番茄褪绿病毒实时荧光定量PCR检测技术的建立

doi:10.3864/j.issn.0578-1752.2018.10.020

摘要/Abstract

摘要：

目的建立一种实时荧光定量PCR（real-time fluorescent quantitative polymerase chain reaction,RT-qPCR）检测番茄褪绿病毒（Tomato chlorosis virus,ToCV）的方法,用于测定田间植株样品及其传毒媒介烟粉虱（Bemesia tabaci）中ToCV的含量。方法首先根据ToCV次要外壳蛋白（minor coat protein,CPm）基因的保守区域序列,运用Primer 3.0在线软件设计ToCV特异性检测引物ToCVqF和ToCVqR,并通过Primer-BLAST进行比对保证检测引物的特异性;其次分别以感染ToCV、番茄黄化曲叶病毒（Tomato yellow leaf curl virus, TYLCV）、番茄斑萎病毒（Tomato spotted wilt virus, TSWV）的番茄样品为模板,应用该对引物进行ToCV基因片段的克隆鉴定;将阳性克隆样品放入含有氨苄青霉素的LB液体培养基中扩大培养,并提取质粒,获得ToCV质粒标准品,将ToCV质粒标准品按10倍浓度梯度稀释,建立标准曲线;同样以按10倍浓度梯度稀释的ToCV质粒标准品为模板,同时运用RT-qPCR和反转录PCR（reverse transcription-polymerase chain reaction,RT-PCR）,比较这两种方法检测ToCV的灵敏度;最后应用该方法对田间采集的8份疑似ToCV感染的番茄样品以及获毒24 h后的单头烟粉虱进行病毒检测,结合扩增曲线、熔解曲线和Ct值对待测样品中的带毒情况和带毒量进行测定。结果在感染ToCV的番茄样品和阳性对照中,引物ToCVqF和ToCVqR均能够特异扩增出ToCV CPm基因片段,目的片段大小为230 bp,而在感染TYLCV、TSWV的番茄样品、健康番茄样品和空白对照中,该对引物则无法扩增出特异性目的条带,说明该对引物特异性较强;构建ToCV的RT-qPCR标准曲线结果表明,随着ToCV质粒标准品模板浓度逐渐降低,Ct值出现递增趋势,并且在质粒标准品浓度2.7×10³—2.7×10⁹ copies/μL范围内,ToCV标准曲线Ct值同质粒标准品浓度之间呈现良好的线性关系,扩增效率为100%,决定系数R²=0.9911,直线方程为y= -3.32×lgx+40.06（y代表循环阈值即Ct值,x代表质粒初始浓度）,该曲线将用于ToCV含量的测定。RT-qPCR灵敏度检测结果表明,该方法能够检测出的ToCV质粒标准品最低浓度为2.7×10³ copies/μL,而常规RT-PCR仅能够检测出2.7×10⁵ copies/μL的病毒样品,通过RT-qPCR法检测ToCV比常规RT-PCR法灵敏100倍。该方法能成功检测出田间植物样品携带ToCV情况,8份疑似ToCV感染的番茄样品中有6份携带ToCV,同常规RT-PCR检测结果一致,结合标准曲线计算出感染ToCV的番茄样品带毒量分别为2.48×10⁵、2.21×10⁵、7.97×10⁴、3.74×10⁷、3.37×10⁷、2.78×10⁶ copies/μL;烟粉虱获毒24 h后,携毒率为100%,单头烟粉虱最高带毒量为2.55×10⁴ copies/μL,最低为6.46×10² copies/μL。结论建立的RT-qPCR检测ToCV的方法特异性强、灵敏度高,可以实现植物样品和媒介昆虫携带ToCV情况和病毒含量的检测,可为ToCV的准确、有效监测和预警提供技术支持。

关键词: 番茄褪绿病毒, 实时荧光定量PCR, 烟粉虱, 检测

Abstract:

