CN116904620A - 一种基于mnp快速检测水稻病原菌的引物组及试剂盒和方法 - Google Patents
一种基于mnp快速检测水稻病原菌的引物组及试剂盒和方法 Download PDFInfo
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Abstract
本发明涉及水稻病原菌分子检测技术领域,公开了一种基于MNP快速检测水稻病原菌的引物组及试剂盒和方法,包括引物对A、引物对B、引物对C和引物对D,所述引物对A为xoo1~xoo20中的至少一对,引物对B为xoc1~xoc14中的至少一对,引物对C为Burkholderia_glumae1~Burkholderia_glumae20中的至少一对,引物对D为Pyricularia_oryzae 1~Pyricularia_oryzae 20中的至少一对;每对引物对均包括正向引物和反向引物;所有引物的碱基序列如SEQ ID No.1~148所示。本方案引物组可一次性的对水稻白叶枯病、谷枯病、水稻细菌性条斑病和稻瘟病的病原菌的多个MNP标记中的至少一个进行检测,根据获得的MNP标记的序列进行水稻病害病原物的定性鉴定、水稻病害病原物各亚种间的遗传变异鉴定、病原菌指纹数据库的构建。
Description
技术领域
本发明涉及水稻病原菌分子检测技术领域,具体涉及一种基于MNP快速检测水稻病原菌的引物组及试剂盒和方法。
背景技术
水稻在种植过程中,经常受到细菌和真菌病原物的危害,导致严重减产、产品质量下降,甚至有些真菌产生毒素,威胁人类身体健康和生命安全。具体的,水稻白叶枯病,谷枯病,水稻细菌性条斑病和稻瘟病是水稻的四大病害。这四种水稻病害的致病菌分别是水稻白叶枯病原菌(Xanthomonas oryzae pv.oryzae,Xoo)、水稻细菌性谷枯病菌(Burkholderia glumae)、稻生黄单胞杆菌稻细条斑致病变种(Xanthomonas oryzaepv.Oryzicola,Xoc)和稻瘟病菌(Pyricularia oryzae Cav.)。水稻发病后,一般秕粒增多,危害严重则影响抽穗灌浆,造成重大损失,一般减产15-25%,严重时可达40-60%;比如,据统计,稻瘟病每年可摧毀供超过6000万人食用的水稻。因此,准确检测水稻样品中的这四类病原菌对水稻种植过程中的病症防治至关重要。
现有水稻病原物的检测方法常需要对水稻样品依次进行分离培养、镜检、免疫学检测或核酸检测,再对每种病原物进行特异性检测,在检测周期、准确性、灵敏度、种/亚种间差异检测等方面存在一个或多个局限。因此,研发一种检测周期短、一次性可检测同一宿主内的多种病原菌、灵敏度高、准确性好的特异性检测技术,不仅能有效弥补现有水稻病原菌检测技术的不足,还能在水稻周期性的病症筛查或出现病症后快速检测水稻样品中病原菌种类,从而便于对水稻病害进行快速筛查和响应,对水稻种植过程中的病害防治具有重要意义。
发明内容
本发明意在提供一种基于MNP快速检测水稻病原菌的引物组及试剂盒和方法,以解决现有技术中水稻样本病原菌的检测过程复杂且准确率低的技术问题。
为达到上述目的,本发明采用如下技术方案:一种基于MNP快速检测水稻病原菌的引物组,包括引物对A、引物对B、引物对C和引物对D,所述引物对A为xoo1~xoo20中的至少一对,引物对B为xoc1~xoc14中的至少一对,引物对C为Burkholderia_glumae1~Burkholderia_glumae20中的至少一对,引物对D为Pyricularia_oryzae 1~Pyricularia_oryzae 20中的至少一对;每对引物对均包括正向引物和反向引物;所述xoo1~xoo20的碱基序列如SEQ ID No.1~40所示,所述xoc1~xoc14的碱基序列如SEQ ID No.41~68所示,所述Burkholderia_glumae1~Burkholderia_glumae20的碱基序列如SEQ IDNo.69~108所示,所述Pyricularia_oryzae 1~Pyricularia_oryzae 20的碱基序列如SEQ ID No.109~148所示。
