CN103571946B - 大豆疫霉特异性的分子检测引物及其应用 - Google Patents
大豆疫霉特异性的分子检测引物及其应用 Download PDFInfo
- Publication number
- CN103571946B CN103571946B CN201310464140.XA CN201310464140A CN103571946B CN 103571946 B CN103571946 B CN 103571946B CN 201310464140 A CN201310464140 A CN 201310464140A CN 103571946 B CN103571946 B CN 103571946B
- Authority
- CN
- China
- Prior art keywords
- phytophthora
- soybean
- soybean phytophthora
- primer
- molecular detection
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Expired - Fee Related
Links
- 244000068988 Glycine max Species 0.000 title claims abstract description 123
- 235000010469 Glycine max Nutrition 0.000 title claims abstract description 123
- 241000233614 Phytophthora Species 0.000 title claims abstract description 104
- 238000001514 detection method Methods 0.000 title claims abstract description 47
- -1 pplication Species 0.000 title description 3
- 201000010099 disease Diseases 0.000 claims abstract description 39
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 claims abstract description 39
- 238000000034 method Methods 0.000 claims abstract description 16
- 108010005730 R-SNARE Proteins Proteins 0.000 claims abstract description 4
- 102000005917 R-SNARE Proteins Human genes 0.000 claims abstract description 4
- 238000012408 PCR amplification Methods 0.000 claims description 23
- 241000196324 Embryophyta Species 0.000 claims description 19
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 14
- 238000004519 manufacturing process Methods 0.000 claims description 11
- 108090000623 proteins and genes Proteins 0.000 claims description 7
- 239000000126 substance Substances 0.000 claims description 4
- 244000005700 microbiome Species 0.000 claims description 3
- 238000011144 upstream manufacturing Methods 0.000 claims description 3
- 238000000246 agarose gel electrophoresis Methods 0.000 claims description 2
- 239000002689 soil Substances 0.000 abstract description 20
- 101150016810 YKT6 gene Proteins 0.000 abstract description 14
- 238000011529 RT qPCR Methods 0.000 abstract description 8
- 230000035945 sensitivity Effects 0.000 abstract description 7
- 238000013461 design Methods 0.000 abstract description 4
- 230000004083 survival effect Effects 0.000 abstract description 3
- 238000012417 linear regression Methods 0.000 abstract description 2
- 108020004414 DNA Proteins 0.000 description 24
- 238000003752 polymerase chain reaction Methods 0.000 description 17
- 238000001962 electrophoresis Methods 0.000 description 10
- 230000003321 amplification Effects 0.000 description 9
- 239000013642 negative control Substances 0.000 description 8
- 238000003199 nucleic acid amplification method Methods 0.000 description 8
- 238000006243 chemical reaction Methods 0.000 description 6
- 239000000047 product Substances 0.000 description 6
- 230000001580 bacterial effect Effects 0.000 description 5
- 239000003153 chemical reaction reagent Substances 0.000 description 5
- 239000013641 positive control Substances 0.000 description 5
- 239000000243 solution Substances 0.000 description 5
- 238000000605 extraction Methods 0.000 description 4
- 244000052769 pathogen Species 0.000 description 4
- 230000001717 pathogenic effect Effects 0.000 description 4
- 241000894007 species Species 0.000 description 4
- 244000052616 bacterial pathogen Species 0.000 description 3
- 238000001502 gel electrophoresis Methods 0.000 description 3
- 238000011081 inoculation Methods 0.000 description 3
- 230000000877 morphologic effect Effects 0.000 description 3
- 239000006228 supernatant Substances 0.000 description 3
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 2
- 241000723346 Cinnamomum camphora Species 0.000 description 2
- 241000948155 Phytophthora sojae Species 0.000 description 2
- 241000233639 Pythium Species 0.000 description 2
- 238000011161 development Methods 0.000 description 2
- 239000012895 dilution Substances 0.000 description 2
- 238000010790 dilution Methods 0.000 description 2