【Objective】 The objective of this study is to develop a real-time fluorescent quantitative PCR (RT-qPCR) method to detect the Tomato chlorosis virus (ToCV) and its concentration in plant and the vector Bemesia tabaci.【Method】 Firstly, the special primers ToCVqF/R were designed using Primer 3.0 for RT-qPCR based on the highly conserved region of the minor coat protein (CPm). The primers specificity was evaluated by Primer-BLAST. Secondly, the fragment was amplified using the primers ToCVqF/R by PCR within the ToCV-infected, Tomato yellow leaf curl virus (TYLCV)-infected, and Tomato spotted wilt virus (TSWV)-infected tomato samples, and cloned into pMD^TM 18-T Vector. The positive clone was selected and multiplied in LB liquid medium containing ampicillin. Furthermore, the recombinant plasmid was extracted from the clone and served as the ToCV standard. Then, the standard curve was generated for quantitative analysis using the ten-fold dilution of the recombinant plasmid. In addition, the detection sensitivity of RT-qPCR was also evaluated and compared to that of reverse transcription-polymerase chain reaction (RT-PCR). Finally, this method was used to detect and quantify ToCV in eight tomato samples with suspected ToCV infection and single B. tabaci after acquiring ToCV for 24 h, according to the amplification curves, melting curves and Ct values.【Result】 The purpose strips could be successfully amplified with the primers ToCVqF/R only in the ToCV-infected tomato sample and positive control, which indicated that the primers was highly specific. Additionally, the sequencing result of the fragment was in according with the target gene CPm. The ToCV standard curve showed that the Ct values gradually increased as the concentration of recombinant plasmid decreased. Among the concentration range of 2.7×10³-2.7×10⁹ copies/μL, the standard curve showed a good linear relationship between Ct values and plasmid concentration. The correlation coefficient of the standard curve was 0.9911, and the amplification efficiency was 100%. The Straight-line equation is y=-3.32×lgx+40.06 (y and x represent the Ct value and plasmid concentration, respectively). The sensitivity of RT-qPCR was 100 times higher than regular RT-PCR. The minimum detectable concentration of ToCV plasmid standard in RT-qPCR assay is 2.7×10³ copies/μL, while that in RT-PCR assay is 2.7×10⁵ copies/μL. This method could successfully detect the ToCV within tomato samples, and the result was consistent with RT-PCR assay. Six of eight tomato samples were infected by ToCV, the viral titers of which were 2.48×10⁵, 2.21×10⁵, 7.97×10⁴, 3.74×10⁷, 3.37×10⁷ and 2.78×10⁶ copies/μL, respectively. After 24 h feeding on ToCV infected tomato plants, 100% of the B. tabaci acquired this virus successfully, and the virus concentration in single whitefly ranged from 6.46×10² to 2.55×10⁴ copies/μL.【Conclusion】 The RT-qPCR method is applicable for accurate and sensitive detection of ToCV both in plants and vector insects, which will provide a technical support for the monitoring and early warning of this virus.

Key words: Tomato chlorosis virus (ToCV), real-time fluorescent quantitative PCR (RT-qPCR), Bemisia tabaci, detection

丁天波, 刘晓蓓, 李洁, 魏可可, 褚栋. 番茄褪绿病毒实时荧光定量PCR检测技术的建立[J]. 中国农业科学, 2018, 51(10): 2013-2022.

TianBo DING, XiaoBei LIU, Jie LI, KeKe WEI, Dong CHU. Development of a Real-Time Fluorescent Quantitative PCR Method for the Detection of Tomato chlorosis virus and Its Application[J]. Scientia Agricultura Sinica, 2018, 51(10): 2013-2022.

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样品号 Sample number	组1 Group 1		组2 Group 2		组3 Group 3
样品号 Sample number	Ct值 Ct value	ToCV含量 Concentration of ToCV (copies/μL)	Ct值 Ct value	ToCV含量 Concentration of ToCV (copies/μL)	Ct值 Ct value	ToCV含量 Concentration of ToCV (copies·μL^-1)
1	30.37	2238	30.35	2270	31.35	1134
2	28.55	7910	31.3	1174	32.16	646
3	30.90	1550	27.69	14363	29.65	3688
4	30.53	2003	29.84	3233	29.76	3418
5	29.85	3211	30.68	1805	28.61	7588
6	29.95	2996	28.68	7228	26.86	25542
7	27.59	15395	28.34	9151	30.4	2192
8	31.02	1426	31.12	1330	27.83	13034
Nc1	No Ct	—	No Ct	—	No Ct	—
Nc2	No Ct	—	No Ct	—	No Ct	—