CN108588249B - 一种用于检测甘薯茎腐病菌的引物对及其检测方法 - Google Patents
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Abstract
本发明公开了一种用于检测甘薯茎腐病菌的引物对及其检测方法,属于生物技术领域。所述的引物对包括上游引物和下游引物,上游引物的核苷酸序列如SEQ ID No.2所示,下游引物的核苷酸序列如SEQ ID No.3所示。本发明通过生物信息学手段获取达旦提狄克氏菌的特异性基因片段,并设计用于PCR扩增该基因片段的特异性引物对,利用该引物对实现对达旦提狄克氏菌的准确鉴定,对甘薯茎腐病菌DNA的最低检测浓度达到0.5ng/μL。利用本发明提供的检测方法可以快速准确鉴定甘薯样品中的甘薯茎腐病菌,无需病原菌培养、性状分析等复杂操作。
Description
技术领域
本发明涉及生物技术领域,具体涉及用于检测甘薯茎腐病菌的引物对及其检测方法。
背景技术
甘薯是世界第六大主粮,中国是世界上最大的甘薯生产国,2006~2010年甘薯种植面积稳定保持在3.70×106hm2左右,占世界甘薯种植面积的45.1%,年平均产量约7.8×107t,占世界甘薯产量的75%左右,总产量在国内仅次于水稻、小麦和玉米,在中国国民经济中占有重要地位。
甘薯细菌性茎腐病于1974年首次在美国发现,后来在日本以及南美洲的委内瑞拉国也有报道。近年来,福建、广东、江西、广西、重庆、河北、河南和浙江等地甘薯相继发生细菌性茎腐病,造成严重损失,如2015年浙江省台州市黄岩区的甘薯种植田中甘薯茎腐病病株率在10%~20%左右,严重的田块高达90%以上,甚至绝收。甘薯茎腐病正严重威胁着我国甘薯产业的健康发展。
甘薯茎腐病典型症状为茎和叶柄上产生褐至黑色水渍状病斑,直至茎基部发黑、变软腐烂,叶发黄,茎和块根维管束呈黑褐色,块根会腐烂、有臭味,田间病薯表面有黑色边的棕色凹陷病斑。病薯、病蔓、田间灌溉水及受污染的器材可作为初侵染源进行传播,潮湿温暖的条件易引发病害。
甘薯茎腐病由达旦提狄克氏菌(Dickeya dadantii)引起。D.dadantii革兰氏染色为阴性,无芽孢和荚膜,能运动,在营养琼脂平板上菌落表面稍凸,不透明,边缘整齐,淡土黄色,表面稍皱缩,无光泽。除甘薯外,D.dadantii还能引起多种作物和观赏植物腐烂,是一种毁灭性的细菌性病害。
目前国内外对甘薯茎腐病菌检测的研究较少。少数研究根据传统的病害症状、病原菌的形态特征、培养性状、生理生化分析和16S rDNA序列分析等方法检测病菌,存在工作量大、鉴定周期长且只能在病害显症后进行鉴定的问题。鉴于甘薯茎腐病严重的危害性和迫切的检疫需求,急需研发特异性强、灵敏度高、成本低的快速检测D.dadantii的方法。
达旦提狄克氏菌(Dickeya dadantii)原属于菊欧文氏菌(Erwiniachrysanthemi)。E.chrysanthemi在被分类到果胶杆菌属(Pectobacterium)改称菊果胶杆菌(P.chrysanthemi)后,又于2005年被分类到Dickeya属,被分成5个种(D.chrysanthemi,D.dadantii,D.dianthicola,D.dieffenbachia和D.zeae),再后D.dieffenbachia被分类为D.dadantii的一个亚种(D.dadantii subsp.dieffenbachia)。现在的Dickeya属包括8个种(D.aquatica,D.chrysanthemi,D.dadantii,D.dianthicola,D.fangzhongdai,D.paradisiaca,D.solani和D.zeae)。用传统的形态学和生理生化分析的方法难以区分Dickeya属的不同种,以前针对E.chrysanthemi的分子检测手段不适用于特异检测D.dadantii。且由于Dickeya属的种间差异较小,针对达旦提狄克氏菌(Dickeya dadantii)很难设计种特异性引物,目前国内外文献未见有相关报道,因此,很难应用PCR的方法对甘薯茎腐病菌进行检测。
发明内容
本发明的目的在于提供一种能够特异性准确鉴定达旦提狄克氏菌(Dickeyadadantii)的方法,以实现对甘薯茎腐病的准确快速检测。
为实现上述目的,本发明采用如下技术方案:
根据NCBI基因组数据库中达旦提狄克氏菌(Dickeya dadantii)及Dickeya属内其他菌株的基因组序列,利用泛基因组分析找出其特异性基因片段,其核苷酸序列如SEQ IDNo.1所示,长度为237bp,并对所找出的基因进行Blast验证其特异性。该基因序列为甘薯茎腐病菌特有。
针对上述特异性基因片段设计引物,本发明提供了一种用于检测甘薯茎腐病菌的引物对,包括上游引物和下游引物,其核苷酸序列分别为:
