| 摘要: |
| 为快速鉴定草莓病毒病种类以预防病毒病扩散，根据草莓镶脉病毒(strawberry vein banding virus，SVBV)、草莓斑驳病毒(strawberry mottle virus，SMoV)、草莓轻型黄边病毒(strawberry mild yellow edge virus，SMYEV)、草莓白化病毒(strawberry pallidosis-associated virus，SPaV)、草莓病毒1(strawberry virus 1，StrV-1)和芸薹黄化病毒(brassica yellows virus，BrYV)的基因保守区域设计特异性引物，建立了能够同时检测这6种病毒的多重PCR方法。该多重PCR反应体系中，2×SanTaq PCR Master Mix用量为10 μL，上下游引物(10 μmol/L)用量为4 μL，其中SVBV为0.25 μL、SMoV为0.50 μL、SMYEV为0.10 μL、SPaV为0.15 μL、StrV-1为0.50 μL和BrYV为0.50 μL，模板1 μL，ddH2O为5 μL，总体积20 μL。多重PCR反应程序设定为:94 ℃预变性5 min，94 ℃变性30 s，53 ℃退火30 s，72 ℃延伸120 s，最后72 ℃延伸10 min，循环数为35。该检测体系的灵敏度可以达到107 copies/μL。该多重PCR方法可以同时实现6种草莓病毒的高效快速检测，为草莓病毒病的早期发现和针对性防治提供了技术支持。 |
| 关键词: 草莓 多重PCR 草莓镶脉病毒 草莓斑驳病毒 草莓轻型黄边病毒 草莓白化病毒 草莓病毒1 芸薹黄化病毒 |
| DOI：10.11841/j.issn.1007-4333.2023.11.10 |
| 投稿时间：2023-03-02 |
| 基金项目:国家重点研发计划项目(2019YFD1001800)；甘肃省科学技术厅科技特派团专项(22CX8NN231) |
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| A multiplex PCR assay for simultaneous detection and identification of six viruses in strawberry |
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LI Su1,XU Tengfei1,HOU Shengfan1,DONG Zhenfei1,ZHAO Xiaoli1,LI Nan2,WANG Hongqing1*
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| (1.College of Horticulture, China Agricultural University, Beijing 100193, China;2.Wander agricultural Science and Technology Development Co., Ltd., Beijing 102211, China) |
| Abstract: |
| In order to rapidly identify strawberry virus disease species and prevent the spread of virus disease, a multiplex reverse transcription polymerase chain reaction system was established for the simultaneous detection and identification of strawberry vein banding virus(SVBV), strawberry mottle virus(SMoV), strawberry mild yellow edge virus(SMYEV), strawberry pallidosis-associated virus(SPaV), strawberry virus 1(StrV-1)and brassica yellows virus(BrYV). Six pairs of specific primers designed on the conserved genomic regions of these viruses. In this multiplex PCR reaction system, the dosage of 2×SanTaq PCR Master Mix was 10 μL, the dosage of upstream and downstream primer(10 μmol/L)was 4 μL, among which SVBV was 0. 25 μL, SMoV was 0. 50 μL, SMYEV was 0. 10 μL, SPaV was 0. 15 μL, StrV-1 was 0. 50 μL, BrYV was 0. 50 μL, template volume was 1 μL, ddH2O was 5 μL, and the total volume was 20 μL. The multiple PCR procedure was pre-denaturation at 94 ℃ for 5 min, denaturation at 94 ℃ for 30 s, annealing at 53 ℃ for 30 s, extension at 72 ℃ for 2 min, and final extension at 72 ℃ for 10 min, with a cycle number of 35, and the sensitivity of this system achieved 107 copies/μL. In conclusion, the proposed multiplex PCR can realize the efficient detection of six strawberry viruses at the same time, which provides technical support for the early prevention and treatment of strawberry virus diseases. |
| Key words: strawberry multiplex PCR SVBV SMoV SMYEV SPaV StrV-1 BrYV |
