial dilution of SStrV plasmid DNA. C is melting curve, and its peak corresponds to a temperature greater than 80℃

图3 荧光定量PCR特异性实验扩增曲线

Fig. 3 Amplification plot of the specificity assay 1：SStrV. 2-6：SCBV、SCMV、SCSMV、SrMV、SCYLV. 7：Blank control

图4 荧光定量PCR灵敏性实验扩增曲线（A）和普通PCR检测方法的敏感性分析（B） A：从左到右分别是由拷贝数为1.31×108 copies /μL SStrv质粒DNA标准品经10倍梯度稀释而构建；B：M：MarkerDS2000；1-8：1.31×108 copies /μL-1.31×10 copies /μL；9：空白对照

Fig. 4 Amplification curve for qPCR sensitivity experiments（A） and sensitivity analysis of conventional PCR（B） A: From left to right were developed with a 10-fold serial dilution of SStrv plasmid DNA; B: M: DSTM 2000（marker）；1-8: 1.31×108 copies /μL-1.31×10 copies /μL. 9: Blank control

表1 荧光定量PCR重复性实验结果

Table 1 Results of qPCR repeatability test

质粒浓度Plasmid concentration/（copies·µL^-1）	组内实验Within-group experiments		组间实验Intergroup experiments
质粒浓度Plasmid concentration/（copies·µL^-1）	Ct值（x±s） Ct value	变异系数（CV） Coefficient variation/%	Ct值（x±s） Ct value	变异系数（CV） Coefficient variation/%
1.31×10⁸	10.94±0.09	0.81	10.89±0.10	0.94
1.31×10⁷	14.51±0.02	0.13	14.44±0.13	0.92
1.31×10⁶	17.90±0.06	0.31	17.80±0.15	0.87
1.31×10⁵	21.35±0.08	0.37	21.23±0.16	0.74
1.31×10⁴	24.47±0.11	0.43	24.53±0.11	0.45
1.31×10³	27.49±0.13	0.48	27.51±0.17	0.63

图5 八个株系不同组织部位SStrV检测图中误差线表示标准偏差，小写字母表示不同组织部位中病毒SStrV载量差异达到（P <0.05）显著水平（使用Duncan法计算）

Fig. 5 Detection of SStrV in different tissue parts of eight lines The error line in the figure refers to the standard deviation. The lowercase letters indicates that the SStrV load of the virus in different tissue sites is significantly different（P <0.05）（Calculated using the Duncan method）

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