CN116083630B - 实时荧光lamp检测三七根腐病致病菌尖孢镰刀菌的引物组 - Google Patents
实时荧光lamp检测三七根腐病致病菌尖孢镰刀菌的引物组 Download PDFInfo
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Abstract
本发明公开了实时荧光LAMP检测三七根腐病致病菌尖孢镰刀菌的引物组,本发明选用尖孢镰刀菌跨膜蛋白的基因为特异性检测位点,设计了6条特异性且在65℃下恒温扩增的实时荧光LAMP检测引物,用于尖孢镰刀菌的快速分子检测,在该LAMP检测体系下,使用10×TE缓冲液裂解样品,通过荧光信号检测样品中是否存在尖孢镰刀菌,并通过建立标准曲线来定量尖孢镰刀菌的数量动态变化,该方法可能用于三七种植土壤以及植株的带菌检测,为病原菌快速分子检测技术提供支持。
Description
技术领域
本发明属于植物病原菌检测技术领域,具体涉及一种利用实时荧光LAMP检测三七根腐病致病菌尖孢镰刀菌的引物组和试剂盒。
背景技术
三七[Panax notoginseng (Burk.).F.H. Chen] 是我国珍贵的中草药,能促进血液循环,防止血瘀,消肿消炎,缓解疼痛,延缓衰老,又被誉为“血管清道夫”。三七中具有药理活性的主要成分为三七总皂苷。由于三七独特的功效,用途不断拓展,市场需求较多。三七主要种植于云南文山州,喜阴湿,种植周期长,病害多发。研究表明三七的主要病害包括根腐病、病毒病、灰霉病等。近年来,病害种类、发病面积及严重程度呈上升趋势,在这些病害中,主要由致病真菌引起的根腐病是最具破坏性的病害之一,可导致药用部位腐烂,甚至全株死亡。
三七根腐病是典型的土传性病害,俗称烂头子,主要包括黄腐型、髓烂型及湿腐型等。根腐病能导致三七根部的腐烂或者干枯,直接导致三七药材的产量及品质下降。传播途径主要是病土、带菌流水污染种子及种苗带菌和传播,其中近距离传播以带菌土壤和带菌流水为主,远距离传播以种苗带菌扩散为主。根腐病的病原体种类多样,包括一些真菌、细菌、线虫等,其中镰刀属真菌尖孢镰刀菌(Fusarium oxysporum)也是重要的根腐病菌之一。
准确识别和检测病原体是有效预防和控制病害的基础。传统鉴定和检测病原菌的步骤包括从感病组织中分离病原体,然后根据形态学特征进行初步鉴定,这种基于形态学的识别不仅困难,而且费时费力,不能满足快速准确识别和诊断病害的需求。聚合酶链式反应PCR技术提高了病原体鉴定的准确性和检测的快速性,但是它们很复杂并且需要昂贵的设备,例如精确的PCR仪器和凝胶成像系统,以及其他分子生物学设备。上述缺点限制了PCR技术在现场检测中的使用和推广。因此,有必要建立一种快速、经济的现场植物病原菌检测新技术。
环介导等温扩增(LAMP)是一种简单、准确、低成本和高效的核酸扩增技术。在LAMP检测中,根据靶基因的6-8个特定区域设计4-6条引物,如果任何一条引物与靶基因不匹配,扩增就无法进行,因此LAMP具有很强的特异性。此外,LAMP使用高活性的链置换DNA聚合酶(Bst DNA聚合酶)在60–65℃的等温温度下有效扩增目标DNA片段,因此,只需简单的水浴或加热块即可满足反应要求,整个反应在一小时内完成,并且可以用肉眼观察结果。这种技术具有高特异性、最低设备要求、操作简单、高灵敏度和反应时间短的优点,并已被广泛用于检测多种植物病原体。因此,开发一种快速准确的现场检测三七根腐病菌尖孢镰刀菌的LAMP检测方法势在必行。
发明内容
为了达到对三七根腐病致病菌尖孢镰刀菌的快速准确检测的要求,及时阻断传播并高效特异防治,避免造成大面积损失,本发明提供了检测三七根腐病菌尖孢镰刀菌的实时荧光LAMP引物组及LAMP检测试剂盒,通过该方法能简单快速准确的检测出三七植株及土壤中尖孢镰刀菌的含量。
本发明实时荧光LAMP检测三七根腐病致病菌尖孢镰刀菌的引物组的的核苷酸序列如下:
TP-FIP:5’-CGACCTATTAAGTGGCACGAGTACCTCACCACTTCCTGTT-3’
TP-BIP:5’-GCTTTGGAGTTCCCAGAACCAATCGACATATCTATCGAAGCAC-3’;
TP-F3:5’-CTCGTCTCAAGCACACTG-3’
TP-B3:5’-AAGAGCACTCCGTCATCT-3’;