- 238000000227 grinding Methods 0.000 description 2
- 239000000463 material Substances 0.000 description 2
- 238000003753 real-time PCR Methods 0.000 description 2
- 238000000926 separation method Methods 0.000 description 2
- 238000011895 specific detection Methods 0.000 description 2
- 206010003645 Atopy Diseases 0.000 description 1
- 241000894006 Bacteria Species 0.000 description 1
- 240000008564 Boehmeria nivea Species 0.000 description 1
- 101150018198 COX1 gene Proteins 0.000 description 1
- 101150071146 COX2 gene Proteins 0.000 description 1
- LZZYPRNAOMGNLH-UHFFFAOYSA-M Cetrimonium bromide Chemical compound [Br-].CCCCCCCCCCCCCCCC[N+](C)(C)C LZZYPRNAOMGNLH-UHFFFAOYSA-M 0.000 description 1
- 241000222201 Colletotrichum capsici Species 0.000 description 1
- 241000195493 Cryptophyta Species 0.000 description 1
- 244000241257 Cucumis melo Species 0.000 description 1
- 235000015510 Cucumis melo subsp melo Nutrition 0.000 description 1
- 238000007400 DNA extraction Methods 0.000 description 1
- 101100275424 Danio rerio mt-co1 gene Proteins 0.000 description 1
- 101100275428 Dictyostelium discoideum cox1/2 gene Proteins 0.000 description 1
- 101710114192 Elicitin Proteins 0.000 description 1
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 1
- 241000233866 Fungi Species 0.000 description 1
- 241000223195 Fusarium graminearum Species 0.000 description 1
- 241000223221 Fusarium oxysporum Species 0.000 description 1
- 101000828633 Homo sapiens Synaptobrevin homolog YKT6 Proteins 0.000 description 1
- 241001330975 Magnaporthe oryzae Species 0.000 description 1
- 241001465754 Metazoa Species 0.000 description 1
- 241001149949 Phytophthora cactorum Species 0.000 description 1
- 241000233616 Phytophthora capsici Species 0.000 description 1
- 241000233618 Phytophthora cinnamomi Species 0.000 description 1
- 241000233633 Phytophthora drechsleri Species 0.000 description 1
- 241000233622 Phytophthora infestans Species 0.000 description 1
- 241000233645 Phytophthora nicotianae Species 0.000 description 1
- 241000233629 Phytophthora parasitica Species 0.000 description 1
- 241000370518 Phytophthora ramorum Species 0.000 description 1
- 108020005120 Plant DNA Proteins 0.000 description 1
- 239000006004 Quartz sand Substances 0.000 description 1
- 206010039509 Scab Diseases 0.000 description 1
- 241000221696 Sclerotinia sclerotiorum Species 0.000 description 1
- 229920002684 Sepharose Polymers 0.000 description 1
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 description 1
- 102100023512 Synaptobrevin homolog YKT6 Human genes 0.000 description 1
- 108010006785 Taq Polymerase Proteins 0.000 description 1
- 241000219793 Trifolium Species 0.000 description 1
- FJJCIZWZNKZHII-UHFFFAOYSA-N [4,6-bis(cyanoamino)-1,3,5-triazin-2-yl]cyanamide Chemical compound N#CNC1=NC(NC#N)=NC(NC#N)=N1 FJJCIZWZNKZHII-UHFFFAOYSA-N 0.000 description 1
- 125000003275 alpha amino acid group Chemical group 0.000 description 1
- 239000007900 aqueous suspension Substances 0.000 description 1
- 239000003795 chemical substances by application Substances 0.000 description 1
- 230000009089 cytolysis Effects 0.000 description 1
- 238000004925 denaturation Methods 0.000 description 1
- 230000036425 denaturation Effects 0.000 description 1
- 230000000249 desinfective effect Effects 0.000 description 1
- 238000003745 diagnosis Methods 0.000 description 1
- 238000009792 diffusion process Methods 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 230000006353 environmental stress Effects 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 230000002538 fungal effect Effects 0.000 description 1
- 238000003306 harvesting Methods 0.000 description 1
- 230000008676 import Effects 0.000 description 1
- 208000015181 infectious disease Diseases 0.000 description 1
- 238000011835 investigation Methods 0.000 description 1
- 239000007788 liquid Substances 0.000 description 1
- 238000002156 mixing Methods 0.000 description 1
- 229910052757 nitrogen Inorganic materials 0.000 description 1
