| 辣椒脉斑驳病毒的多基因联合检测与鉴定 |
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| 引用本文： | 杨宏凯,杨晶文,沈建国,蔡伟,高芳銮.辣椒脉斑驳病毒的多基因联合检测与鉴定[J].中国农业科学,2020,53(16):3412-3420. |
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| 作者姓名： | 杨宏凯 杨晶文 沈建国 蔡伟 高芳銮 |
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| 作者单位： | 1福建农林大学植物病毒研究所福建省植物病毒学重点实验室,福州 3500022福州海关技术中心,福州 3500013榕城海关综合技术服务中心,福建福清350300 |
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| 基金项目： | 国家重点研发计划(2016YFF02032003);福建省自然科学基金(2019J01653);福清市科技计划(FQ201802) |
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| 摘 要： | 【目的】辣椒脉斑驳病毒（Chilli veinal mottle virus,ChiVMV）是辣椒生产上危害严重的病毒,也是口岸检疫的重要病毒之一。本文旨在建立一种快速检测ChiVMV的多基因联合检测方法,实现ChiVMV的快速、准确鉴定。【方法】利用DAS-ELISA、RT-PCR方法对印度进境辣椒进行检测以确定是否携带有ChiVMV。根据ChiVMV外壳蛋白（coat protein,CP）和圆柱状内含体蛋白（cytoplasmic inclusion protein,CI）保守区域分别设计引物,筛选两个基因（CP和CI）的特异性引物组合,通过设定不同的引物用量组合以及对退火温度等条件进行优化,探索最佳的反应条件,建立多基因联合检测方法。利用建立的多基因联合检测体系对包含ChiVMV在内的辣椒病毒进行检测,以验证该体系特异性。同时对ChiVMV阳性样品的不同浓度cDNA进行扩增,测定其检测灵敏度。此外,对田间辣椒样本进行检测,以验证体系的实际应用效果。【结果】通过对印度进境辣椒的DAS-ELISA检测,发现样品提取液与ChiVMV呈阳性反应;利用CP861-F/CP861-R特异性引物对进行RT-PCR,扩增出与预期大小相一致的特异性片段,确认该批辣椒样品携带有ChiVMV。多基因联合检测体系中,应用引物对CP337-F/CP337-R、CI655-F/CI655-R分别扩增出长度为337、655 bp的特异性目的片段。经优化后的反应体系和程序为cDNA 2 μL、CP337-F/CP337-R各0.625 μL（10 μmol·L-1）、CI655-F/CI655-R各1.375 μL（10 μmol·L-1）、2×PCR Master Mix 12.5 μL、ddH2O 6.5 μL,退火温度50℃,共35个循环。建立的多基因联合检测方法具有良好的特异性和灵敏度,两个基因检测灵敏度均为10-4数量级。实际应用检测结果表明,该方法可从感染ChiVMV的样品中同时扩增出CP和CI的特异性目的片段。【结论】建立的多基因联合检测方法具有特异性强、灵敏度高、重复性好的特点,可为辣椒脉斑驳病毒的快速检测提供可靠的技术支持。
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| 关 键 词： | 辣椒脉斑驳病毒 外壳蛋白基因 圆柱状内含体蛋白基因 多基因联合检测 |
| 收稿时间： | 2019-10-27 |
Multi-Gene-Based PCR Detection and Identification of Chilli veinal mottle virus |
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| YANG HongKai,YANG JingWen,SHEN JianGuo,CAI Wei,GAO FangLuan.Multi-Gene-Based PCR Detection and Identification of Chilli veinal mottle virus[J].Scientia Agricultura Sinica,2020,53(16):3412-3420. |
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| Authors: | YANG HongKai YANG JingWen SHEN JianGuo CAI Wei GAO FangLuan |
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| Institution: | 1Fujian Key Laboratory of Plant Virology, Institute of Plant Virology, Fujian Agriculture and Forestry University, Fuzhou 3500022Technology Center of Fuzhou Customs District, Fuzhou 3500013Comprehensive Technical Service Center of Rongcheng Customs District, Fuqing 350300, Fujian |
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| Abstract: | 【Objective】Chilli veinal mottle virus (ChiVMV), one of the most destructive pathogens causing server losses to chilli production, is an important plant virus in port quarantine. The objective of this study is to establish a fast and accurate multi-gene-based PCR detection method for ChiVMV.【Method】DAS-ELISA and RT-PCR were used to detect the infected chilli samples imported from India. Two pairs of specific primers were designed from the conserved regions of ChiVMV coat protein (CP) and cytoplasmic inclusion protein (CI), respectively. The multi-gene-based PCR detection method was established after optimizing parameters including primer dosage and annealing temperature. The established multi-gene-based PCR detection system was also used to detect chilli viruses including ChiVMV to verify the specificity of this system, and the different concentrations of cDNA in ChiVMV positive samples were amplified to determine its sensitivity. In addition, the utility of the system was tested by detecting ChiVMV in plant samples infected by this virus.【Result】All samples reacted positive on the DAS-ELISA test. RT-PCR amplifications of the ELISA-positive subsamples all generated expected fragments of 861 bp in size, using the specific primer pair of CP861-F/CP861-R. These results indicated that all samples were infected by ChiVMV. The specific target fragments of 337 and 655 bp were respectively amplified using the primer pairs of CP337-F/CP337-R and CI655-F/CI655-R in the multi-gene-based PCR detection system, whose optimized reaction system is cDNA 2 μL, CP337-F/CP337-R 0.625 μL (10 μmol·L-1), CI655-F/CI655-R 1.375 μL (10 μmol·L-1), 2×PCR Master Mix 12.5 μL, ddH2O 6.5 μL, annealing temperature 50℃, and for 35 cycles. The established multi-gene-based PCR detection system had good specificity and sensitivity, and two targeted fragments could be detected after the total DNA was diluted to 10-4. Other ChiVMV-infected samples were successfully detected by this method, generating two expected fragments of CP and CI, respectively.【Conclusion】The established multi-gene-based PCR detection method has strong specificity, high sensitivity, excellent repeatability, which is useful in the detection and identification of ChiVMV in port quarantine. |
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| Keywords: | Chilli veinal mottle virus (ChiVMV) coat protein gene (CP) cytoplasmic inclusion protein gene (CI) multi-gene-based PCR detection |
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