松材线虫和拟松材线虫双重RPA检测研究  

Duplex-RPA Detection for Bursaphelenchus xylophilus and Bursaphelenchus mucronatus

作　　者：方圆 吴迅 林宇 王海燕 吴慧平[1] 鞠玉亮 FANG Yuan;WU Xun;LIN Yu;WANG Hai-yan;WU Hui-ping;JU Yu-liang(Key Laboratory of Biology and Sustainable Management of Plant Disease and Pests of Anhui Higher Education Institutes,School of Plant Protection,Anhui Agricultural University,Hefei 230036;Tianjin Customs District of the People’s Republic of China,Tianjin 300461)

机构地区：[1]安徽农业大学植物保护学院/植物病虫害生物学与绿色防控安徽普通高校重点实验室,合肥230036 [2]天津海关动植物与食品检测中心,天津300461

出　　处：《生物技术通报》2021年第7期183-190,共8页Biotechnology Bulletin

基　　金：国家自然科学基金项目(31801714);安徽省自然科学基金项目(1808085QC80);安徽高校自然科学研究重点项目(KJ2018A0147)。

摘　　要：旨在建立一种以重组酶聚合酶扩增技术(RPA)为基础的快速检测方法,用于松材线虫和拟松材线虫的同步检测鉴定。根据松材线虫和拟松材线虫ITS区差异序列,分别设计松材线虫和拟松材线虫特异性上游引物Bx-rpa-F、Bm-rpa-F和两者通用下游引物Bxm-rpa-R。优化3条引物在反应体系中的最佳配比,建立双重RPA检测体系,并对其特异性和灵敏度进行分析。研究结果显示,双重RPA在37℃恒温条件反应30 min,即可完成对松材线虫和拟松材线虫核酸的同步扩增。Bx-rpa-F/Bm-rpa-F/Bxm-rpa-R对松材线虫的扩增片段为346 bp,对拟松材线虫的扩增产物为189 bp,且三者配比为5∶3∶8时,双重RPA扩增效果最好。双重RPA对松材线虫和拟松材线虫具有较高的检测特异性,对松材线虫的检测极限为10 pg/μL,相当于1/100条线虫,对拟松材线虫的检测极限为100 pg/μL,相当于1/10条线虫,其灵敏度低于常规PCR技术,但其满足对单条线虫检测的需求。双重RPA从松木样品中同步检测到松材线虫和拟松材线虫,操作简便、检测效率高、特异性强、灵敏度高、对仪器设备要求低,为松材线虫的检疫鉴定提供新方法。The aim of this work is to develop a rapid recombinase polymerase amplification(RPA)method for simultaneously detecting Bursaphelenchus xylophilus and Bursaphelenchus mucronatus.The forward RPA primer Bx-rpa-F,Bm-rpa-F and the common reverse RPA primer Bxm-rpa-R were designed according to the sequence variation of ITS from B.xylophilus and B.mucronatus.The concentration of the three RPA primers in duplex-RPA was optimized,duplex-RPA detection system was established,and its specificity and sensitivity were analyzed.The results showed that duplex-RPA simultaneously amplified DNA extracted from B.xylophilus and B.mucronatus at 37℃within 30 min.The RPA product of B.xylophilus amplified with Bx-rpa-F/Bxm-rpa-R was 346 bp,and that of B.mucronatus with Bm-rpa-F/Bxm-rpa-R was 189 bp.In addition,duplex-RPA presented the best amplification efficiency when the concentration of Bx-rpa-F/Bm-rpa-F/Bxm-rpa-R was 5∶3∶8.Duplex RPA had high specificity and sensitivity for the detection of B.xylophilus and B.mucronatus.The detection limit of duplex-RPA for B.xylophilus was 10 pg/μL,which was equivalent to 1/100 single nematode;while the detection limit of duplex-RPA for B.mucronatus was 100 pg/μL,which was equivalent to 1/10 single nematode.The sensitivity was lower than that by conventional method,however,it met the requirement for detecting an individual nematode.In conclusion,duplex-RPA may simultaneously detect B.xylophilus and B.mucronatus from infected pine wood samples.Moreover,duplex-RPA has the advantages of simple operation,high detection efficiency,strong specificity,high sensitivity and low requirements for instruments and equipment,which provides a new method for quarantine and identification of B.xylophilus.

关 键 词：松材线虫 重组酶聚合酶技术 特异性 灵敏度 实用性 

分 类 号：S763.18[农业科学—森林保护学]