- 239000002773 nucleotide Substances 0.000 description 1
- 125000003729 nucleotide group Chemical group 0.000 description 1
- 239000000843 powder Substances 0.000 description 1
- 238000001556 precipitation Methods 0.000 description 1
- 238000012797 qualification Methods 0.000 description 1
- 238000002864 sequence alignment Methods 0.000 description 1
- 238000002791 soaking Methods 0.000 description 1
- 125000006850 spacer group Chemical group 0.000 description 1
- 238000003892 spreading Methods 0.000 description 1
- 238000003860 storage Methods 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
- 238000012795 verification Methods 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
- C12Q1/04—Determining presence or kind of microorganism; Use of selective media for testing antibiotics or bacteriocides; Compositions containing a chemical indicator therefor
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6844—Nucleic acid amplification reactions
- C12Q1/6851—Quantitative amplification
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6844—Nucleic acid amplification reactions
- C12Q1/686—Polymerase chain reaction [PCR]
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q2545/00—Reactions characterised by their quantitative nature
- C12Q2545/10—Reactions characterised by their quantitative nature the purpose being quantitative analysis
- C12Q2545/114—Reactions characterised by their quantitative nature the purpose being quantitative analysis involving a quantitation step
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q2561/00—Nucleic acid detection characterised by assay method
- C12Q2561/113—Real time assay
Landscapes
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Organic Chemistry (AREA)
- Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Biophysics (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Immunology (AREA)
- Microbiology (AREA)
- Molecular Biology (AREA)
- Analytical Chemistry (AREA)
- Physics & Mathematics (AREA)
- Biotechnology (AREA)
- Biochemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Toxicology (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
本发明以一个细胞存活必须的R-SNARE蛋白编码基因Ykt6为靶标,设计出大豆疫霉特异性的分子检测引物,该引物具有较高的灵敏度,与Ykt6疫霉属通用引物Ykt6F/Ykt6R配套使用,采用套式PCR的方法,可以将灵敏度提高1000倍,足以检测出微量大豆疫霉,该方法特异性和灵敏度高,可对大豆疫霉菌丝、卵孢子和游动孢子进行检测。同时,利用大豆疫霉特异性开发出实时定量PCR的检测方法及线性回归方程,可以准确、定量的检测大豆中侵染的大豆疫霉菌的量,检测出土壤中残存的大豆疫霉卵孢子的含量,为病害的预测预报提供有力数据,为进出口检疫等提供技术支持。
Description
技术领域
本发明属于生物技术领域。具体涉及一种用于大豆疫霉特异性的分子检测引物及其应用。
背景技术
大豆是世界上重要经济农作物,大豆疫霉(Phytophthora
sojae)侵染大豆而引起幼苗猝死和成株的根腐病是世界大豆生产上的主要病害之一,每年给世界大豆产业带来巨额经济损失。大豆疫霉菌可以在大豆生长的各个时期侵染大豆,在潮湿多雨的季节病害发生尤其严重,由于大豆疫霉以同宗配合的方式进行有性生殖,在发病植株上可以产生大量的卵孢子,植物收获后残留在土壤中的卵孢子就成为大豆疫霉的初侵染源,据报道大豆疫霉的卵孢子可以在土壤中存活5年以上。因此大豆疫霉根腐病一旦发生将很难消除。大豆疫霉是我国对外公布的A1类进境植物检疫对象。1948年,美国的印第安那州首次发现了该病原菌的存在,并与1955年公开报道,该病害在美国、巴西等世界大豆主要生产国家和地区均发生严重,每年给全球大豆生产造成的直接经济损失达到10-20亿美元。
1989年沈崇尧等首次在我国东北地区发现了大豆疫霉根腐病的存在,
1993年苏彦纯等首次报道了我国黑龙江大豆主产区发生大豆疫霉根腐病,此后在我国北方的大豆主产区陆续发现该病害的发生,近年在以生产鲜食毛豆为主的福建大豆产区也发生了大豆疫霉根腐病。黑龙江省植保站1997、1998两年对黑龙江省大豆疫病发生情况进行普查,结果表明仅黑龙江省发病面积已超过 30万公顷。由此可见,该病害已经对我国的大豆生产产业带来巨大危害,是一个急需解决的问题。
近年来,随着我国大豆需求量的加大,从美国等国家进口的商品大豆日趋增多,每年进口大豆的数量几乎超过了国产大豆年生产量的总和,而这些国家大豆疫霉病害的发生普遍较严重。由于大豆疫霉是典型的土传病害,卵孢子能在土壤中长期生存,而进境大豆中所夹带的土壤极有可能携带大豆疫霉病菌,增加了大豆疫病传播扩散的风险,这种严峻的形势下,快速准确地检测病原菌和诊断病害是控制病害成灾的主要措施之一。同时采用定量检测的方法,检测土壤中残存的病原物含量,可以为预报预测提供具体的数据。
大豆疫霉的传统检测方法是用大豆叶碟从土壤中进行诱捕和平板培养或者将二者相结合,传统方法在大豆疫霉检测及菌株分离中发挥了重要作用。但大豆疫霉病原菌与其他疫霉菌的形态特征非常相似,以形态特征为基础的传统检测方法需要20天左右才能确定进口大豆中是否带菌,而且该方法要求操作者具备专业的疫霉分离、形态学鉴定知识和丰富的经验,从而导致经常出现漏检,无法满足田间疫情调查和海关检疫的要求。时间长,技术要求高,导致效率低。
从聚合酶链式反应(PCR)发明以来,便以其快速灵敏的特性成为动植物病原物检测最重要的方法。伴随着各个病原物基因组测序的完善,很多特异性的核酸序列被用来作为PCR反应特异性靶标,可以更加准确地对病原物进行鉴定。发展分子检测最重要的一点就是选用合适的分子检测靶标。目前广泛采用的靶标是基于核糖体转录间隔区域(ITS)序列,ITS序列具有物种间高度变异和种内保守的特性,这使得ITS序列可以在物种的各个分类水平上进行区分。但是,伴随着现代分子生物学的快速发展,各个物种基因组序列的不断公布,发现很多不同种的ITS序列具有很高的相似性,使用ITS序列很难将这些种区分开。而且由于ITS在基因组中是多拷贝的,并不适合开发定量检测方法。因此必须开发出更多的分子检测靶标以满足植物病原菌分子检测的需要。随着疫霉基因组学的发展,不断有新的分子靶标被发现并用于分子检测。elicitin基因被用来作为疫霉菌检测的标靶,Ras家族相关的编码基因Ypt1被广泛用于疫霉菌的分子检测,线粒体编码基因Cox1、Cox2和可能编码储存蛋白的Lpv基因分别作为橡树疫霉(P. ramorum)和樟疫霉(P.