TP-LooF:5’-GCTGGCAAGATTGAACGC-3’
TP-LooR:5’-CAGGACAATGATCAAAGTCGC-3’;
上述引物组以尖孢镰刀菌的跨膜蛋白(Transmembrane Protein)基因(NCBI登录号JK747097.1)为特异性检测位点。
本发明还提供了一种含有上述引物组的实时荧光LAMP检测三七根腐病致病菌尖孢镰刀菌的试剂盒,其包括上述引物组以及LAMP检测所需的常规检测试剂;
实时荧光LAMP检测体系包括:
(1)25μL LAMP扩增体系为:10× Isothermal Amplification Buffer II 2.5μL、MgSO4 1μL (6 mM)、dNTP Mix 4μL (1.4mM)、TP-F3 1μL (5μM)、TP-B3 1μL (5μM)、TP-FIP1μL (40μM)、TP-BIP 1μL (40μM)、TP-LoopF 0.4μL (5μM)、TP-LoopB 0.4μL (5μM)、Bst3.0 DNA Polymerase 4U 0.5μL、20×Eva Green 1.25μL,DNA模板1μL,加入Nuclease-freeWater 补足到25μL;
(2)10×TE缓冲液:含有100mM Tris-HCl、10mM EDTA的水溶液,pH8.0,用于裂解样本以提取DNA。
实时荧光LAMP检测试剂盒还包含了质粒标准品,质粒是由跨膜蛋白基因插入T载体(pGEM-T-TP)构建而成。
利用上述试剂盒检测三七根腐病菌尖孢镰刀菌的方法如下:
(1)采用10×TE缓冲液提取制备实时荧光LAMP的反应模板;
(2)配制实时荧光LAMP的扩增体系;
(3)在步骤(1)提取的模板中加入步骤(2)中配制的实时荧光LAMP扩增体系,混匀后在65℃恒温进行实时荧光LAMP反应,反应条件为:65℃ 30s,45个循环,反应结束后进行熔解曲线分析(95℃反应0.05s;65℃反应0.05s,按0.5℃/step的升温速率递增到95℃);
(4)实时荧光LAMP标准曲线的建立
制备浓度在0.2pg/μL -20ng/μL的尖孢镰刀菌质粒标准品溶液,对不同浓度的溶液进行实时荧光LAMP反应,反应体系与扩增步骤同(2)和(3),每个循环反应结束后收集并记录荧光信号,以各浓度对应的质粒质量对数值作为纵坐标,Ct值为横坐标,得到线性回归方程,并建立标准曲线;
(5)结果诊断:若出现荧光信号则判定所检测样品中存在尖孢镰刀菌,并将得到的Ct值代入尖孢镰刀菌的标准曲线中,计算检测样品中尖孢镰刀菌的含量。
本发明的优点在于:
(1)准确性高。本发明是根据基因序列在真菌种内的高度保守性和属种间可变性的特点,设计对三七根腐病致病菌尖孢镰刀菌具有特异扩增作用的LAMP引物,只在根腐病病株的腐烂根部及其土壤中特异性扩增出尖孢镰刀菌,说明本发明所设计的引物用于检测三七根腐病菌尖孢镰刀菌准确可靠;
(2)灵敏度高。本发明将设计的特异引物进行实时荧光LAMP分析,对三七根腐病致病菌尖孢镰刀菌的检测灵敏度在DNA水平上可达到0.2pg/μL;
(3)适用性广、实用性好。本发明三七根腐病致病菌尖孢镰刀菌的实时荧光LAMP检测方法,采用10×TE缓冲液处理样品,能简便快速提取样品DNA进行检测,也能对感病的三七根部、叶片、种苗以及土壤样品进行检测,可实现三七根腐病的早期检测,即在病害显症前进行检测,防治病害的爆发流行。
附图说明
图1为常规PCR验证尖孢镰刀菌TP基因特异性的扩增凝胶图,其中泳道Marker为2000 bp DNA Marker,泳道1为尖孢镰刀菌,泳道2为茄腐镰刀菌,泳道3为交链格孢,泳道4为毁灭柱孢菌,泳道5为厚垣镰刀菌,泳道6为草茎点霉,泳道7为木贼镰刀菌,泳道8为空白对照(无DNA模板);
图2为LAMP快速验证引物特异性的扩增曲线图(上图)以及熔解曲线图(下图),其中七种病原真菌为:尖孢镰刀菌、茄腐镰刀菌、木贼镰刀菌、交链格孢、毁灭柱孢菌、草茎点霉、厚垣镰刀菌;
图3为本发明利用尖孢镰刀菌TP基因片段构建质粒的实时荧光LAMP的扩增曲线图,其中1-6号分别为质粒浓度20ng/μL、2ng/μL、0.2ng/μL、0.02ng/μL、0.002ng/μL、0.0002ng/μL的实时荧光LAMP扩增曲线图;
