CN116497141A - 一种用于检测苦荞叶斑病菌木樨黑孢霉的荧光定量pcr引物对和探针与试剂盒及其应用 - Google Patents
一种用于检测苦荞叶斑病菌木樨黑孢霉的荧光定量pcr引物对和探针与试剂盒及其应用 Download PDFInfo
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Abstract
本发明公开了一种用于检测苦荞叶斑病菌木樨黑孢霉的荧光定量PCR引物对和探针与试剂盒及其应用,所述试剂盒包括引物对和Taqman探针,所述的引物对包括正向引物TefF1和反向引物TefR,其碱基序列分别为5’‑CGT CAC CGT CAT TGG TAA GT‑3’、5’‑TCT GAA GGC GAA GTG GGT TA‑3’。所述探针TefP的5'端标记荧光报告基团,3'端标记荧光淬灭基团,碱基序列为5’‑ACG GCT CCT TGC ACC TGT TCA C‑3’。所述引物对、Taqman探针与qPCR MIX反应液、阳性对照和阴性对照组成检测试剂盒。所述试剂盒能够对苦荞麦叶斑病菌木樨黑孢霉进行早期检测,具有特异、灵敏、准确等优点,在苦荞麦叶斑病菌木樨黑孢霉的早期预测预报和综合治理等方面具有重要意义。
Description
技术领域
本发明属于生物技术领域,涉及一种基于荧光定量PCR技术检测苦荞麦叶斑病菌木樨黑孢霉的试剂盒,还涉及检测苦荞麦叶斑病菌木樨黑孢霉的方法。
背景技术:
黑孢霉(Nigrosporasp.)是植物的内生菌或腐生菌,可危害重要的经济作物、水果和观赏植物,主要危害叶片。例如,N. aurantiaca引起烟草叶斑病,N. oryzae引起蓝莓叶斑病。此外,黑孢霉的个别种类甚至可以危害人或其他哺乳动物。近年新发现一种苦荞麦叶斑病,由木樨黑孢霉(Nigrospora osmanthi)引起。由于苦荞麦叶斑病病原有多种,如交链格孢(Alternaria alternata)、Peyronellaea calorpreferensi、Didymella rhei等,病原不同,症状也不同。为了表述方便,本发明中由木樨黑孢霉引起的苦荞麦叶斑病,以下简称木樨黑孢霉叶斑病。木樨黑孢霉叶斑病染病初期,叶片出现圆形或卵圆形、浅黄至浅褐色斑点,而后斑点逐渐扩大,合并形成圆形或不规则大病斑,病斑周围有红褐色边缘,最终导致叶片枯萎脱落,严重影响苦荞麦的产量和品质。目前该病尚没有较好的针对性防治措施。因此,建立早期快速检测方法对预防和控制苦荞麦木樨黑孢霉叶斑病具有十分重要的意义。
实时荧光定量PCR(以下简称qPCR)是指在PCR反应体系中加入荧光化学物质,利用荧光信号积累实时监测PCR扩增产物并进行定量分析的方法。该方法较普通PCR方法更加灵敏可靠,尤其是Taqman荧光探针法qPCR在检测的特异性和灵敏度又有大幅度提高,常用于单核苷酸多态性(SNP)、病毒和病原菌检测。目前尚未有基于qPCR技术检测苦荞麦木樨黑孢霉叶斑病的报道。
发明内容:
有鉴于此,本发明的目的是提供一种用于检测苦荞叶斑病菌木樨黑孢霉的荧光定量PCR引物对和探针与试剂盒及其应用。
为实现本发明的目的,本发明采用的技术方案:一种用于检测苦荞叶斑病菌木樨黑孢霉的荧光定量PCR引物对和探针,所述引物对包括核苷酸序列如SEQ ID NO:1所示的正向引物TefF和核苷酸如SEQ ID NO:2所示的反向引物TefR;所述探针TefP序列的5'端为FAM荧光报告基团标记,3'端为TAMRA淬灭基团标记,所述核苷酸序列如SEQ ID NO:3所示。
一种用于检测苦荞叶斑病菌木樨黑孢霉的试剂盒,包括引物对和Taqman探针,所述引物对包括核苷酸序列如SEQ ID NO:1所示的正向引物TefF和核苷酸如SEQ ID NO:2所示的反向引物TefR;所述探针TefP序列的5'端为FAM荧光报告基团标记,3'端为TAMRA淬灭基团标记,所述核苷酸序列如SEQ ID NO:3所示。
所述的试剂盒还包括qPCR MIX反应液、阳性对照和阴性对照;所述qPCR MIX反应液包含以下含量的组分:5U Taq酶、2.5 mmol/L dNTPs、25 mmol/L MgCl2和2×PCRbuffer;所述阳性对照为含有木樨黑孢霉TEF1-α基因片段的质粒模板;所述阴性对照为无菌双蒸水。