cinnamomi)检测的靶标。
本发明旨在寻找大豆疫霉中新的分子检测靶标基因,并利用该基因设计出大豆疫霉特异性的检测引物,开发出大豆疫霉实时定量PCR的检测方法。Ykt6p是一个细胞存活必须的R-SNARE蛋白,Ykt6在真核生物的进化上非常保守,将不同物种中的Ykt6氨基酸序列进行比对分析,结果表明,在系统进化上疫霉和植物及藻类在亲缘关系上比真菌更近,这与疫霉的分类地位完全符合。由此我们将Ykt6作为一个新的分子检测靶标,通过序列比对分析设计出了大豆疫霉特异性的分子检测引物,并在此基础上开发出了大豆疫霉实时定量PCR的检测方法。
发明内容
本发明提供了一种大豆疫霉特异性扩增引物及其大豆疫霉实时定量PCR的检测方法 。
本发明的技术方案为:
大豆疫霉特异性的分子检测引物,是以R-SNARE蛋白编码基因Ykt6为靶标,设计出的引物,其特征在于:所述引物的1对引物序列为:
上游 (20bp) PsYkt6-F:
TGCGTGGCCGTGAATTGATC
下游 (20bp) PsYkt6-R:
CTGCAGAGCGACAACAATCG。
所述的大豆疫霉特异性的分子检测引物在大豆疫霉分子检测上的应用。
一种检测大豆疫霉的方法,其特征在于提取待检微生物的DNA,以提取的DNA 为模板,利用权利要求1 所述的分子检测引物进行 PCR扩增 ;扩增产物进行琼脂糖凝胶电泳,在紫外光下检测结果,如果存在分子量约为399bp的条带,则证明所检病原为大豆疫霉。
所述的大豆疫霉特异性的分子检测引物在实时定量荧光PCR分子检测中的应用,其特征在于:基于已定量的标准DNA样品做出的标准曲线方程,标准品DNA浓度的对数值与Ct值之间的相关系数R2>0.99,定量线性回归方程为y = -3.567x + 28.879,其中y为Ct值,x为大豆疫霉菌 DNA 浓度常用对数值,为定量未知样品的DNA提供了理论依据。
所述的大豆疫霉特异性的分子检测引物在实时定量荧光PCR分子检测中的应用,其特征在于:对发病植株、污染田块、水源进行快速PCR分子检测。具体方案如下:
以Ykt6基因为靶标,设计的大豆疫霉特异性扩增引物为:
上游 (20bp) PsYkt6
-F: TGCGTGGCCGTGAATTGATC
下游 (20bp)
PsYkt6-R: CTGCAGAGCGACAACAATCG。
用上述引物进行PCR扩增,所用试剂及浓度为:25mmol Tris.Cl(PH 8.3)、125mmol KCl、3.75 mmol MgCl2、0.25mmol dNTPs、大豆疫霉特异性引物PsYkt6-F/PsYkt6-R各 1μmol、0.1mg BSA、Taq DNA聚合酶50U,取1-5μL待检测物DNA溶液作为反应模板,取去离子水加入定容至25μL,混匀后PCR扩增,程序为(1)94℃变性3分钟,(2)94℃变性30秒;(3)56 ℃退火30秒;(4)72℃延伸30秒;(5)程序2-4循环32 次,最后72℃延伸5分钟。扩增产物的电泳检测。取10μL
PCR扩增产物,在1%(质量体积比)的琼脂糖凝胶上进行电泳,电压为120V,20分钟后在紫外光下检测结果。如果存在分子量约为399bp的条带,则证明所检病原为大豆疫霉。
本发明引物的特异性验证。选用3株不同地区来源的大豆疫霉菌株,提取基因组作为实验模板,大豆疫霉特异性引物PsYkt6-F/PsYkt6-R进行PCR扩增,可以扩增出1条约399bp大小的电泳条带,而其它疫霉种菌株、腐霉属菌株和真菌菌株没有扩增条带,结果表明PsYkt6-F/PsYkt6-R可以作为大豆疫霉的特异性引物,从而区别于其它物种。
本发明引物的灵敏度验证。提取大豆疫霉标准菌株P6497 DNA,从1ng/µl开始10倍向下稀释至10ag/µl,各个浓度梯度取1µl为PCR模板,大豆疫霉特异性引物PsYkt6-F/PsYkt6-R进行PCR扩增,取10µl电泳产物进行电泳检测,该特异性引物可以检测到10pg量的基因组。用Ykt6疫霉通用引物Ykt6F (AGAGTTGCTGCGCCAGGATGG)/Ykt6R(GTCTTGGACAACACGGCGGTG)进行第一轮PCR扩增,取1µl产物作为下一轮PCR扩增的模板,以大豆疫霉特异性引物PsYkt6-F/PsYkt6-R进行第二轮PCR扩增,取10 µl电泳产物进行电泳检测,结果表明套式PCR可以检测到10fg量的基因组,即其灵敏度可以检测到1个卵孢子和1个游动孢子。
用Ykt6大豆疫霉特异性引物进行实时定量荧光PCR扩增。选用TAKARA公司的SYBR PREMIX Ex
Taq荧光定量试剂进行定量扩增。将大豆疫霉标准菌株的DNA进行10倍梯度稀释,得到模板DNA浓度分别为100
ng/μL,10 ng/μL,1 ng/μL,100 pg/μL,10 pg/μL,1 pg/μL。分别用1μL上述DNA作为定量PCR扩增模板,其它试剂及浓度分别为大豆疫霉特异性引物PsYkt6-F/PsYkt6-R各 0.2μmol、SYBR PREMIX Ex
Taq (2×) 12.5μL,ROX Reference Dye (50×) 0.4μL,定容至25μL。使用ABI公司的实时定量PCR扩增仪,扩增程序为(1)95℃预变性30秒,(2)95 ℃ 5秒;(3)60 ℃ 30秒;(4)程序2-3循环40 次。基于已定量的标准DNA样品做出的标准曲线为定量未知样品的DNA提供了理论依据。标准品DNA的对数值与Ct值之间的相关系数R2>0.99
(0.991),定量线性回归方程为y =