本方案的原理及优点是:
本方案通过将水稻中常见病症的病原菌的特异性核苷酸引物对混合在一起形成引物组,并利用该引物组对水稻白叶枯病、谷枯病、条斑病和稻瘟病的病原菌的多个MNP标记中的至少一个进行检测,根据获得的MNP标记的序列进行水稻病害病原物的定性鉴定、水稻病害病原物各亚种间的遗传变异鉴定、病原菌指纹数据库的构建;一方面,可一次性对水稻样品中的四种病原菌同时进行定性检测,从而实现水稻病症对应病原菌的快速筛查;另一方面,还能鉴定出水稻病害病原菌各亚种间的遗传变异,从而便于针对不同病原菌亚种引发的病症进行针对性给药防治水稻病害,进而防止病原菌的大面积侵染。
优选的,所述引物组包括xoo1~xoo20、xoc1~xoc14、Burkholderia_glumae1~Burkholderia_glumae20和Pyricularia_oryzae 1~Pyricularia_oryzae 20,每条引物的浓度为0.4μM。
有益效果:本方案将4种病原菌的多对引物混合在一起,可进一步通过检测位点数推测水稻样品中不同病原菌的浓度,从而针对病原菌的不同感染程度针对性给药,进一步实现水稻病害防治的精确性,降低水稻病害防治成本。
优选的,一种基于MNP快速检测水稻病原菌的试剂盒,包括上述引物组。
优选的,还包括多重PCR预混液。
优选的,一种基于MNP快速检测水稻病原菌的方法,包括如下步骤:
S1、样本制备:从水稻样本中分离得到水稻病原菌或制备水稻样本;
S2、DNA提取:提取上述水稻病原菌或水稻样本的DNA,获得水稻病原菌DNA;
S3、PCR扩增:利用上述试剂盒扩增S2所得水稻病原菌DNA的特异性核苷酸片段,获得扩增体系;
S4、高通量测序:对S3所得扩增体系进行测序,获得样本的引物标记位点种类和数目,根据检测所得引物标记种类判断待测样品中的水稻病原菌种类;根据检测所得引物标记数目判断待测样品中水稻;
所述水稻病原菌为:水稻白叶枯病原菌(X.oryzae pv.oryzae,Xoo)、水稻细菌性谷枯病菌(B.glumae)、稻生黄单胞杆菌稻细条斑致病变种(X.oryzaepv.Oryzicola,Xoc)和稻瘟病菌(P.oryzae Cav.)。
优选的,利用水稻病原菌DNA进行PCR扩增时,扩增体系中水稻病原菌DNA的浓度不低于0.05ng/μL。
优选的,所述水稻样本的制备方法为:采集水稻样本,经无菌水冲洗、晾干后,进行液氮研磨,获得所述水稻样本。
优选的,所述水稻样本为水稻谷粒、叶鞘、叶片、茎秆、穗颈或小穗梗中的任意一种或多种组合。
本方案通过将水稻中常见病症的病原菌的特异性核苷酸引物对混合在一起形成引物组,每条引物的终浓度为0.4μM,并利用该引物组对水稻白叶枯病、谷枯病、条斑病和稻瘟病的病原菌的多个MNP标记中的至少一个进行检测,根据获得的MNP标记的序列进行水稻病害病原物的定性鉴定、水稻病害病原物各亚种间的遗传变异鉴定、病原菌指纹数据库的构建;一方面,可一次性对水稻样品中的四种病原菌同时进行定性检测,从而实现水稻病症对应病原菌的快速筛查;另一方面,还能鉴定出水稻病害病原菌各亚种间的遗传变异,从而便于针对不同病原菌亚种引发的病症进行针对性给药防治水稻病害,进而防止病原菌的大面积侵染。
具体实施方式
下面通过具体实施方式进一步详细说明,但本发明的实施方式不限于此。若未特别指明,下述实施例所用的技术手段为本领域技术人员所熟知的常规手段;所用的实验方法均为常规方法;所用的材料、试剂等,均可从商业途径得到。
本发明实施例使用的水稻白叶枯病原菌、水稻细菌性谷枯病菌ATCC33617和稻生黄单胞杆菌稻细条斑致病变种来源于中国检验检疫科学研究院植检所,稻瘟病菌为中国农业大学赠予。
实施例