所述试剂盒在苦荞麦叶斑病菌木樨黑孢霉检测方面的应用。
采用所述试剂盒检测苦荞麦叶斑病菌木樨黑孢霉的方法,包括以下步骤:
1)提取木樨黑孢霉基因组DNA;
2)以木樨黑孢霉DNA为模板,采用通用引物EF1-728F/EF-2(现有技术Carbone I,et al. A method for designing primer sets for speciation studies infilamentous ascomycetes. Mycologia, 1999, 91(3): 553-556;O’Donnell K, et al.Multiple evolutionary origins of the fungus causing Panama disease of banana:concordant evidence from nuclear and mitochondrial gene genealogies. PNAS,1998, 95(5): 2044-2049.)进行PCR扩增,获得TEF1-α基因片段,切胶纯化,连接至pBM16ATopo载体上,转化大肠杆菌DH5a,获得阳性克隆,测序验证。阳性克隆扩繁后,提取质粒。测定质粒吸光值A260,将A260转化成DNA浓度,再将DNA浓度转化成拷贝数,进行梯度稀释,并以稀释后的DNA模板进行qPCR扩增,绘制标准曲线,并建立标准曲线方程;
3)提取待测样品苦荞麦叶片基因组DNA;
4)以待测样品苦荞麦叶片DNA为模板,用所述引物对、探针进行qPCR检测,得到样品的扩增曲线。根据所述样品的Ct值和扩增曲线判断检测结果,当Ct值小于32且出现“S”型曲线则为阳性,即检测样品为含有苦荞麦叶斑病菌木樨黑孢霉;反之,则为阴性。将所述样品的Ct值代入标准曲线方程计算出样品中病原菌的DNA量或拷贝数。
本发明根据木樨黑孢霉的TEF1-α基因序列设计特异性引物对和TaqMan探针,建立了苦荞麦叶斑病菌木樨黑孢霉的qPCR扩增体系,可定性鉴定和定量检测苦荞麦叶斑病菌木樨黑孢霉。
由于采用了上述技术方案,本发明具有如下的优点:
1)特异性强:本发明中的引物对、TaqMan探针根据苦荞麦叶斑病菌木樨黑孢霉TEF1-α基因序列而设计,充分考虑了木樨黑孢霉与其近似种在TEF1-α序列上的细微差异,经检验,即使是在进化上最接近的Nigrosporalacticolonia和Nigrosporaaurantiaca也不能被检出。
2)灵敏度高:本发明对目标菌的检测下限为0.255 fg/ml、9.95 copies/µl。
3)准确度高:在人工接种的苦荞麦植株中,采用本发明的技术,接种后1 d就能够检测出目标病原菌,被检测出携带目标病原菌的植株在接种后3~5天陆续发病,准确性好。
4)应用范围广:适用于苦荞麦叶斑病菌木樨黑孢霉的定量检测、早期诊断与防治;适用于苦荞麦品种针对于目标病原的抗性鉴定。
综上所述,采用本发明,能够特异、灵敏、准确地检测苦荞麦叶斑病菌木樨黑孢霉,为该病害的早期预测和防治提供技术支撑。
附图说明:
图1显示本发明实施例2中特异性检测结果。其中,CK为阴性对照,N.lacticolonia和N.aurantiaca为木樨黑孢霉(N. osmanthi)的近似种,作为参比菌株。
图2显示本发明实施例3中灵敏度检测结果。其中,1为阴性对照的扩增曲线;2~9分别代表pBM16-NoTEF质粒浓度依次为5.1 ng、510 pg、51.0 pg、5.1 pg、510 fg、51.0 fg、5.1 fg、0.51 fg的扩增曲线。
图3显示本发明实施例3中所建立的标准曲线。其中,y代表Ct值,x代表pBM16-NoTEF质粒DNA拷贝数的log10值。
图4 显示本发明实施例4中在接种苦荞麦1天后对目标病原菌的实测结果。其中,21为木樨黑孢霉基因组DNA的扩增曲线; 1~19为对应编号的接种植株叶片DNA样品的扩增曲线; 20为对应编号的未接种对照叶片DNA样品的扩增曲线。
具体实施方式:
以下实施例用于说明本发明,但不用来限制本发明的范围。
主要材料、试剂和方法:
供试病原菌:木樨黑孢霉(NigrosporaOsmanthi LS-2)为湖南科技大学生命科学与健康学院植物分子生物学实验室于2020年从苦荞麦叶斑病病叶上分离并鉴定,并于中国典型培养物保藏中心(CCTCC)保藏,地址:中国武汉,武汉大学,保藏编号CCTCC NO:M2022315。保藏日期:2022年3月25日,病原菌接种在固体PDA培养基上,培养条件为温度28℃。
参比菌株:Nigrospora lacticolonia (CGMCC3.18123)和Nigrospora aurantiaca (CGMCC3.18130)从国家菌种保藏中心(CGMCC)获得。
供试苦荞麦品种:‘平苦一号’由山西农业大学农学院提供。在湖南科技大学生物园温室盆栽种植,培养条件为温度20~30℃、相对湿度60%~80%,待生长至4~5叶供试。
主要试剂和仪器分别如下表1、2:
表1 主要试剂
表2 主要仪器
仪器 | 型号 | 生产厂家 |
霉菌培养箱 | MJ-250F-I | 上海一恒公司 |
PCR 仪 | Applied Biosystems 2720 | Thermo Fisher公司 |
超微量分光光度计 | NanoPhotometer-NP80 | IMPLEN GMBH公司 |
实时荧光定量PCR系统 | CFX96 Touch Real-Time PCR | Bio-Rad公司 |
病原菌和荞麦叶片基因组DNA提取:病原菌DNA提取采用Biospin公司出品的真菌基因组DNA提取试剂盒,按说明书进行。苦荞麦叶片基因组采用改良的CTAB法,按现有技术(李金璐, 等. 一种改良的植物DNA提取方法.植物学报, 2013, 48(1): 72-78.)进行。提取的DNA于-20℃保存备用。
qPCR反应体系如表3。
表3 qPCR反应体系
组分 | 体积 (µL) |
2×Taqman Mix | 10 |
TefF (10 µmol/L) | 1.2 |
TefR (10 µmol/L) | 1.2 |
TefP (10 µmol/L) | 0.8 |
Taq聚合酶 | 0.15 |
ddH2O | 5.65 |
DNA模板 | 1 |
总计 | 20 |
qPCR扩增程序:采用两步法。程序为:94℃,预变性2 min;94℃变性10 s;57℃退火延伸40 s;42个循环。每循环延伸结束时收集荧光信号。所述荧光定量PCR检测优选设置阳性对照孔和阴性对照。
实施例1 引物及Taqman探针的设计
从NCBI (https://www.ncbi.nlm.nih.gov/)下载木樨黑孢霉LS-2、木樨黑孢霉其他菌株及其同属相似种的翻译延伸因子1-α(TEF1-α)基因序列,采用ClustalW程序进行多重序列比对,筛选适合于特异性引物设计的多态性位点,应用Primer Express 5.0软件设计qPCR引物及Taqman探针。正向引物序列为TefF:5’-CGTCACCGTCATTGGTAAGT-3’(SEQ IDNO:1);反向引物序列为TefR:5’-TCTGAAGGCGAAGTGGGTTA-3’(SEQ ID NO:2)。预期扩增产物大小为76 bp。Taqman特异性探针序列为TefP:5’-ACGGCTCCTTGCACCTGTTCAC-3’(SEQ IDNO:3)。探针5’端含有FAM报告基团,3’端含有淬灭基团TAMRA。引物和探针均由昆明捷腾生物有限公司合成。
实施例2 特异性检测
以基因组DNA为模板,在相同条件下对木樨黑孢霉LS-2及其在TEF1-α同源性最高的近似种Nigrosporalacticolonia和Nigrosporaaurantiaca进行qPCR检测,只有木樨黑孢霉LS-2有明显的扩增曲线生成,两个近似种与空白对照(无菌双蒸水)均无扩增(附图1),说明设计的引物和探针有较高的特异性。
实施例3 灵敏度检测及标准曲线的建立
以木樨黑孢霉LS-2 DNA为模板,采用通用引物EF1-728F/EF-2进行普通PCR扩增,获得TEF1-α基因片段,切胶纯化,连接至pBM16A Topo载体上,获得pBM16A-NoTEF阳性克隆。测定质粒吸光值A260,按以下公式将A260转化成DNA浓度和拷贝数:
对于ds DNA而言,1 A260 ≈ 50 mg/ml ≈ 50 ng/ml