-3.567x + 28.879,其中y为Ct值,x为大豆疫霉菌 DNA
浓度常用对数(log10)值。在基因组DNA浓度为10
pg/μL时,实时定量 PCR仍然能够特异性的产生扩增曲线。而在基因组DNA浓度为1
pg/μL时,实时定量PCR不能够特异性的产生扩增曲线。
本发明以一个细胞存活必须的R-SNARE蛋白编码基因Ykt6为靶标,设计出大豆疫霉特异性的检测引物,该引物具有较高的灵敏度,与Ykt6疫霉属通用引物Ykt6F/Ykt6R配套使用,采用套式PCR的方法,可以将灵敏度提高1000倍,足以检测出微量大豆疫霉,该方法特异性和灵敏度高,可对大豆疫霉菌丝、卵孢子和游动孢子进行检测。同时,利用大豆疫霉特异性开发出实时定量PCR的检测方法及线性回归方程,可以准确、定量的检测大豆中侵染的大豆疫霉菌的量,检测出土壤中残存的大豆疫霉卵孢子的含量,为病害的预测预报提供有力数据,为进出口检疫等提供技术支持。
1.
具体实施方式及方法
下面结合实施例对本发明作进一步说明,但本发明的实质并不限于下述实施例。
实施例
1
大豆感病植株的检测结果:
1)发病植株DNA的提取:将有水浸状病斑的大豆叶片或根茎部位用70%酒精消毒后,用液氮研磨后取少量粉末,采用CTAB法提取基因组。也可使用碱裂解法快速提取DNA,方法如下,取一段发病的植株组织,每毫克组织加入10
µl 0.5 M NaOH,充分研磨后转移至1.5 ml的EP管中,12000 rpm离心5 分钟,取1 µl直接用于PCR扩增。
2)普通PCR扩增验证:取1µl
DNA溶液作为反应模板,以本发明大豆疫霉特异性碱基序列PsYkt6-F/PsYkt6-R为引物,以上文提到的试剂及用量进行PCR扩增,取扩增产物10
µl用于凝胶电泳检测,结果表明,发病的植株都能扩增出特异性的目的条带,说明该病害是由大豆疫霉所引起的,而在健康植株及以水为模板的阴性对照中没有扩增条带。
3)实时定量荧光PCR检测:取1 µl
DNA溶液作为反应模板,以本发明大豆疫霉特异性碱基序列PsYkt6-F/PsYkt6-R为引物,以上文提到的试剂及用量进行实时定量荧光PCR扩增,每个样品对应的平均Ct值分别为24.568(样品1)、21.594(样品2)、28.618(样品3)、22.341(样品4),带入线性方程,计算出植物体中含有的大豆疫霉含量分别为16.17
ng/μL、110.24 ng/μL、1.18 ng/μL、68.06 ng/μL。此方法可用于植物抗性鉴定中定量检测不同抗性水平的大豆对大豆疫霉抗侵染的能力。
实施例
2
土壤中残留大豆疫霉卵孢子的检测结果:
1)土壤中微生物DNA的提取:将每个土样晾干碾碎,然后分别称取0.5g土样,采用FastDNA®SPIN
试剂盒(Q-Biogene
Ltd, USA)进行DNA的提取。
2)普通PCR扩增验证:取1μLDNA溶液作为反应模板,以本发明大豆疫霉特异性碱基序列PsYkt6-F/PsYkt6-R引物及Ykt6疫霉属通用引物Ykt6F/ Ykt6R配套使用,采用套式PCR进行扩增,取扩增产物10μL用于凝胶电泳检测,结果表明,在14份从不同地区大豆田中采集的土壤中,有11份能够检测出目的条带,说明含有大豆疫霉的卵孢子,人工接种含有大豆疫霉卵孢子的土壤中也同样能扩增出目的条带,而在不含有大豆疫霉土壤的阴性对照及以水为模板的阴性对照中没有扩增条带。
3)实时定量荧光PCR检测:取1μLDNA溶液作为反应模板,以本发明大豆疫霉特异性碱基序列PsYkt6-F/PsYkt6-R为引物,以上文提到的试剂及用量进行实时定量荧光PCR扩增,每个样品对应的平均Ct值分别为32.946(样品1)、31.568(样品2),28.653(样品3),31.172(样品4),33.371(样品5),26.532(人工接种的卵孢子的土壤,阳性对照),带入线性方程,计算出植物体中含有的大豆疫霉含量分别为0.072
ng/μL、0.176 ng/μL、1.157 ng/μL、0.228 ng/μL、0.055 ng/μL、4.549 ng/μL。此方法可用于定量检测土壤中含有卵孢子的量,为疫情的预报预测提供依据。
实施例
3
大豆疫霉污染水源中游动孢子的检测结果:
1)游动孢子的富集及DNA提取:大豆疫霉在有水膜的环境下能够形成孢子囊并释放大量游动孢子,是再侵染的重要途径。取大豆疫霉污染水源500mL,在5000g的离心力下离心20min,倒去上清液,沉淀的游动孢子用100μL水悬浮,转入1.5mL离心管,加入0.05g石英砂,涡旋震荡10秒后, 2000rpm离心5分钟后取上清用于PCR扩增。
2)普通PCR扩增验证:取1μL游动孢子上清液作为反应模板,以本发明大豆疫霉特异性碱基序列PsYkt6-F/PsYkt6-R引物及Ykt6疫霉属通用引物Ykt6F/Ykt6R配套使用,采用套式PCR进行扩增,取扩增产物10μL用于凝胶电泳检测,结果表明,在发生大豆疫霉病害田块周围的水中能够检测出目的条带,说明含有大豆疫霉的游动孢子孢子,人工接种含有大豆疫霉游动孢子的水样作为阳性对照,在没有大豆疫霉污染的水中及以水为模板的阴性对照中没有扩增条带。
2.
附图说明
图 1. 大豆疫霉特异性引物PsYkt6-F/PsYkt6-R的验证。1-3、分离自不同地区的大豆疫霉菌株、5.辣椒疫霉、6恶疫霉、7.隐地疫霉、8.烟草疫霉、9.棕榈疫霉、10.瓜类疫霉、11.掘氏疫霉、12.荔枝疫霉、13.苎麻疫霉、14.致病疫霉、15.苜蓿疫霉、16.樟疫霉、17.寄生疫霉、18-20.腐霉属、19.禾谷镰刀菌、20.尖孢镰刀菌、21.稻瘟病菌、22.辣椒炭疽病菌、23.油菜菌核病菌、24.以水为模板的阴性对照。