本发明提供了一种基于MNP快速检测水稻病原菌的引物组,包括引物对A、引物对B、引物对C和引物对D,引物对A为xoo1~xoo20中的至少一对,引物对B为xoc1~xoc14中的至少一对,引物对C为Burkholderia_glumae1~Burkholderia_glumae20中的至少一对,引物对D为Pyricularia_oryzae 1~Pyricularia_oryzae 20中的至少一对;本方案的引物组包含xoo1~xoo20、xoc1~xoc14、Burkholderia_glumae1~Burkholderia_glumae20和Pyricularia_oryzae 1~Pyricularia_oryzae 20中的全部;每对引物对均包括正向引物和反向引物;xoo1~xoo20的碱基序列如SEQ ID No.1~40所示,xoc1~xoc14的碱基序列如SEQ ID No.41~68所示,Burkholderia_glumae1~Burkholderia_glumae20的碱基序列如SEQ ID No.69~108所示,Pyricularia_oryzae 1~Pyricularia_oryzae 20的碱基序列如SEQ IDNo.109~148所示。
本方案74对引物的筛选方法如下:
基于NCBI的水稻白叶枯病菌X.oryzae pv.oryzae(Xoo),条斑病菌(X.oryzaepv.Oryzicola,Xoc)、谷枯病菌B._glumae和稻瘟病菌P._oryzae不同分离株的基因组完整或部分序列,通过序列比对,利用四种菌的参考基因组数据和全基因组测序数据,遵守扩增区多态性高、引物区保守、扩增区尽量选择单拷贝区域等引物筛选的通用原则;引物在参考基因组上的扩增长度不超过250bp等其它特殊要求,一共筛选到74个MNP标记位点,其中白叶枯病菌Xoo的MNP标记位点数量20个,条斑病菌Xoc的MNP标记位点数量14个,谷枯病菌Burkholderia_glumae的MNP标记位点数量20个,稻瘟病菌Pyricularia_oryzae的MNP标记位点数量20个,所得引物的碱基序列如表1所示。
表1MNP标记引物序列
本发明实施例提供了一种基于MNP快速检测水稻病原菌的试剂盒,该试剂盒包括上述引物组和多重PCR预混液。
本发明实施例还提供了一种基于MNP快速检测水稻病原菌的方法,包括如下步骤:
S1、样本制备:从水稻样本中分离得到水稻病原菌,或制备水稻样本;
水稻样本为水稻谷粒、叶鞘或叶片中的任意一种;水稻样本的制备方法为:采集水稻样本,经无菌水冲洗、晾干后,进行液氮研磨,获得水稻样本。
S2、DNA提取:提取上述水稻病原菌或水稻样本的DNA,获得水稻病原菌DNA;
S3、PCR扩增:利用权利要求4的试剂盒扩增S2所得水稻病原菌DNA的特异性核苷酸片段,获得扩增体系;利用水稻病原菌DNA进行PCR扩增时,扩增体系中水稻病原菌DNA的浓度不低于0.05ng/μL。
S4、高通量测序:对S3所得扩增体系进行测序,获得样本的引物标记位点种类和数目,根据检测所得引物标记种类判断待测样品中的水稻病原菌种类;根据检测所得引物标记数目判断待测样品中水稻;
水稻病原菌为:水稻白叶枯病原菌(X.oryzae pv.oryzae,Xoo)、水稻细菌性谷枯病菌(B.glumae)、稻生黄单胞杆菌稻细条斑致病变种(X.oryzaepv.Oryzicola,Xoc)和稻瘟病菌(P.oryzae Cav.)。
实验例1:水稻样本中白叶枯病原菌的检测
从3个不同来源的水稻样品(具体为水稻叶片或叶鞘)里分离出3个水稻白叶枯X.oryzae pv.oryzae(Xoo)菌株,用细菌基因组提取试剂盒(DP302,天根生化科技有限公司)提取DNA,得到3个试验样本BLL110、JXO111和LSL122,每个样本用表1列的引物构建3个文库。
文库构建时采用本发明实施例提供的试剂盒对样本进行扩增,得到扩增产物,将该扩增产物连接上购买的商业化样本标签,获得适用于高通量测序的文库,文库按照等摩尔量进行混合,混合后进行高通量测序,获得测序数据。对于每一个样品的测序数据分析,首先利用Bowtie2(version 2.1.0)将测序片段比对到每一种病原物的参考基因组上,若该测序片段与参考序列相同,则认为该参考序列对应的病原菌MNP标记检出,最后统计检测到的每一个病原物MNP标记的数目。结果分析具体如表2所示。
表2试剂盒鉴定水稻白叶枯饼菌的分析结果
