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中国生物防治学报 ›› 2017, Vol. 33 ›› Issue (6): 796-802.DOI: 10.16409/j.cnki.2095-039x.2017.06.012

• 研究论文 • 上一篇    下一篇

蝗虫微孢子TaqMan探针Real-time PCR检测新方法

扈鸿霞, 郑秋英, 叶小芳, 赵贝, 季荣   

  1. 新疆师范大学生命科学学院/新疆特殊环境物种多样性应用与调控实验室/中亚区域跨境有害生物联合控制国际研究中心/新疆师范大学 生命科学学院, 乌鲁木齐 830054
  • 收稿日期:2017-04-21 出版日期:2017-12-08 发布日期:2017-12-16
  • 通讯作者: 季荣,博士,教授,E-mail:jirong@xjnu.edu.cn
  • 作者简介:扈鸿霞,博士,副教授,E-mail:huhongxia111@126.com
  • 基金资助:
    新疆维吾尔自治区高校科研计划科学研究重点项目(20140705135643406);新疆维吾尔自治区教育厅重点实验室项目(XJKJT1322-016-01);新疆维吾尔自治区天山创新团队计划

Establishment of TaqMan Real-time PCR Method for Detection of Paranosema locustae

HU Hongxia, ZHENG Qiuying, YE Xiaofang, ZHAO Bei, JI Rong   

  1. Xinjiang Key Laboratory of Species Diversity Application and Regulation/International Research Center for Cross-Border Pest Management in Central Asia/College of Life Sciences, Xinjiang Normal University, Urumqi 830054, China
  • Received:2017-04-21 Online:2017-12-08 Published:2017-12-16

摘要: 蝗虫微孢子已被广泛运用于蝗虫防治。本研究采用蝗虫微孢子保守性高的小核糖体亚单位RNA为目的基因,将其克隆到pMD18-T载体上构建重组质粒,制备质粒标准品。设计一对特异性引物和TaqMan探针,并对引物浓度、探针浓度分别进行了优化,建立了蝗虫微孢子TaqMan实时荧光定量PCR检测方法。该检测方法的灵敏度达1.29×105 copies/L,各浓度质粒标准品的组内和组间重复的变异系数均小于1%。这里建立的蝗虫微孢子TaqMan探针荧光定量PCR检测方法较快速、简便和准确,适用于生产中蝗虫微孢子的快速检测。

关键词: 蝗虫微孢子, Real-time PCR, TaqMan探针

Abstract: Paranosema locustae, an entomopathogen of locusts, is widely used in biocontrol of locusts. The small subunit ribosomal RNA gene of P. locustae was used as the target molecule for designing specific primers and TaqMan probe. pMD18-T, the constructed recombinant plasmid was used to prepare standard agent for fluorescent quantitative PCR amplification. Through optimization of PCR reaction system, linear range of the detection method was determined, and sensitivity and repeatability of the method were evaluated. Finally, a fluorescent quantitative PCR detection method was established successfully. The detectable sensitivity of this method reached 1.29×105 copies/L. Three repeated detection trials showed that the variation coefficients within batches and between batches were both less than 1%. These results indicated that the established fluorescent quantitative PCR based on TaqMan probe technology is an accurate, sensitive and easy to operate method for P. locustae detection.

Key words: Paranosema locustae, fluorescent quantitative PCR, TaqMan probe

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