图 2. 大豆疫霉特异性引物PsYkt6-F/PsYkt6-R的灵敏度验证。以大豆疫霉标准菌株DNA 1ng/µl开始10倍向下稀释至10ag/µl,各个浓度梯度取1µl为PCR模板,大豆疫霉特异性引物PsYkt6-F/PsYkt6-R进行PCR扩增,取10µl电泳产物进行电泳检测,该特异性引物可以检测到100pg量的基因组。用Ykt6疫霉通用引物Ykt6F/ Ykt6R进行PCR扩增,取1µl产物作为下一轮PCR扩增的模板,以大豆疫霉Ykt6的特异性引物PsYkt6-F/PsYkt6-R进行PCR扩增,取10µl电泳产物进行电泳检测,结果表明套式PCR可以检测到10fg量的基因组。
图 3. 将大豆疫霉标准菌株的DNA进行10倍梯度稀释,得到模板DNA浓度分别为100
ng/μL,10 ng/μL,1 ng/μL,100 pg/μL,10 pg/μL,1 pg/μL。分别用1μL上述DNA作为定量PCR扩增模板,标准品DNA的对数值与Ct值之间的相关系数R2=0.991,定量线性回归方程为y =
-3.567x + 28.879。其中y为Ct值,x为大豆疫霉菌 DNA
浓度常用对数( log10)值。
图4. 大豆疫霉特异性引物PsYkt6-F/PsYkt6-R对发病植株的PCR检测。1.阳性对照,2、3为发病植株,4、5为健康植株,6.阴性对照
图5. 大豆疫霉特异性引物PsYkt6-F/PsYkt6-R对发病田块土壤的PCR检测,1.阳性对照,2-15,发病田块土壤中的PCR扩增结果。16、17,没有发病的田块土壤的PCR结果, 6.阴性对照。
图6. 大豆疫霉特异性引物PsYkt6-F/PsYkt6-R对污染河水中游动孢子的PCR检测,1.阳性对照,2-3,污染河水中的检测结果。4,没有污染的水中检测结果 5.阴性对照。
Claims (5)
1.大豆疫霉特异性的分子检测引物,是以R-SNARE蛋白编码基因Ykt6为靶标,设计出的引物,其特征在于:所述引物的1对引物序列为:
上游 (20bp)
PsYkt6 -F: TGCGTGGCCGTGAATTGATC
下游 (20bp)
PsYkt6-R: CTGCAGAGCGACAACAATCG。
2.如权利要求1所述的大豆疫霉特异性的分子检测引物在大豆疫霉分子检测上的应用。
3.一种检测大豆疫霉的方法,其特征在于提取待检微生物的DNA,以提取的DNA 为模板,利用权利要求1 所述的分子检测引物进行 PCR扩增 ;扩增产物进行琼脂糖凝胶电泳,在紫外光下检测结果,如果存在分子量约为399bp的条带,则证明所检病原为大豆疫霉。
4.根据权利要求1所述的大豆疫霉特异性的分子检测引物在实时定量荧光PCR分子检测中的应用,其特征在于:基于已定量的标准DNA样品做出的标准曲线方程,标准品DNA浓度的对数值与Ct值之间的相关系数R2>0.99 ,定量线性回归方程为y = -3.567x + 28.879,其中y为Ct值,x为大豆疫霉菌 DNA 浓度常用对数值,为定量未知样品的DNA提供了理论依据。
5.根据权利要求4所述的大豆疫霉特异性的分子检测引物在实时定量荧光PCR分子检测中的应用,其特征在于:对发病植株、污染田块、水源进行快速实时定量荧光PCR分子检测。
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN201310464140.XA CN103571946B (zh) | 2013-09-30 | 2013-09-30 | 大豆疫霉特异性的分子检测引物及其应用 |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN201310464140.XA CN103571946B (zh) | 2013-09-30 | 2013-09-30 | 大豆疫霉特异性的分子检测引物及其应用 |
Publications (2)
Publication Number | Publication Date |
---|---|
CN103571946A CN103571946A (zh) | 2014-02-12 |
CN103571946B true CN103571946B (zh) | 2016-03-02 |
Family
ID=50044654
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CN201310464140.XA Expired - Fee Related CN103571946B (zh) | 2013-09-30 | 2013-09-30 | 大豆疫霉特异性的分子检测引物及其应用 |
Country Status (1)
Country | Link |
---|---|
CN (1) | CN103571946B (zh) |
Families Citing this family (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN104195259B (zh) * | 2014-09-17 | 2016-11-30 | 天津出入境检验检疫局动植物与食品检测中心 | 同步检测草莓疫霉和树莓疫霉的三重pcr引物及其扩增方法和用途 |
