CN116377119A - 一种短柄镰刀菌的pcr检测方法及其应用 - Google Patents
一种短柄镰刀菌的pcr检测方法及其应用 Download PDFInfo
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Abstract
本发明公开了一种短柄镰刀菌的PCR检测方法及其应用,属于生物检测鉴定技术领域。该PCR检测方法中使用的引物组包括上游引物和下游引物;所述上游引物的序列如SEQ ID NO.1所示;所述下游引物的序列如SEQ ID NO.2所示。本发明PCR检测方法具备很好的特异性,从23个测试菌株中仅能扩增出短柄镰刀菌,并且可以对低至10pg/μL的病原菌基因组DNA进行检测。因此,本发明所设计的引物和检测方法可以快速、特异、灵敏性鉴定短柄镰刀菌。
Description
技术领域
本发明属于生物检测鉴定技术领域,尤其涉及一种短柄镰刀菌的PCR检测方法及其应用。
背景技术
番茄在我国各地区种植广泛,随着种植面积逐渐扩大,复种指数连年提高,土传病害的发生与危害亦呈逐年加重的趋势。常见的番茄土传病害包括青枯病、枯萎病、根腐病、黄萎病、褐色根腐病和立枯病等,其优势病原菌分别为茄科劳尔氏菌(Ralstoniasolanacearum)、尖孢镰刀菌(Fusarium oxysporum)、腐皮镰刀菌(Fusarium solani)、大丽轮枝菌(Verticillium dahliae)、番茄棘壳孢(Pyrenochaeta lycopersici)和立枯丝核菌(Rhizoctonia solani),给我国各地茄科类作物产区造成了严重的经济损失。近期,在番茄土传病害的调查中,对短柄镰刀菌在中国引起番茄枯萎症状病害进行首次报道(Jun Liu,Yi Si Deng, Wei Chang, and Hua Wang. 2023. First report of tomato wilt causedby Fusarium brachygibbosum in China. https://doi.org/10.1094/PDIS-01-23-0076-PDN)。该病菌对番茄种植可能会构成严重威胁,并有可能给番茄产业造成巨大的经济损失。因此,该病原菌的快速鉴定将有助于研究有效的病害管理策略,防止番茄严重减产。然而,短柄镰刀菌的PCR分子快速检测研究还未见报道。
随着分子生物学技术的快速发展,许多基于分子生物学的病原菌分子检测方法相继被开发。与传统的基于分离培养与形态观察的检测方法相比,病原菌的分子鉴定方法准确、高效。
发明内容
本发明的目的之一在于提供一种用于短柄镰刀菌PCR检测的引物组,所述引物组包括上游引物和下游引物;所述上游引物的序列如SEQ ID NO.1所示;所述下游引物的序列如SEQ ID NO.2所示。
本发明的目的之二在于提供一种检测短柄镰刀菌的试剂,所述试剂含有上述引物组SEQ ID NO.1和SEQ ID NO.2。
本发明的目的之三在于提供一种检测短柄镰刀菌的试剂盒,所述试剂盒中含有上述引物组SEQ ID NO.1和SEQ ID NO.2或上述试剂。
本发明的目的之四在于提供一种短柄镰刀菌的PCR鉴定方法,所述鉴定方法以样品总DNA为模板,利用上述引物组SEQ ID NO.1和SEQ ID NO.2进行PCR扩增,反应结束后根据琼脂糖凝胶电泳判定结果。
优选地,PCR的反应程序为:94℃预变性5min;进入循环,94℃变性30s,60℃退火30s,72℃延伸30s,35个循环;最后延伸72℃10min。
