CN104651490B - 一种番茄溃疡病菌快速检测的方法及其专用引物 - Google Patents
一种番茄溃疡病菌快速检测的方法及其专用引物 Download PDFInfo
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Abstract
本发明公开了一种番茄溃疡病菌快速检测的方法及其专用引物,专用引物是由序列表中的序列1、序列2、序列3和序列4的核苷酸序列组成的引物组。检测番茄溃疡病菌的方法,是以番茄溃疡病菌标准菌株DNA为模板,在由权利要求1所述的序列表中序列1、序列2、序列3和序列4的核苷酸序列组成的引物组的引导下进行恒温PCR扩增,扩增产物经琼脂糖凝胶电泳检测在100 bp‑1000bp之间有梯度条带,扩增产物用SYBR Green I直接染色可呈现橘黄色。本发明提供了的一组快速扩增番茄溃疡病菌基因组特定区段的引物,该引物将在田间及检疫部门对番茄溃疡病菌快速检测及鉴定中发挥重要作用。
Description
技术领域
本发明涉及一种快速检测番茄溃疡病菌的方法及其专用引物。
背景技术
番茄溃疡病(Bacterial canker of tomato)是番茄(Lycopersicon esculentumMill.)生产中最为严重的病害之一,该病自从1909年首次在美国密执安州的温室番茄上发现以来,现已广泛分布于美国各番茄产区,并逐渐成为世界性病害。目前,在美洲、欧洲、亚洲、非洲和大洋洲的60多个国家都有番茄溃疡病发生危害的报道。该病害一旦发生没有有效措施控制,严重威胁着番茄的产量、品质以及采后果实的抗性,造成严重经济损失。
我国有关番茄溃疡病的记载始于1954年,目前该病在我国北京、黑龙江、吉林、辽宁、内蒙古、新疆、河北、山西、山东、上海、安徽、海南、广西等省、市、自治区都有发生,使许多地区番茄生产受到了不同程度的影响,近年来该病每年均有发生,且呈逐年扩散和加重的趋势。其中尤以甘肃、新疆、内蒙古、河北等省区为甚。2011年河北省望都县植保站对全县番茄大棚进行了系统调查,发现番茄溃疡病病棚率达75%以上。
引起该病的病原菌为密执安棒状杆菌密执安亚种(Clavibacter michiganensissubsp.michiganensis,Cmm),被列入我国检疫对象的名单。使用无菌种子和无菌苗是控制番茄细菌性溃疡病的首要措施,因此对番茄细菌性溃疡病的早期检测是控制该病传播和发生的有效手段。
环介导等温扩增(loop-mediated isothermal amplification,LAMP)技术是Notomi等于2000年开发的一种新的恒温体外核酸扩增技术,该技术利用能够识别靶DNA上6个特定区域的4条引物和有链置换活性的Bst(Bacillus stearo thermophilus)DNA聚合酶,使模板两端引物结合处循环出现环状单链结构,在等温条件下使引物顺利与模板结合并进行链置换扩增反应。由于LAMP独特的核酸扩增机制,它的产物是由多重复靶序列的茎-环状DNA和花椰菜状DNA所组成的混合物,在琼脂糖凝胶电泳上会呈现出LAMP所特有的阶梯状条带。扩增产物不仅可以通过电泳检测,还可以添加SYBR Green I、溴化乙锭(EB)或其他核酸染色剂进行显色后观察。该技术具有高特异性、快速灵敏、高效、操作简便等特点,建立稳定、灵敏度高的LAMP反应体系,可以为番茄溃疡病菌的快速检测提供便捷服务。
本发明根据具有亚种特异性的番茄溃疡病菌ITS序列,设计了一组用于特异性检测番茄溃疡病菌的LAMP引物,并对反应温度和反应体系中甜菜碱、MgSO4、Bst DNA聚合酶、dNTPs等试剂的使用浓度进行了优化,最终建立了番茄溃疡病病菌的LAMP检测方法,此体系在具备水浴锅和荧光染料的条件下,1h左右的反应时间里就能够有效地检测到目的菌,而PCR扩增结合琼脂糖凝胶电泳检测通常需要花费3-4h甚至更长时间,因此在专业技术和实验设备不足的县市级植保植检部门,本发明建立的检测方法更具备实用性。
发明内容
本发明的目的是提供一组可用于检测番茄溃疡病菌的引物。
本发明的另一个目的是提供一种可用于快速检测番茄溃疡病菌的方法。
本发明所提供的用于检测番茄溃疡病菌的引物,是由序列表中的序列1、序列2、序列3和序列4的核苷酸序列组成的引物对。
将由序列表中序列1、序列2、序列3和序列4的核苷酸序列组成的引物组命名为cmm-LAMP,序列表中SEQ ID No:1由42个碱基组成,SEQ ID No:2由41个碱基组成,SEQ IDNo:3由19个碱基组成,SEQ ID No:4由18个碱基组成。
本发明所提供的快速检测番茄溃疡病菌的方法,是以待测番茄溃疡病菌标准菌株DNA为模板,在由序列表中序列1、序列2、序列3和序列4的核苷酸序列组成的引物组cmm-LAMP的引导下进行等温PCR扩增,扩增产物经琼脂糖凝胶电泳检测可在100bp-1000bp之间产生梯度条带,用SYBR Green I染色后可在扩增管中呈现橘黄色。