CN105316411A (zh) * | 2015-11-13 | 2016-02-10 | 无锡艾科瑞思产品设计与研究有限公司 | 一种用于检测大豆疫霉的试剂盒及其检测方法 |
CN112501332B (zh) * | 2020-09-11 | 2022-04-12 | 安徽省农业科学院植物保护与农产品质量安全研究所 | 一种小麦全蚀病菌特异性分子检测引物及其检测方法和应用 |
Citations (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN101705293A (zh) * | 2009-12-04 | 2010-05-12 | 天津出入境检验检疫局动植物与食品检测中心 | 大豆疫霉pcr检测特异引物对 |
CN103014152A (zh) * | 2012-11-21 | 2013-04-03 | 安徽省农业科学院植物保护与农产品质量安全研究所 | 一种用于检测大豆疫霉的lamp引物、试剂盒及其检测方法 |
CN103103255A (zh) * | 2012-11-21 | 2013-05-15 | 安徽省农业科学院植物保护与农产品质量安全研究所 | 一种用于检测辣椒疫霉的引物及其检测试剂盒 |
-
2013
- 2013-09-30 CN CN201310464140.XA patent/CN103571946B/zh not_active Expired - Fee Related
Patent Citations (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN101705293A (zh) * | 2009-12-04 | 2010-05-12 | 天津出入境检验检疫局动植物与食品检测中心 | 大豆疫霉pcr检测特异引物对 |
CN103014152A (zh) * | 2012-11-21 | 2013-04-03 | 安徽省农业科学院植物保护与农产品质量安全研究所 | 一种用于检测大豆疫霉的lamp引物、试剂盒及其检测方法 |
CN103103255A (zh) * | 2012-11-21 | 2013-05-15 | 安徽省农业科学院植物保护与农产品质量安全研究所 | 一种用于检测辣椒疫霉的引物及其检测试剂盒 |
Non-Patent Citations (2)
Title |
---|
Genome-wide identification of Phytophthora sojae SNARE genes and functional characterization of the conserved SNARE PsYKT6;Wei Zhao等;《Fungal Genetics and Biology》;20101123;第48卷;241–251 * |
大豆疫霉的分子检测;陈长卿等;《西北农林科技大学学报(自然科学版)》;20050831;第33卷(第8期);73-77 * |
Also Published As
Publication number | Publication date |
---|---|
CN103571946A (zh) | 2014-02-12 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
Liu et al. | Evidence for the plant recruitment of beneficial microbes to suppress soil‐borne pathogens | |
Zhou et al. | A mycovirus modulates the endophytic and pathogenic traits of a plant associated fungus | |
Bai et al. | Global and endemic Asian lineages of the emerging pathogenic fungus Batrachochytrium dendrobatidis widely infect amphibians in China | |
Arantes et al. | Environmental mapping of Paracoccidioides spp. in Brazil reveals new clues into genetic diversity, biogeography and wild host association | |
Zhang et al. | Diversity and cold adaptation of culturable endophytic fungi from bryophytes in the Fildes Region, King George Island, maritime Antarctica | |
Kowalchuk et al. | Rhizosphere fungal communities are influenced by Senecio jacobaea pyrrolizidine alkaloid content and composition | |
Bockelmann et al. | Quantitative PCR reveals strong spatial and temporal variation of the wasting disease pathogen, Labyrinthula zosterae in northern European eelgrass (Zostera marina) beds | |