图4为以各浓度对应的质粒质量对数值作为纵坐标,Ct值为横坐标建立的标准曲线图(左图)和Ct值(右图);
图5为应用本发明的LAMP引物检测三七田间样品的实时荧光LAMP扩增曲线图(上图)以及熔解曲线图(下图),上图中1-3为根腐病病株,4-6为根腐病病株的根际土壤样品。
具体实施方式
下面通过附图和实例对本发明进一步说明,但本发明保护范围不局限于所述内容,本实例中方法如无特殊说明均按常规方法操作,所用试剂如无特殊说明的则采用常规试剂或按常规方法配置试剂;
实施例中所用菌种为尖孢镰刀菌(F. oxysporum)、茄腐镰刀菌(Fusarium solani)、交链格孢(Alternaria alternata)、毁灭柱孢菌(Cylindrocarpon destructans)、厚垣镰刀菌(F. chlamydosporum)、草茎点霉(Phoma herbarum)、木贼镰刀菌(Fusarium equiseti);检测样品采自云南省文山州三七种植基地;
实施例1:尖孢镰刀菌跨膜蛋白基因特异性分析
根据前期研究结果选择尖孢镰刀菌跨膜蛋白基因(JK747097.1)作为检测靶点,并设计特异PCR引物;上游引物F为CAGCCTTGACCCAGCTCGTA,下游引物R为TACTCCGCGCAAGAGCACTC,引物委托昆明硕擎生物科技有限公司合成。
采用以下几种病原真菌作为筛选模板,尖孢镰刀菌、茄腐镰刀菌、交链格孢、毁灭柱孢菌、厚垣镰刀菌、草茎点霉、木贼镰刀菌;采用CTAB法提取真菌菌丝的基因组DNA,采用紫外分光光度计检测DNA提取的纯度和浓度。
以上述几种病原真菌的基因组DNA为模板进行PCR,以检测引物的特异性,PCR反应体系(20μL)如下:2×GS Taq PCR Mix 12.5μL、无菌双蒸水9.5μL、上游引物F (10μM) 1μL、下游引物R (10μM) 1μL、DNA模板1μL;PCR扩增条件为:95℃ 3 min;94℃ 30 s, 56℃ 30s, 72℃ 30 s, 28个循环;72℃ 5min。PCR产物通过1.4%琼脂糖凝胶电泳检测,结果如图1所示,这对引物只在以尖孢镰刀菌DNA为模板的PCR扩增中产生了300bp左右的扩增产物,可初步认定本实验设计的引物具有种间特异性,利用本引物只在尖孢镰刀菌中产生特异的条带,而在其他三七病原真菌中不引起有效扩增,因而能特异地将尖孢镰刀菌与其他病原菌区分开来。
进一步将PCR产物进行T-A克隆,选择pGEM®-T easy Vector System (Promega,USA)作为克隆载体,与尖孢镰刀菌的PCR扩增产物进行连接,将连接产物转入大肠杆菌DH5α感受态细胞中,随后挑选阳性克隆测序,并对测序结果进行生物信息学分析。利用BLAST(http://blast.ncbi.nlm.nih.gov/Blast.cgi)在线分析工具对测序得到的序列与GenBank中的基因序列进行同源性分析,结果显示扩增序列与尖孢镰刀菌跨膜蛋白基因(JK747097.1)有99%的相似性。
实施例2:特异性实时荧光LAMP检测方法的建立
利用LAMP Designer软件设计LAMP引物组(TP-F3、TP-B3、TP-FIP、TP-BIP、TP-LoopF、TP-LoopB),引物由北京擎科生物科技有限公司合成。
25μL LAMP扩增体系为:10× Isothermal Amplification Buffer II 2.5μL、MgSO4 1μL (6mM)、dNTP Mix 4μL (1.4mM)、TP-F3 1μL (5μM) 、TP-B3 1μL (5μM)、TP-FIP1μL (40μM)、TP-BIP 1μL (40μM)、TP-LoopF 0.4μL (5μM)、TP-LoopB 0.4μL (5μM)、Bst3.0 DNA Polymerase 4U 0.5 μL、20×Eva Green 1.25μL,DNA模板1μL,加入Nuclease-free Water 补足到25μL。反应条件为65℃ 30s,共40个循环,反应结束后进行熔解曲线分析(95℃ 0.05s,65℃ 0.05s,按0.5℃/step的升温速率递增到95℃)。
采用CTAB法提取真菌DNA,以ddH2O和6种真菌(茄腐镰刀菌、厚垣镰刀菌、木贼镰刀菌、毁灭柱孢菌、交链格孢、草茎点霉)基因组DNA模板作为对照,每个真菌模板设置3次重复,以验证本发明中LAMP引物检测尖孢镰刀菌的特异性;
