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中国农学通报

中国农学通报 ›› 2023, Vol. 39 ›› Issue (9): 128-133.doi: 10.11924/j.issn.1000-6850.casb2022-0351

• 植物保护·农药 • 上一篇    下一篇

斜纹夜蛾成虫带核型多角体病毒的巢式PCR检测

蒲小明1(), 邓海滨2, 路征3, 张景欣1, 孙大元1, 沈会芳1, 刘平平1, 林壁润1, 杨祁云1   

  1. 1 广东省农业科学院植物保护研究所/农业农村部华南果蔬绿色防控重点实验室/广东省植物保护新技术重点实验室，广州 510640
    2 广东省烟草科学研究所，广东韶关 512029
    3 中国烟草总公司广东省公司，广州 510610
  • 收稿日期:2022-04-07 修回日期:2022-08-15 出版日期:2023-03-25 发布日期:2023-03-23
  • 通讯作者: 杨祁云，女，1966出生，广东揭阳人，研究员，本科，学士，研究方向：农作物病虫害防控。通信地址：510640 广东省广州市天河区金颖路7号，Tel：020-85161048，E-mail：839034017 @qq.com。
  • 作者简介:

    蒲小明，男，1981年出生，湖南芷江人，研究员，博士，研究方向：农作物病虫害防控。通信地址：510640 广东省广州市天河区金颖路7号，Tel：18565358036，E-mail：。

  • 基金资助:
    广东省烟草科技项目“浈江流域上游主要作物病虫害绿色防控研究与应用”(201802); “性诱剂结合生物农药防控烟草夜蛾科害虫技术研究与应用”(201908); 广东省现代农业产业绿色发展共性关键技术研发创新团队建设项目“烟草病虫害绿色防控技术”(2021KJ112); 广东丝苗米跨县集群产业园（汕尾市）项目“水稻现代植保与营养关键技术研究与应用”(15702); 广东省博罗蔬菜产业园科技支撑项目“蔬菜种植关键植保技术推广应用”(15713)

Spodoptera litura Nucleopolyhedrovirus (SpltNPV) in Spodoptera litura Adults: Nested PCR Detection

PU Xiaoming1(), DENG Haibin2, LU Zheng3, ZHANG Jingxin1, SUN Dayuan1, SHEN Huifang1, LIU Pingping1, LIN Birun1, YANG Qiyun1   

  1. 1 Institute of Plant Protection, Guangdong Academy of Agricultural Sciences, Key Laboratory of Green Prevention and Control on Fruits and Vegetables in South China, Ministry of Agriculture and Rural Affairs, Guangdong Provincial Key Laboratory of High Technology for Plant Protection, Guangzhou 510640
    2 Tobacco Research Institute of Guangdong Province, Shaoguan, Guangdong 512029
    3 Guangdong Branch of China Tobacco Corporation, Guangzhou 510610
  • Received:2022-04-07 Revised:2022-08-15 Online:2023-03-25 Published:2023-03-23

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摘要：

旨在建立一种快速、可靠的斜纹夜蛾成虫带核型多角体病毒(Spodoptera litura Nucleopolyhedrovirus, SpltNPV)的分子检测方法，以克服目前常规PCR技术很难检测到斜纹夜蛾成虫带病毒的缺陷。以SpltNPV的蜕皮甾体尿苷二磷酸葡萄糖转移酶(ecdysteroid UDP- glucosyltransferase, egt)基因为检测对象，基于该基因序列设计了巢式PCR引物，其中一对外侧引物egt-F/R和一对内侧引物egt1-F/R，并研究斜纹夜蛾成虫带毒的巢式PCR检测可靠性。结果表明：基于egt基因的巢式PCR方法可以特异性扩增SpltNPV的DNA，引物egt-F/R的扩增检测最低限为10-7 μg/μL；且基于引物egt-F/R的两轮普通PCR无法检测出成虫带毒，而采用巢式PCR方法可有效扩增出目的egt片段，巢式PCR检测方法的灵敏度比两轮普通PCR检测方法提高2个数量级。因此，本研究成功建立了基于SpltNPV的egt基因的巢式PCR检测技术，该方法为斜纹夜蛾成虫带毒、传毒等流行病学研究提供了可靠的技术支撑。

关键词: 斜纹夜蛾核型多角体病毒, 成虫, egt, 巢式PCR, 检测

Abstract:

The aim of this study is to establish a rapid and reliable method for molecular detecting of SpltNPV in Spodoptera litura adults, in order to overcome the defect of the conventional PCR technology. In this paper, the ecdysteroid UDP-glucosyltransferase (egt) gene of Spodoptera litura was used as the detection object. Nested PCR primers were designed based on the egt gene sequences, including a pair of outer primers egt-F/R and a pair of inner primers egt1-F/R. And the reliability of nested PCR detection was studied on Spodoptera litura adults. The results showed that the nested PCR method had strong specificity for SpltNPV DNA amplification, and the minimum limit for amplification detection of primer egt-F/R was 10-7 μg/μL. The results also indicated that two rounds of conventional PCR amplification could not detect the SpltNPV by the primers of egt-F/R, and the nested PCR method could effectively amplify the target fragment of gene egt. In addition, the sensitivity of the nested PCR method was 2 orders of magnitude higher than that of the two rounds of the conventional PCR detection method. Therefore, the nested PCR method for detecting SpltNPV was established for Spodoptera litura adults in this study. Moreover, the method can provide technical support for the epidemiological study on the infection and transmission of the virus in Spodoptera litura adults.

Key words: Spodoptera litura Nucleopolyhedrovirus (SpltNPV), adult, egt, nested PCR, detection