木毒蛾核型多角体病毒的PCR快速检测技术  

Establishment of PCR detection for Lymantria xylina multiple nucleopolyhedrovirus

作　　者：刘用垄 孙悦 陈佳宁 曲良建[2] 张飞萍[1] 王荣[1] LIU Yonglong;SUN Yue;CHEN Jianing;QU Liangjian;ZHANG Feiping;WANG Rong(College of Forestry,Fujian Agriculture and Forestry University,Fuzhou,Fujian 350002,China;Research Institute of Forest Ecology,Environment and Protection,Chinese Academy of Forestry,Beijing 100091,China)

机构地区：[1]福建农林大学林学院,福建福州350002 [2]中国林业科学研究院森林生态环境与保护研究所,北京100091

出　　处：《生物安全学报》2023年第1期80-84,共5页Journal of biosafety

基　　金：国家自然科学基金青年科学基金项目(31600522);福建省自然科学基金(2017J0106)。

摘　　要：【目的】木毒蛾是福建沿海地区防护林树种木麻黄的主要害虫之一,具有成为国际危险性有害生物的潜在可能性。木毒蛾核型多角体病毒(Lymantria xylina multiple nucleopolyhedrovirus,LyxyMNPV)是高效控制木毒蛾的优良天敌资源,具有高度安全性。建立LyxyMNPV的PCR快速检测技术,有利于该虫防治技术及木毒蛾核型多角体病毒的进一步研究。【方法】根据LyxyMNPV基因组中的特有基因gp131设计特异性引物,利用PCR法扩增该目的基因片段并测序检验,建立LyxyMNPV的分子快速检测技术体系。【结果】以GAL为引物的PCR检测技术对LyxyMNPV基因组的灵敏度可达1 fg·mL^(-1),对多角体悬液浓度检测最低量可达5.0 PIB·mL^(-1),可明确区分LyxyMNPV和其他7种昆虫核型多角体病毒。该体系同时适用于感病木毒蛾的不同虫态(卵、幼虫、蛹和成虫)的检测,以及环境样本(包括土壤、树枝和寄生蜂)的检测。【结论】成功建立了具有高度特异性、灵敏性的LyxyMNPV分子快速检测技术,为LyxyMNPV的快速检测提供分子生物学证据。【Aim】The casuarina moth,Lymantria xylina,is one of the main pests of Casuarina equisetifolia,in the coastal areas of Fujian Province.It has the potential to become an international quarantine pest.L.xylina multiple nucleopolyhedrovirus(LyxyMNPV)is an excellent resource for the control of L.xylina as it is a natural enemy of this pest and is very safe for use in the environment.The establishment of PCR detection for LyxyMNPV is conducive to the study and control of L.xylina.【Method】A pair of specific primers was designed according to the previously reported unique gene gp131 of LyxyMNPV.The target gene fragment was amplified using PCR and sequenced to establish a rapid PCR detection system.【Result】The detection limit of our method was 1 fg·mL^(-1)for LyxyMNPV DNA or 5.0 PIB·mL^(-1)for polyhedrons.It had high specificity in distinguishing LyxyMNPV from the other seven insect nucleopolyhedroviruses,and was detectable in different developmental stages of infected L.xylina,including eggs,larvae,pupae,and adults.This system has also been successfully used for virus detection in the soil,host plant branches,and parasitic wasps of L.xylina.【Conclusion】A highly specific and sensitive molecular rapid PCR detection method was successfully established,which provided convincing and microscopic molecular evidence for the rapid detection of LyxyMNPV.

关 键 词：聚合酶链式反应 木毒蛾核型多角体病毒 灵敏检测 生物防治 

分 类 号：S763[农业科学—森林保护学]