结果见图2,图中显示只有以尖孢镰刀菌基因组DNA为模板的LAMP反应中出现扩增曲线,三个重复得到三条重合的扩增曲线,其Ct值分别为26.37、26.57、26.40,熔解曲线峰型单一,峰值均在89℃左右,在模板分别为茄腐镰刀菌、厚垣镰刀菌、木贼镰刀菌、毁灭柱孢菌、交链格孢、草茎点霉的LAMP反应中未检测到扩增曲线和熔解曲线,说明本发明引物组适用于三七根腐病菌尖孢镰刀菌的实时荧光LAMP检测,也表明所建立的LAMP 引物以及检测体系只能对尖孢镰刀菌的基因组DNA 进行特异性扩增,不能与其余6种菌株的DNA发生特异性反应,该实时荧光LAMP 检测体系可以将三七根腐病菌尖孢镰刀菌与其它病原菌区分开来,具有种的特异性。
实施例3:特异性实时荧光LAMP检测方法的灵敏度测试
将实施例1制备的携带尖孢镰刀菌跨膜蛋白基因片段的重组质粒(pGEM-T-TP)作为标准品,并测定质粒浓度,然后进行10倍的浓度梯度稀释,共设置6个梯度,分别为20ng/μL、2ng/μL、0.2ng/μL、0.02ng/μL、0.002ng/μL和0.0002ng/μL,稀释介质为无核酸酶水;对6个梯度的标准品进行LAMP反应灵敏度测试;实时荧光LAMP反应体系和反应条件同实施例2,实时荧光扩增曲线见图3;结果显示本发明中LAMP检测方法的灵敏度是0.2pg/μL;
以各梯度浓度对应的质粒质量对数值作为纵坐标,Ct值为横坐标,在Excel软件中生成标准曲线,得到线性回归方程为y = -0.3294x + 6.6034,R2=0.9963,建立的标准曲线见图4。
实施例4:三七根腐病菌尖孢镰刀菌实时荧光LAMP快速检测方法的应用
为了确定实施例2建立的快速检测方法是否适用于三七及土壤样品中尖孢镰刀菌的检测,在文山州三七栽培基地中采集了一系列根腐病样品,1-3号为根腐病病株,4-6号为根腐病病株的根际土壤样品,7-9号为发生叶斑病的三七叶片,10-12叶斑病病株的根际土壤,13号为无模板阴性对照;
用无菌剪刀剪下0.01g病株样品,土壤样品用电子天平称量0.01g,放置于含有50μL 10× TE缓冲液的离心管中,用牙签进行充分混合,静置5min;期间配制25μL LAMP扩增体系:10×Isothermal Amplification Buffer II 2.5μL、MgSO4 1μL (6mM)、dNTP Mix 4μL(1.4mM)、TP-F3 1μL (5μM)、TP-B3 1μL (5μM)、TP-FIP 1μL (40μM)、TP-BIP 1μL (40μM)、TP-LoopF 0.4μL (5μM)、TP-LoopB 0.4μL (5μM)、Bst 3.0 DNA Polymerase 4U 0.5μL、20×Eva Green 1.25μL,取静置后的10×TE缓冲液裂解液1μL作为反应模板,加入Nuclease-freeWater补足到25μL;反应条件为65℃ 30s, 共45个循环。
结果见图5,结果显示三七根腐病及其根际土壤样本中可以检测到尖孢镰刀菌的存在(扩增曲线1-6),样品出现明显的“S”型扩增曲线,而三七叶斑病病株及其土壤样本中未见明显扩增;上述实验结果显示,本实验设计的引物以及建立的实时荧光LAMP快速检测体系能够诊断三七及其土壤样品中是否含有尖孢镰刀菌。
Claims (2)
1.实时荧光LAMP检测三七根腐病致病菌尖孢镰刀菌的引物组,其特征在于,引物组的核苷酸序列如下:
TP-FIP:5’-CGACCTATTAAGTGGCACGAGTACCTCACCACTTCCTGTT-3’
TP-BIP:5’-GCTTTGGAGTTCCCAGAACCAATCGACATATCTATCGAAGCAC-3’;
TP-F3:5’-CTCGTCTCAAGCACACTG-3’
TP-B3:5’-AAGAGCACTCCGTCATCT-3’;
TP-LoopF:5’-GCTGGCAAGATTGAACGC-3’
TP-LoopB:5’-CAGGACAATGATCAAAGTCGC-3’。
2.含有权利要求1所述的实时荧光LAMP检测三七根腐病致病菌尖孢镰刀菌的引物组的试剂盒。
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