如表2所示,对病症为白叶枯病的水稻样本BLL110、JXO111和LSL122,本方案试剂盒均能稳定的检测出水稻白叶枯病原菌Xoo的20个标记位点,说明本方案试剂盒能对样品中水稻白叶枯病菌进行准确鉴定。
实验例2:水稻样本中水稻细菌性条斑病菌的检测
从出现水稻条斑病病症的2个水稻样本(具体为水稻叶片)里分离出病原菌,用细菌基因组提取试剂盒提取DNA,得到2个试验样本CYC601和YNB0-1,用表1中引物构建文库并进行二代测序,测序结果具体如表3所示。
表3试剂盒鉴定水稻病原菌的分析结果
如表3所示,对病症为水稻细菌性条斑病的水稻样本CYC601和YNB0-1,本方案试剂盒均检测获得水稻细菌性条斑病菌Xoc,说明本方案试剂盒能对样品中水稻细菌性条斑病菌进行准确鉴定,且水稻样本CYC601中还包含谷枯病菌Burkholderia_glumae。具体的,本方案试剂盒能准确鉴定样品CYC601和YNB0-1的水稻水稻细菌性条斑病菌Xoc的13个标记位点。而水稻样本YNB0-1中还侵染了少量的谷枯病菌,具体能检测出谷枯病菌Burkholderia_glumae的4个标记位点。
实验例3:水稻样本中谷枯病菌的检测
在水稻细菌性谷枯病菌ATCC33617中加入5%稻白叶枯病原菌和1%稻生黄单胞杆菌稻细水稻细菌性条斑病致病变种,用于验证MNP标记法的特异性;同时挑选2个发病水稻的病灶组织(具体为水稻叶鞘)。用细菌基因组提取试剂盒对上述3个样本提取DNA,得到3个试验样本(ATCC33617、Slu-10和Slu-7),用表1中引物构建文库并进行二代测序,测序结果具体如表4所示。
表4试剂盒鉴定水稻病原菌的分析结果
如表4所示,对病症为谷枯病的水稻样本Slu-10和Slu-7,本方案试剂盒均检测获得稻瘟菌Pyricularia_oryzae和水稻白叶枯病菌xoo,说明本方案试剂盒能对样品中稻瘟菌和水稻白叶枯病菌进行准确鉴定,且水稻样本Slu-10和Slu-7均包含这两种病原菌。具体的,本方案试剂盒能鉴定出谷枯病菌Burkholderia_glumae的11个MNP标记位点和水稻白叶枯病菌xoo的3个MNP标记位点;水稻样本Sul-7与Slu-10来源于同一块田的水稻,鉴定结果显示水稻样本Sul-7可鉴定出白叶枯病菌xoo的19个MNP标记位点和谷枯病菌Burkholderia_glumae的11个MNP标记位点。而样品Sul-7与Slu-10中水稻白叶枯病菌和谷枯病菌检出标记数目的差异源于水稻样品中病原菌侵染浓度的差异。
另外,混有其它细菌DNA的样品ATCC33617,可鉴定出白叶枯病菌xoo的10个MNP标记位点、谷枯病菌Burkholderia_glumae的9个位点,以及细菌性条斑病菌Xoc的1个位点,检测出1个位点的原因可能是细菌性条斑病菌Xoc的含量较低引起的。
实验例4:水稻样本中稻瘟病菌的检测
从3株不同来源的水稻组织(叶片、茎秆、穗颈、小穗梗和谷粒)中分离、纯化得到稻瘟病菌株FJNP-8、GDQJ和HNSD;另取2个发病水稻样本LNPJ和YNGN,共计5个待测水稻样本(详见表5)。用植物基因组提取试剂盒提取DNA,得到5个试验样本,用表1中引物构建文库并进行二代测序,测序结果具体如表5所示。
表5试剂盒鉴定水稻病原菌的分析结果
如表5所示,对稻瘟病菌株FJNP-8、GDQJ和HNSD,本方案试剂盒均检测获得稻瘟菌Pyricularia_oryzae的20个MNP位点,说明本方案试剂盒能对样品中稻瘟菌进行准确鉴定。
而水稻样本LNPJ提取所得DNA,能检出稻瘟菌Pyricularia_oryzae的20个MNP位点、水稻白叶枯病菌Xoo的17个位点、水稻细菌性条斑病菌xoc的10个位点以及谷枯病菌Burkholderia_glumae的6个位点,说明水稻样本LNPJ中已侵染4种病原菌,应尽快给药处理;且因白叶枯病菌Xoo、水稻细菌性条斑病病菌xoc和谷枯病菌Burkholderia_glumae的检出位点较多,给药时应针对性加重给药浓度。
