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林业科学 ›› 2013, Vol. 49 ›› Issue (11): 98-102.doi: 10.11707/j.1001-7488.20131113

• 论文与研究报告 • 上一篇    下一篇

枣实蝇特异引物PCR鉴定技术

程晓甜1, 阿地力·沙塔尔1, 张伟3, 温俊宝2, 李新泉1, 陈梦4   

  1. 1. 新疆农业大学林学与园艺学院 新疆教育厅干旱区林业生态与产业技术重点实验室 乌鲁木齐 830052;
    2. 北京林业大学省部共建森林培育与保护教育部重点实验室 北京 100083;
    3. 新疆出入境检验检疫局技术中心 乌鲁木齐 830083;
    4. 新疆维吾尔自治区林业有害生物防治检疫局 乌鲁木齐 830000
  • 收稿日期:2013-06-01 修回日期:2013-09-20 出版日期:2013-11-25 发布日期:2013-11-26
  • 通讯作者: 阿地力·沙塔尔
  • 基金资助:

    新疆维吾尔自治区科技计划项目“特色林果重大病虫害持续高效绿色防控技术研究”(201130102-3);新疆维吾尔自治区重点学科森林培育资助项目。

Species-specific PCR Primers for Identification of Carpomyia vesuviana

Cheng Xiaotian1, Adili Shataer1, Zhang Wei3, Wen Junbao2, Li Xinquan1, Chen Meng4   

  1. 1. Key Lab.of Forestry Ecology and Industry Technology in Arid Region, Education Department of Xinjiang College of Forestry and Horticulture, Xinjiang Agricultural University Urumqi 830052;
    2. Key Laboratory for Silviculture and Forest Conservation of Ministry of Education, Beijing Forestry University Beijing 100083;
    3. Xinjiang Entry-Exit Inspection and Quarantine Bureau Urumqi 830083;
    4. Master Station of Prevention and Quarantine of Forestry Plant Diseases and Insect Pests in Xinjiang Urumqi 830000
  • Received:2013-06-01 Revised:2013-09-20 Online:2013-11-25 Published:2013-11-26

摘要:

在设计的枣实蝇15对引物中,利用种特异引物PCR (SS-PCR)和琼脂糖凝胶电泳技术筛选出1对枣实蝇的特异性引物,引物的特异性用桔小实蝇、瓜实蝇、南瓜实蝇、番石榴实蝇和桃果实蝇等5种实蝇来验证。SS-PCR方法的检测灵敏度用40,20,10,1,0.1,0.01,0.001 ng·μL-1等7个不同浓度系列的枣实蝇DNA模板来检测。结果表明,SS-PCR方法的检测限度达0.01 ng·μL-1以下,最适模板DNA浓度为1~20 ng·μL-1。所设计的引物对枣实蝇不同虫态均能进行特异性扩增,结果准确可靠。该技术体系可用于枣实蝇的鉴定识别与检测监测,对阻止其进一步扩散蔓延具有重要意义。

关键词: 种特异引物PCR, 枣实蝇, 琼脂糖凝胶电泳, 鉴定

Abstract:

In this study SS-PCR and agarose gel electrophoresis technique were used to select a pair of specific primers from the 15 pairs of primers of designed for Carpomyia vesuviana. Five fruit fly species, Bactrocera dorsalis, B.cucurbitae, B.tau, B. correcta and B.zonata, were used to determine the primer specificity. A series of genomic DNA dilution of C.vesuviana, 40, 20, 10, 1, 0.1, 0.01, and 0.001 ng·μL-1, were used to detect the sensitivity of SS-PCR. The results showed that the detection limit of SS-PCR was less than 0.01 ng·μL-1, and the optimum template DNA concentration was between 1 ng and 20 ng·μL-1. The designed primers could be used for amplifying the DNA of C.vesuviana at different stages. The developed SS-PCR method can be used for identification, inspection and monitoring of C.vesuviana, which would have an important significance in preventing the insect from spreading any further.

Key words: SS-PCR, Carpomyia vesuviana, agarose gel electrophoresis, identification

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