上游引物(SEQ ID No.2):5'-CATATCAACCAGACCAGCCGTT-3';
下游引物(SEQ ID No.3):5'-CGGCCTGCTTTAAACAACGTATTA-3'。
利用上述引物扩增的目标片段长度为167bp。
本发明还提供了一种包含所述的引物对的检测试剂盒。该试剂盒除了包括上述引物对,还包括dNTP、Taq聚合酶、PCR buffer、阳性对照DNA、阴性对照DNA。
本发明提供的引物对能够特异性地扩增出达旦提狄克氏菌(Dickeya dadantii)的特异性基因片段,因此,可以应用于检测鉴定达旦提狄克氏菌。对于农业生产中田间病害的早期诊断及甘薯茎腐病菌的监控、防治具有重要的实用价值。
本发明还提供了一种检测甘薯茎腐病菌的方法,包括以下步骤:
(1)提取待检样品DNA;
(2)采用所述的引物对,建立PCR反应体系,进行PCR扩增;
(3)对PCR产物进行检测和判断,
如扩增产物中出现167bp的条带,则说明待检样品中具有甘薯茎腐病菌Dickeyadadantii;否则,说明待检样品中不具有甘薯茎腐病菌。
所述的待检样品为甘薯块茎、甘薯苗、甘薯茎。
作为优选,所述PCR反应体系以25μL体系计,包括:2×Taq PCRMasterMix 12.5μL、10μmol/L上下游引物各1μL、DNA模板1μL,灭菌双蒸水9.5μL。
作为优选,所述PCR扩增条件:94℃预变性3min;94℃变性30s,56℃退火30s,72℃延伸10s,35个循环;72℃延伸5min,4℃终止反应。
本发明具备的有益效果:
本发明通过生物信息学手段获取达旦提狄克氏菌的特异性基因片段,并设计用于PCR扩增该基因片段的特异性引物对,利用该引物对实现对达旦提狄克氏菌的准确鉴定,对甘薯茎腐病菌DNA的最低检测浓度达到0.5ng/μL。
利用本发明提供的检测方法可以快速准确鉴定甘薯样品中的甘薯茎腐病菌,无需病原菌培养、性状分析等复杂操作。
附图说明
图1为以菌体DNA为模板进行PCR检测引物特异性的电泳结果图;其中,M为DNA分子量标准,1泳道为Dickeya dadantii CZ1501,2泳道为D.dadantii 898,3泳道为D.dadantii3937,4泳道为D.fangzhongdai,5泳道为D.dieffenbachiae,6泳道为D.chrysanthemi,7泳道为D.solani,8泳道为D.aquatica,9泳道为D.dianthoicola,10泳道为D.zeae,11泳道为Brenneria rubrifaciens,12泳道为Pectobacterium cacticida,13泳道为P.cypripedii,14泳道为Erwinia rhapontici,15泳道为E.tasmaniensis,16泳道为Pantoea anthophila,17泳道为P.eucalypti,18泳道为Acidovorax avenae subsp.citrulli,19泳道为A.avenaesubsp.avenae,20泳道为水代替DNA的阴性对照。
图2为以梯度稀释的甘薯茎腐病菌DNA为模板进行PCR检测的电泳结果图;M为DNA分子量标准,1-8泳道DNA浓度依次为500ng/μL、50ng/μL、5ng/μL、0.5ng/μL、0.05ng/μL、0.005ng/μL、0.0005ng/μL、0.00005ng/μL的甘薯茎腐病菌DNA为模板。
图3为对模拟样品进行PCR检测的电泳结果图;其中,M为DNA分子量标准,1泳道为健康甘薯苗样品,2泳道为模拟甘薯茎腐病样品。
具体实施方式
下面结合附图和实施例对本发明进行详细说明,但本发明所保护范围不限于此。
实施例中涉及的实验仪器及试剂:
细菌基因组DNA提取试剂盒(TIANGEN公司,北京,中国);2×TSINGKE Master Mix(杭州擎科生物科技有限公司);DL2000DNA Marker(杭州擎科生物科技有限公司);凝胶电泳仪(bio-rad);PCR仪(BIOER);BIOWEST凝胶琼脂,购自杭州擎科生物科技有限公司;凝胶成像系统(bio-rad)。
实施例1
特异性基因序列筛选及引物设计
1)特异性基因筛选
根据NCBI基因组数据库中甘薯茎腐病菌基因组序列,利用泛基因组分析找出其特异性基因,并对所找出的基因进行本地Blast验证其特异性。特异性基因序列如SEQ IDNo.1所示。
2)引物设计
针对特异性基因序列通过在线设计特异性寡核苷酸引物工具Primer-BLAST设计引物,上游引物如SEQ ID No.2所示,下游引物如SEQ ID No.3所示,具体生产引物的公司为杭州擎科生物技术有限公司。
实施例2
分子鉴定方法的建立
1)待测样品的DNA提取