另外,从水稻样本YNGN提取出来的DNA,检出稻瘟菌Pyricularia_oryzae的20个MNP位点,水稻白叶枯病菌Xoo的14个位点,谷枯病菌Burkholderia_glumae的1个位点,说明水稻样本YNGN已感染这3种病菌,应尽快给药处理;且因白叶枯病菌Xoo和稻瘟菌Pyricularia_oryzae的检出位点较多,给药时应针对性加重给药浓度对其进行灭杀治疗;而谷枯病菌Burkholderia_glumae的检出位点较少,给药时应降低给药浓度对其进行预防,实现预防和治疗的结合。
以上所述的仅是本发明的实施例,方案中公知的具体技术方案和/或特性等常识在此未作过多描述。应当指出,对于本领域的技术人员来说,在不脱离本发明技术方案的前提下,还可以作出若干变形和改进,这些也应该视为本发明的保护范围,这些都不会影响本发明实施的效果和专利的实用性。本申请要求的保护范围应当以其权利要求的内容为准,说明书中的具体实施方式等记载可以用于解释权利要求的内容。
Claims (8)
1.一种基于MNP快速检测水稻病原菌的引物组,其特征在于:包括引物对A、引物对B、引物对C和引物对D,所述引物对A为xoo1~xoo20中的至少一对,引物对B为xoc1~xoc14中的至少一对,引物对C为Burkholderia_glumae1~Burkholderia_glumae20中的至少一对,引物对D为Pyricularia_oryzae 1~Pyricularia_oryzae 20中的至少一对;每对引物对均包括正向引物和反向引物;所述xoo1~xoo20的碱基序列如SEQ ID No.1~40所示,所述xoc1~xoc14的碱基序列如SEQ ID No.41~68所示,所述Burkholderia_glumae1~Burkholderia_glumae20的碱基序列如SEQ ID No.69~108所示,所述Pyricularia_oryzae1~Pyricularia_oryzae 20的碱基序列如SEQ ID No.109~148所示。
2.根据权利要求1所述的一种基于MNP快速检测水稻病原菌的引物组,其特征在于:所述引物组包括xoo1~xoo20、xoc1~xoc14、Burkholderia_glumae1~Burkholderia_glumae20和Pyricularia_oryzae 1~Pyricularia_oryzae 20,每条引物的浓度为0.4μM。
3.一种基于MNP快速检测水稻病原菌的试剂盒,其特征在于:包括如权利要求1或2任一项所述的引物组。
4.根据权利要求3所述的一种基于MNP快速检测水稻病原菌的试剂盒,其特征在于:还包括多重PCR预混液。
5.一种基于MNP快速检测水稻病原菌的方法,其特征在于:包括如下步骤:
S1、样本制备:从水稻样本中分离得到水稻病原菌,或制备水稻样本;
S2、DNA提取:提取上述水稻病原菌或水稻样本的DNA,获得水稻病原菌DNA;
S3、PCR扩增:利用权利要求4所述的试剂盒扩增S2所得水稻病原菌DNA的特异性核苷酸片段,获得扩增体系;
S4、高通量测序:对S3所得扩增体系进行测序,获得样本的引物标记位点种类和数目,根据检测所得引物标记种类判断待测样品中的水稻病原菌种类;根据检测所得引物标记数目判断待测样品中水稻;
所述水稻病原菌为:水稻白叶枯病原菌(X.oryzae pv.oryzae,Xoo)、水稻细菌性谷枯病菌(B.glumae)、稻生黄单胞杆菌稻细条斑致病变种(X.oryzae pv.Oryzicola,Xoc)和稻瘟病菌(P.oryzae Cav.)。
6.根据权利要求5所述的一种基于MNP快速检测水稻病原菌的方法,其特征在于:利用水稻病原菌DNA进行PCR扩增时,扩增体系中水稻病原菌DNA的浓度不低于0.05ng/μL。
7.根据权利要求6所述的一种基于MNP快速检测水稻病原菌的方法,其特征在于:所述水稻样本的制备方法为:采集水稻样本,经无菌水冲洗、晾干后,进行液氮研磨,获得所述水稻样本。
8.根据权利要求7所述的一种基于MNP快速检测水稻病原菌的方法,其特征在于:所述水稻样本为水稻谷粒、叶鞘、叶片、茎秆、穗颈或小穗梗中的任意一种或多种组合。
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