更优选地,PCR反应体系总体积25ul,其中包括:2.5μL 10×PCR缓冲液,1.5μL25mM Mg2+,2μL 10mM dNTP,0.125ul 5U/μL Taq DNA聚合酶,浓度为10μM的上游引物SEQ IDNO.1 1μL,浓度为10μM的下游引物SEQ ID NO.2 1μL,DNA模板1μL,ddH2O 15.875μL。
更优选地,所述DNA模板提取自番茄。
本发明的目的之五在于提供上述引物组SEQ ID NO.1和SEQ ID NO.2或上述试剂或上述试剂盒在鉴定短柄镰刀菌中的应用。
与现有技术相比,本发明具有如下有益效果:
本发明的优点在于设计的引物组合可以在番茄样品中快速检测到短柄镰刀菌的有无。PCR检测具备很好的特异性,从23个测试菌株中仅能扩增出短柄镰刀菌,并且可以对低至10pg/μL的病原菌基因组DNA进行检测。因此,本发明所设计的引物和检测方法可以快速、特异、灵敏性鉴定短柄镰刀菌。
附图说明
图1为实施例2中PCR引物对短柄镰刀菌特异性检测的琼脂糖凝胶电泳图,其中M:2000bp DNA Marker;NC:无菌水,阴性对照;PC:短柄镰刀菌DNA,阳性对照;泳道1:尖孢镰刀菌DNA;泳道2:轮枝镰刀菌DNA;泳道3:层出镰刀菌DNA;泳道4:禾谷镰刀菌DNA;泳道5:假禾谷镰刀菌DNA;泳道6:腐皮镰刀菌DNA;泳道7:变红镰刀菌DNA;泳道8:木贼镰刀菌DNA;泳道9:亚洲镰刀菌DNA;泳道10:藤仓镰刀菌DNA;泳道11:锐顶镰刀菌DNA;泳道12:交互链格孢菌DNA;泳道13:齐整小核菌DNA;泳道14:核盘菌DNA;泳道15:禾谷丝核菌DNA;泳道16:桃拟茎点霉DNA;泳道17:茶拟盘多毛孢DNA;泳道18:大丽轮枝菌DNA;泳道19:葡萄座腔菌DNA;泳道20:灰葡萄孢DNA;泳道21:瓜蔓枯病菌DNA;泳道22:瓜类炭疽病菌DNA。
图2为实施例3中PCR引物对短柄镰刀菌灵敏性检测的琼脂糖凝胶电泳图,其中M:2000bp DNA Marker;泳道1:10ng/μL;泳道2:1ng/μL;泳道3:100pg/μL;泳道4:10pg/μL;泳道5:1pg/μL;泳道6:100fg/μL;泳道7:10fg/μL。
图3为实施例4中PCR检测田间番茄样品的琼脂糖凝胶电泳图,其中M:2000bp DNAMarker;PC:短柄镰刀菌DNA,阳性对照;NC:无菌水,阴性对照;泳道1-22:田间番茄样品。
具体实施方式
实施例1
短柄镰刀菌分子检测方法的建立
1、引物设计
从NCBI(National Center for Biotechnology Information)数据库中下载短柄镰刀菌Fusarium brachygibbosum HN-1(GenBank Accession No. MU249523.1),尖孢镰刀菌Fusarium oxysporum(GenBank Accession No. NC_030986), F. proliferatum FP-A8(GenBank Accession No. MRDB01000001.1), F. verticillioides 7600 (GenBankAccession No. CM000579.1), F. equiseti D25-1(GenBank Accession No.QOHM01000001.1), F. solani JS-169 (GenBank Accession No. NGZQ01000001.1), F.incarnatum MOD1-FUNGI18 (GenBank Accession No. RBBZ01000100.1) and F.asiaticum KCTC 16664 (GenBank Accession No. CP088257.1)的全基因组序列,对所有全基因组序列进行多基因组比对分析,确定了一个21bp碱基序列,该序列位于编码氨甲酰磷酸合成酶的基因上,以此序列作为短柄镰刀菌PCR反应的上游特异性引物(Fb-F),并在上游引物序列后设计特异下游引物(Fb-R)。在NCBI数据库中对设计的特异上下游引物进行比对, 初步鉴定引物序列的特异性,最终获得短柄镰刀菌PCR的特异性检测引物,引物由上海生工生物有限公司合成:
