CN110016515B - 一种运用pcr引物检测猕猴桃果腐病菌的方法 - Google Patents
一种运用pcr引物检测猕猴桃果腐病菌的方法 Download PDFInfo
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Abstract
一种运用PCR引物检测猕猴桃果腐病菌的方法本发明涉及使用PCR技术检测植物及植物产品中猕猴桃果腐病菌(Neofabraea actinidiae)的方法，属于植物病原真菌检测领域。本发明设计合成了猕猴桃果腐病菌的特异性引物，用于对猕猴桃果腐病菌基因组DNA的特异检测。本发明建立了一种快速简便、特异性强、准确可靠的猕猴桃果腐病菌的分子检测方法，一个工作日内完成整个检测。
Description
技术领域
本发明涉及使用PCR技术检测植物及植物产品中猕猴桃果腐病菌Neofabraea actinidiae(P.R.Johnst., M.A.Manning & X.Meier)的方法，属于植物病原真菌检测领域。
背景技术
猕猴桃果腐病菌Neofabraea actinidiae(P.R.Johnst., M.A.Manning &X.Meier)，隶属于子囊菌门（Ascomycota），盘菌亚门（Pezizomycotina），锤舌菌纲（Leotiomycetes），柔膜菌目（Helotiales），皮盘菌科（Dermataceae），明孢盘菌属（Neofabraea）。无性世代为Cryptosporiopsis actinidiae，它与引起苹果牛眼果腐病的N. malicorticis, N. perennans, N. alba, N. kienholzii互为近似种，其中N. malicorticis以其异名Pezicula malicorticis（苹果树炭疽病菌）被列入我国进境植物检疫性有害生物名录。
关于猕猴桃果腐病菌Neofabraea actinidiae的文献资料较少，现有资料显示该病菌在新西兰有分布，危害猕猴桃多个重要品种，导致果实腐烂，造成严重的经济损失。尤其是在黄金猕猴桃上发病严重。R. A. Fullerton等（2007）研究认为病菌主要在花期侵染，潜伏在花和果实上，采摘后的果实经过12-18周的长期低温贮存后表现症状。除猕猴桃外，该病菌在苹果、柿子树、红桉树以及天然林上也有侵染报道。
近年来，猕猴桃作为高端水果，国内市场需求旺盛，进口量大幅增长，猕猴桃原产于中国，作为猕猴桃主要产区，我国陕西、四川、河南、湖南、湖北、贵州、浙江、江西、安徽、福建、广西等省份均有种植，栽培面积居世界首位，品种资源丰富。目前，新西兰猕猴桃占据进口市场，货物经冷链运输，装卸货时往往不会表现症状。一旦病菌随进境果实传入国内，将会对我国水果产业及农业生态造成难以估量的损失。
鉴于猕猴桃果腐病菌传入我国的风险性及病菌危害的严重性，为保护我国农林业生产安全，维护国家利益，进一步规范这一重要水果病害的检疫，提高口岸疫情检测的准确性和验放速度，防止该病菌传入我国，建立一套快速、准确、完整的猕猴桃果腐病菌的检测方法迫在眉睫。
发明内容
本发明收集明孢盘属8种真菌（见表1），建立了快速简便、特异性强、准确可靠的明孢盘属真菌分子检测方法，能将猕猴桃果腐病菌准确鉴定出来。该方法检测快速，方法可靠，整个过程在一个工作日内完成，可有效在口岸检测中推广应用。
具体技术方案如下：
本发明目的是提供一种检测猕猴桃果腐病菌基因组DNA的PCR方法，它包括如下步骤：
（1）植物基因组DNA的提取；
（2）猕猴桃果腐病菌的特异引物，序列如下：
NACF1：GGA ATA TGG AAG TTC TTG AGG AAT AC
NACR1：GGA TCT CAC AAT GCG TCC AT
该引物用于猕猴桃果腐病菌基因组DNA特异性检测，扩增片段约为582bp。
与现有技术相比，本发明的有益效果是：本发明建立了快速简便、特异性强、准确可靠的猕猴桃果腐病菌分子检测方法，能将猕猴桃果腐病菌与明孢盘属其他种区别开来。该方法检测快速，方法可靠，整个过程在一个工作日内完成，可有效在口岸检测中推广应用。
下面结合附图与具体实施方式对本发明作进一步详细描述。
附图说明