挑取待检测菌株单菌落于5mL NA液体培养基中,180r·min-1,30℃摇床中震荡培养12h,至浓度为108CFU·mL-1,离心收集菌体,利用TIANGEN细菌基因组DNA提取试剂盒提取细菌基因组DNA,紫外分光光度计Nanodrop 2000检测提取DNA质量和浓度。-20℃备用。
2)PCR扩增体系和方法的建立
PCR扩增反应体系为25μL体系,具体为:1μL DNA模板、1μL上游引物:Dda-F、1μL下游引物:Dda-R、12.5μL Taq PCR Mix、9.5μL ddH2O。
PCR扩增程序为:94℃预变性3min;94℃变性30s,56℃退火30s,72℃延伸10s,35个循环;72℃延伸5min,4℃终止反应。
扩增结束后,将PCR扩增产物用1%的琼脂糖在0.5×TBE缓冲液中进行电泳检测。
3)特异性试验
分别提取3株甘薯茎腐病菌及20株参比菌株DNA,分别取1μL作为模板进行PCR扩增反应,扩增体系及程序如2)。扩增产物电泳结果如图1。
由图1可知,只有甘薯茎腐病菌有预期大小(167bp)的目的条带,而其他参比菌株及无菌水对照均无条带扩增出,表明建立的甘薯茎腐病菌的分子鉴定方法具有良好的特异性。
4)灵敏度检测
对浓度为500ng/μL的甘薯茎腐病菌基因组DNA 10倍梯度稀释8个稀释度,各取1μL的甘薯茎腐病菌DNA为模板进行PCR扩增反应,扩增体系及程序如2)。扩增产物电泳结果如图2。
由图2可知,本发明的分子检测方法的最低检测浓度为0.5ng/μL。说明建立的甘薯茎腐病菌的PCR检测方法具有较高的灵敏度。
5)模拟样品检测
采用针刺接种法接种菌浓度为108CFU·mL-1的甘薯茎腐病菌至甘薯茎部,24小时后,同时采用本发明方法,对甘薯茎腐病模拟样品进行检测,检测结果如图3所示。对该样品进行病原细菌分离,结果与分子鉴定方法一致。
序列表
<110> 浙江大学
<120> 一种用于检测甘薯茎腐病菌的引物对及其检测方法
<160> 3
<170> SIPOSequenceListing 1.0
<210> 1
<211> 237
<212> DNA
<213> 达旦提狄克氏菌(Dickeya dadantii )
<400> 1
atgggcgttc ataatattac cggctatttt gatatttatt tctcatatca accagaccag 60
ccgttctggt ctggccgatt tatcatgcca ttgaagcaaa ccagcaacgt tctgttgcct 120
gctggaaata aaccgtggca ggataaagaa ttacagatac gagctaaagt tcatgcctgt 180
ttgaataata cgttgtttaa aagcaggccg gcgttcgcag cgaactcgat cgcctaa 237
<210> 2
<211> 22
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 2
catatcaacc agaccagccg tt 22
<210> 3
<211> 24
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 3
cggcctgctt taaacaacgt atta 24
Claims (5)
1.引物对在鉴定达旦提狄克氏菌(Dickeya dadantii)中的应用,其特征在于,所述引物对包括上游引物和下游引物,其核苷酸序列分别为:
上游引物:5'-CATATCAACCAGACCAGCCGTT-3';
下游引物:5'-CGGCCTGCTTTAAACAACGTATTA-3'。
2.一种检测甘薯茎腐病菌的方法,其特征在于,包括以下步骤:
(1)提取待检样品DNA;
(2)采用引物对,建立PCR反应体系,进行PCR扩增;
所述引物对包括上游引物和下游引物,其核苷酸序列分别为:
上游引物:5'-CATATCAACCAGACCAGCCGTT-3';
下游引物:5'-CGGCCTGCTTTAAACAACGTATTA-3';
(3)对PCR产物进行检测和判断,
如扩增产物中出现167bp的条带,则说明待检样品中具有甘薯茎腐病菌Dickeyadadantii;否则,说明待检样品中不具有甘薯茎腐病菌。
3.如权利要求2所述的方法,其特征在于,所述的待检样品为甘薯块茎、甘薯苗、甘薯茎。
4.如权利要求2所述的方法,其特征在于,所述PCR反应体系,以25μL体系计,包括:2×Taq PCR MasterMix 12.5μL、10μmol/L上下游引物各1μL、DNA模板1μL,灭菌双蒸水9.5μL。
5.如权利要求2所述的方法,其特征在于,所述PCR扩增条件:94℃预变性3min;94℃变性30s,56℃退火30s,72℃延伸10s,35个循环;72℃延伸5min,4℃终止反应。
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