上游引物:
Fb-F(SEQ ID NO.1): CAATTGCTGCCACTCGACCTG;
下游引物
Fb-R(SEQ ID NO.2):TATTGTGGTGAGGAGGAGTCG。
2、病原菌基因组DNA的提取
根据TIANGEN的植物基因组提取试剂盒说明书进行提取。
3、短柄镰刀菌的PCR分子检测
以样品DNA为模板进行PCR扩增。PCR反应体系总体积25ul,包括:2.5μL 10×PCR缓冲液,1.5μL Mg2+(浓度25mM),2μL dNTP(浓度10mM),0.125ul Taq DNA聚合酶(5U/μL),上游引物Fb-F 1μL(浓度10μM)和下游引物Fb-R 1μL(浓度10μM),短柄镰刀菌DNA模板1μL,ddH2O15.875μL。PCR反应程序为:94℃预变性5min;进入循环,94℃变性30s,60℃退火30s,72℃延伸30s,35个循环;最后延伸72℃10min;完成扩增,扩增产物在4℃保存。取5μL PCR产物在1%m/v琼脂糖凝胶中电泳,经溴化乙锭染色后在紫外光下观测扩增产物大小,扩增得到283bpDNA条带为短柄镰刀菌。
实施例2
短柄镰刀菌PCR检测引物的特异性验证
1、待测样品DNA提取
采用上述方法提取短柄镰刀菌,尖孢镰刀菌,轮枝镰刀菌,层出镰刀菌,禾谷镰刀菌,假禾谷镰刀菌,腐皮镰刀菌,变红镰刀菌,木贼镰刀菌,亚洲镰刀菌,藤仓镰刀菌,锐顶镰刀菌,交互链格孢菌,齐整小核菌,核盘菌,禾谷丝核菌,桃拟茎点霉,茶拟盘多毛孢,大丽轮枝菌,葡萄座腔菌,灰葡萄孢,瓜蔓枯病菌,瓜类炭疽病菌的DNA;
2、特异性检测
分别以上述供试真菌的基因组DNA为模板进行PCR扩增。PCR反应体系总体积25ul,包括:2.5μL 10×PCR缓冲液,1.5μL Mg2+(浓度25mM),2μL dNTP(浓度10mM),0.125ul TaqDNA聚合酶(5U/μL),上游引物Fb-F 1μL(浓度10μM)和下游引物Fb-R 1μL(浓度10μM),短柄镰刀菌DNA模板1μL,ddH2O 15.875μL。PCR反应程序为:94℃预变性5min;进入循环,94℃变性30s,60℃退火30s,72℃延伸30s,35个循环;最后延伸72℃10min;完成扩增,扩增产物在4℃保存。取5μL PCR产物在1%m/v琼脂糖凝胶中电泳,经溴化乙锭染色后在紫外光下观测扩增产物大小。如图1所示,多重PCR只能特异性的扩增得到283bpDNA条带的短柄镰刀菌,而其他菌株均没有扩增条带。
实施例3
短柄镰刀菌PCR检测引物的灵敏性验证
1、待测样品DNA提取
采用上述方法提取短柄镰刀菌DNA。
2、灵敏性检测