其中,等温扩增体系为:25μL总体系中含模板DNA 2μL,Betaine甜菜碱0.4mol/L,MgSO42.0mmol/L,Bst DNA聚合酶4U,dNTPs 1.0mmol/L,引物1、引物2各1.5μL,引物3、引物4各0.5μL,1×Isothermal Amplification Buffer,其余用无菌蒸馏水补充反应体系至25.0μL。反应程序为:65℃温育40min,80℃灭活10min。
检测扩增产物的方法有两种:
其一为对扩增产物进行1.5%琼脂糖凝胶电泳,然后用Goldview显色。具体方法为:取5μL扩增产物,加入0.5μL变性载样指示剂(98%无离子甲酰胺,10mM EDTA pH8.0,0.25%溴酚蓝),在浓度为1.5%的琼脂糖凝胶中电泳分离,检测扩增产物中是否在100bp-1000bp之间产生梯度条带。
其二为在扩增产物中,按1/100的比例加入用10×TBE稀释的SYBR Green I染料,呈现橘黄色为阳性,说明模板中含有番茄溃疡病菌基因组DNA,无颜色反应色为阴性,说明模板中不含有番茄溃疡病菌基因组DNA。
本发明提供了一组快速扩增番茄溃疡病菌基因组特定区段的引物cmm-LAMP,该引物将在田间及检疫部门对番茄溃疡病菌快速检测及鉴定中发挥重要作用。
下面结合具体实施例对本发明做进一步详细说明。
下述实施例中所用方法如无特别说明均为常规方法,引物由上海生工生物工程公司合成。番茄溃疡病菌标准菌株由中国农业大学李建强教授提供,并由河北农业大学生物防治研究室收集保存。
实施例1、番茄溃疡病菌模板DNA的获得
用改良523培养基培养番茄溃疡病菌,25℃振荡培养3天,收集菌体后加无菌水100μL,95℃加热10min,1000r/min离心5min制成模板。
实施例2、番茄溃疡病菌LAMP扩增及检测
反应体系为:25μL总体系中含模板DNA 2μL,Betaine甜菜碱0.4mol/L,MgSO42.0mmol/L,Bst DNA聚合酶4U,dNTPs 1.0mmol/L,引物1、引物2各1.5μL,引物3、引物4各0.5μL,1×Isothermal Amplification Buffer。
反应程序程序为:65℃温育40min,80℃灭活20min。
检测方法为:取5μL LAMP扩增产物用1.5%琼脂糖凝胶电泳检测扩增结果(见图1)。
SYBR Green I染料用10×TBE稀释,按1/100的比例加入剩余的扩增产物中,观察颜色变化(见图2)。
附图说明
图1为番茄溃疡病菌LAMP扩增产物经1.5%琼脂糖凝胶电泳检测结果。
图2为番茄溃疡病菌LAMP扩增产物经SYBR Green I染色结果。
<110> 河北农业大学
<120>一种番茄溃疡病菌快速检测的方法及其专用引物
<160> 4
SEQ ID No. 1
<210> 1
<211> 42
<212> DNA
<213> 人工序列
<400> 1
GATCCACCGGAAAACCGGTGAAACCGCCCGTATATTGAGAAC
SEQ ID No. 2
<210> 1
<211> 41
<212> DNA
<213> 人工序列
<400> 2
ATTCTGTCTCCCTTCGGGGAGGATCCACCGTTTGCCCTTAG
SEQ ID No. 3
<210> 1
<211> 19
<212> DNA
<213> 人工序列
<400> 3
TTTCCGTCGTCCTGTTGTG
SEQ ID No. 4
<210> 1
<211> 18
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<213> 人工序列
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CGTCCTTCTTCGGCTCCA
Claims (3)
1.一种番茄溃疡病菌密执安棒状杆菌密执安亚种快速检测的方法,其特征在于,是以待测菌株DNA为模板,在由序列表中序列1、序列2、序列3和序列4的核苷酸序列组成的引物组的引导下进行恒温PCR扩增,扩增产物经琼脂糖凝胶电泳检测在100bp-1000bp之间有梯度条带或经SYBR Green I染色显示阳性结果即表示待测菌株模板中含有密执安棒状杆菌密执安亚种;
25 μL总体系中含模板DNA 2 μL,Betaine甜菜碱0.4 mol/L,MgSO4 2.0 mmol/L,BstDNA聚合酶4U,dNTPs 1.0 mmol/L,引物1、引物2各1.5 μL,引物3、引物4各0.5 μL,1×Isothermal Amplification Buffer,其余用无菌蒸馏水补充反应体系至25.0 µL,反应程序为:65℃温育40 min,80℃灭活10 min。
2.根据权利要求1所述的方法,其特征在于,检测扩增产物的方法为对扩增产物进行1.5 %琼脂糖凝胶电泳,然后用Goldview显色。
3.根据权利要求1所述的方法,其特征在于,检测扩增产物的方法为在扩增产物中按1/100的比例加入用10×TBE稀释的SYBR Green I 染料。
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