Busby et al. | Leaf endophytes and Populus genotype affect severity of damage from the necrotrophic leaf pathogen, Drepanopeziza populi | |
Cui et al. | Multiplex PCR assay for simultaneous detection of six major bacterial pathogens of rice | |
Tsror et al. | Characterization of D ickeya strains isolated from potato grown under hot‐climate conditions | |
CN103571946B (zh) | 大豆疫霉特异性的分子检测引物及其应用 | |
CN105063219A (zh) | 番石榴炭疽病菌特异性pcr检测引物及其检测方法 | |
Keča et al. | Ecology and distribution of Armillaria species in Norway | |
Ajani et al. | First reports of P seudo‐nitzschia micropora and P. hasleana (B acillariaceae) from the S outhern H emisphere: Morphological, molecular and toxicological characterization | |
Shaheen et al. | Genetics and genomics of Fusarium wilt of chilies: A review | |
Feng et al. | The correlation of virulence, pathogenicity, and itraconazole resistance with SAP activity in Candida albicans strains | |
CN103014152A (zh) | 一种用于检测大豆疫霉的lamp引物、试剂盒及其检测方法 | |
Alkan et al. | Monitoring of host suitability and defense-related genes in wheat to Bipolaris sorokiniana | |
Wang et al. | The newly identified Trichoderma harzianum partitivirus (ThPV2) does not diminish spore production and biocontrol activity of its host | |
CN104232634B (zh) | 一种松树蜂特异性ss-coⅰ引物对及快速分子检测方法 | |
CN105524986A (zh) | 一种快速检测亚洲柑桔黄龙病菌的lamp检测方法 | |
Salamon et al. | Expression patterns of miR398, miR167, and miR159 in the interaction between bread wheat (Triticum aestivum L.) and pathogenic Fusarium culmorum and beneficial Trichoderma fungi | |
Lin et al. | Molecular detection assays for rapid field-detection of rice sheath blight | |
CN105603067A (zh) | 基于多重pcr快速鉴定我国外来入侵福寿螺的特异引物对及其应用 | |
Rostami et al. | Characterization, pathogenicity and host range studies of Paecilomyces formosus associated with dieback of Christ's thorn trees (Paliurus spina‐christi Mill.) in Iran |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
C06 | Publication | ||
PB01 | Publication | ||
SE01 | Entry into force of request for substantive examination | ||
SE01 | Entry into force of request for substantive examination | ||
C14 | Grant of patent or utility model | ||
GR01 | Patent grant | ||
CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20160302 |