使用NanoDrop超微量分光光度计测定上述基因组DNA的浓度,分别用ddH2O依次梯度稀释至10ng/μL、1ng/μL、100pg/μL、10pg/μL、1pg/μL、100fg/μL和10fg/μL。分别以上述不同浓度的DNA为模板进行PCR扩增。PCR反应体系总体积25ul,包括:2.5μL 10×PCR缓冲液,1.5μL Mg2+(浓度25mM),2μL dNTP(浓度10mM),0.125ul Taq DNA聚合酶(5U/μL),上游引物Fb-F 1μL(浓度10μM)和下游引物Fb-R 1μL(浓度10μM),短柄镰刀菌DNA模板1μL,ddH2O15.875μL。PCR反应程序为:94℃预变性5min;进入循环,94℃变性30s,60℃退火30s,72℃延伸30s,35个循环;最后延伸72℃10min;完成扩增,扩增产物在4℃保存。取5μL PCR产物在1%m/v琼脂糖凝胶中电泳,经溴化乙锭染色后在紫外光下观测扩增产物大小。如图2所示,灵敏性验证结果显示多重PCR检测短柄镰刀菌的DNA浓度的最低限制为10pg/μL。
实施例4
田间番茄样品的实际检测
1、待测植物材料总DNA的提取
取番茄根系约0 .5g,在液氮中充分研磨成粉末,然后根据TIANGEN的植物基因组提取试剂盒说明书进行提取。
2、田间番茄样品的病原菌检测
以上述番茄样品DNA为模板进行PCR扩增。PCR反应体系总体积25ul,包括:2.5μL10×PCR缓冲液,1.5μL Mg2+(浓度25mM),2μL dNTP(浓度10mM),0.125ul Taq DNA聚合酶(5U/μL),上游引物Fb-F 1μL(浓度10μM)和下游引物Fb-R 1μL(浓度10μM),短柄镰刀菌DNA模板1μL,ddH2O 15.875μL。PCR反应程序为:94℃预变性5min;进入循环,94℃变性30s,60℃退火30s,72℃延伸30s,35个循环;最后延伸72℃10min;完成扩增,扩增产物在4℃保存。取5μL PCR产物在1%m/v琼脂糖凝胶中电泳,经溴化乙锭染色后在紫外光下观测扩增产物大小。如图3所示,扩增得到283bpDNA条带为短柄镰刀菌,22份田间番茄样品中分别有14份被鉴定出短柄镰刀菌。
以上所述的实施例仅是对本发明的优选方式进行描述,并非对本发明的范围进行限定,在不脱离本发明设计精神的前提下,本领域普通技术人员对本发明的技术方案做出的各种变形和改进,均应落入本发明权利要求书确定的保护范围内。
Claims (8)
1.一种用于短柄镰刀菌PCR检测的引物组,其特征在于,所述引物组包括上游引物和下游引物;所述上游引物的序列如SEQ ID NO.1所示;所述下游引物的序列如SEQ ID NO.2所示。
2.一种检测短柄镰刀菌的试剂,其特征在于,所述试剂含有权利要求1中所述的引物组。
3.一种检测短柄镰刀菌的试剂盒,其特征在于,所述试剂盒中含有权利要求1中所述的引物组或权利要求2中所述的试剂。
4.一种短柄镰刀菌的PCR鉴定方法,其特征在于,所述鉴定方法以样品总DNA为模板,利用权利要求1所述的引物组进行PCR扩增,反应结束后根据琼脂糖凝胶电泳判定结果。
5.根据权利要求4所述的PCR鉴定方法,其特征在于,PCR的反应程序为:94℃预变性5min;进入循环,94℃变性30s,60℃退火30s,72℃延伸30s,35个循环;最后延伸72℃10min。
6.根据权利要求5所述的PCR鉴定方法,其特征在于,PCR反应体系总体积25ul,其中包括:2.5μL 10×PCR缓冲液,1.5μL 25mM Mg2+,2μL 10mM dNTP,0.125ul 5U/μL Taq DNA聚合酶,浓度为10μM的上游引物SEQ ID NO.1 1μL,浓度为10μM的下游引物SEQ ID NO.2 1μL,DNA模板1μL,ddH2O 15.875μL。
7.根据权利要求6所述的PCR鉴定方法,其特征在于,所述DNA模板提取自番茄。
8.权利要求1所述的引物组或权利要求2所述的试剂或权利要求3所述的试剂盒在鉴定短柄镰刀菌中的应用。
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