图1猕猴桃果腐病菌基因组DNA特异引物的扩增
具体实施方式
实施例1：植物材料基因组DNA的提取
本实验猕猴桃果腐病菌菌株源于天津出入境检验检疫局动植食中心植检实验室。共计9株，相关信息见表1。
表1 供试材料代码、拉丁名、中文名及收集年份
用70 %酒精对病果表面消毒，挑取病斑处霉层及病健交界处果肉组织，置PDA培养基上20 ℃ 12 h光照黑暗交替培养，5d后观察培养结果。可疑菌落用PDA进行分离纯化，取适量菌丝块，液氮充分研磨，用德国QIAGEN公司Dneasy plant mini kit植物基因提取试剂盒提取DNA，并将DNA溶于100 μL 1× TE缓冲液中，置于-20℃保存。
实施例2：特异引物的设计
人工合成猕猴桃果腐病菌的特异引物，引物序列如下：
NACF1：GGA ATA TGG AAG TTC TTG AGG AAT AC
NACR1：GGA TCT CAC AAT GCG TCC AT
实施例3：猕猴桃果腐病菌特异引物的PCR扩增
1. 反应混合液的配制
反应体系配置见表2。
表2 猕猴桃果腐病菌特异性扩增体系
2. PCR反应程序
预变性：94ºC，3min
变性：94ºC，30s
退火：60ºC，6s
延伸：72ºC，30s
循环数：35个
延伸：72ºC，7min。
3. 结果分析
对9个实验材料的基因组DNA进行特异性引物NACF1 /NACR1的扩增，以添加无菌水为模板作为阴性对照。扩增产物经1.5 %琼脂糖凝胶电泳，EB染色均能观察到特定目的大小带，引物扩增的片段应约为582 bp，只有猕猴桃果腐病菌显示特定大小的目的带（见图中1、2泳道），N. perennans, N. alba, N. kienholzii, N. malicorticis, N. krawtzewii , N. illicii, N. citricarpa 和阴性对照无相应产物（见图中3～10泳道，图中1～10泳道分别为N. actinidiae, N. actinidiae, N. perennans, N. alba, N. kienholzii, N. malicorticis, N.krawtzewii , N. illicii, N. citricarpa和阴性对照），M泳道为2000bp DNA marker。
序列表
<110> 天津出入境检验检疫局动植物与食品检测中心
<120> 一种运用PCR引物检测猕猴桃果腐病菌的方法
<160> 2
<170> SIPOSequenceListing 1.0
<210> 1
<211> 26
<212> DNA
<213> 猕猴桃果腐病菌(Neofabraea actinidiae)
<400> 1
ggaatatgga agttcttgag gaatac 26
<210> 2
<211> 20
<212> DNA
<213> 猕猴桃果腐病菌(Neofabraea actinidiae)
<400> 2
ggatctcaca atgcgtccat 20
Claims (3)
1.一种运用PCR引物检测猕猴桃果腐病菌的方法，其特征在于，猕猴桃果腐病菌PCR特异性引物对，序列如下：
NACF1：GGA ATA TGG AAG TTC TTG AGG AAT AC
NACR1：GGA TCT CAC AAT GCG TCC AT
扩增片段为582bp，用于猕猴桃果腐病菌基因组DNA特异性扩增检测。
2.根据权利要求1所述的一种运用PCR引物检测猕猴桃果腐病菌的方法，其特征在于，所述方法包括如下步骤：
（1）植物基因组DNA的提取；
（2）建立猕猴桃果腐病菌基因组DNA的PCR扩增体系，按照反应程序进行扩增；
（3）扩增产物的琼脂糖凝胶电泳检测。
3.权利要求1或2所述的一种运用PCR引物检测猕猴桃果腐病菌的方法在特异检测猕猴桃果腐病菌上的应用。
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| 进境新西兰猕猴桃中猕猴桃果腐病菌的检测与鉴定;张露茜等;《植物检疫》;20170715(第04期);第46-49页 * |
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