拷贝数= [6.02×1023 (拷贝/mol)× DNA质量(g)]/[质粒bp数×660(g/mol)],式中660为每个碱基对(bp)的平均分子量。
将起始浓度为51 ng/µL的pBM16A-NoTEF阳性质粒用无菌蒸馏水进行10倍梯度稀释,稀释成1×10-1、1×10-2、1×10-3、1×10-4、1×10-5、1×10-6、1×10-7和1×10-8,以稀释后的DNA模板进行qPCR扩增,绘制标准曲线,建立标准曲线方程。
经检测,在20 µl的反应体系中检测下限为0.51 fg,对应拷贝数的检测下限为9.95copies/µl,结果如附图2。
经检测,pBM16-TEF阳性质粒的浓度与Ct值具有很强的相关性:模板浓度越高,其Ct值越小。标准曲线方程为:y=-3.325x + 53.515 (R2=0.998),扩增效率E=99.9%,结果如附图3。
实施例4 利用qPCR检测苦荞麦接种后叶片中的病原菌
收集木樨黑孢霉的分生孢子,用含0.7%葡萄糖和0.05%吐温20的无菌水配制成104孢子/mL的孢子悬液。选取20株长势基本一致的、处于四叶期的健康苦荞麦植株,其中1~19号为处理组,用小塑料喷壶均匀喷洒上述孢子悬液;20号为对照组,均匀喷洒含0.7%葡萄糖和0.05%吐温20的无菌水。处理组与对照组在温室的不同区隔中严格分开培养,培养条件如前述,同时观察病情。接种后1天采用直径为6 mm的打孔器取样。每株苦荞麦取第2、3叶,每叶打1个孔,同株同一取样时间点的叶圆片混合,用无菌水清洗叶圆片3次,用前述改良的CTAB法提取基因组DNA。用木樨黑孢霉引物对及探针进行qPCR扩增,反应体系和程序如前述。实验设置3个生物学重复和2个技术重复。
观察发病情况,发现上述接种的19株苦荞麦叶片在接种后3~5天内,出现木樨黑孢霉叶斑病的典型病斑。qPCR扩增结果显示,这19株接种的植株均能够在接种后1天即可检测到目标病原菌(附图4),表明此方法具有较高的准确性和灵敏度,与肉眼诊断相比,至少能够提前2天预测木樨黑孢霉叶斑病的发生。
以上所述仅是本发明的优选实施方式,应当指出,对于本技术领域的普通技术人员来说,在不脱离本发明原理的前提下,还可以做出若干改进和润饰,这些改进和润饰也应视为本发明的保护范围。
Claims (5)
1. 一种用于检测苦荞叶斑病菌木樨黑孢霉的荧光定量PCR引物对和探针,其特征在于,所述引物对包括核苷酸序列如SEQ ID NO:1所示的正向引物TefF和核苷酸如SEQ IDNO:2所示的反向引物TefR;所述探针TefP序列的5'端为FAM荧光报告基团标记,3'端为TAMRA淬灭基团标记,所述核苷酸序列如SEQ ID NO:3所示。
2.一种用于检测苦荞叶斑病菌木樨黑孢霉的试剂盒,其特征在于,包括引物对和Taqman探针,所述引物对包括核苷酸序列如SEQ ID NO:1所示的正向引物TefF和核苷酸如SEQ ID NO:2所示的反向引物TefR;所述探针TefP序列的5'端为FAM荧光报告基团标记,3'端为TAMRA淬灭基团标记,所述核苷酸序列如SEQ ID NO:3所示。
3.根据权利要求2所述的试剂盒,其特征在于,还包括qPCR MIX反应液、阳性对照和阴性对照;所述qPCR MIX反应液包含以下含量的组分:5U Taq酶、2.5 mmol/L dNTPs、25mmol/L MgCl2和2×PCR buffer;所述阳性对照为含有木樨黑孢霉TEF1-α基因片段的质粒模板;所述阴性对照为无菌双蒸水。
4.根据权利要求2或3所述试剂盒在苦荞麦叶斑病菌木樨黑孢霉检测方面的应用。
5.根据权利要求4所述试剂盒在苦荞麦叶斑病菌木樨黑孢霉检测方面的应用,特征在于,检测方法包括以下步骤:
1)提取木樨黑孢霉基因组DNA;
2)以木樨黑孢霉基因组DNA为模板,采用通用引物EF1-728F/EF-2进行普通PCR扩增,获得TEF1-α基因片段,切胶纯化,连接至pBM16A Topo载体上,选择阳性克隆,提取质粒;测定质粒吸光值A260,将A260转化成DNA浓度,再将DNA浓度转化成拷贝数,进行梯度稀释,并以稀释后的DNA模板进行荧光定量PCR扩增,绘制标准曲线,并建立标准曲线方程;
3)提取待测样品苦荞麦叶片基因组DNA;
4)以待测样品苦荞麦叶片基因组DNA为模板,用所述引物对、Taqman探针进行荧光定量PCR检测,得到样品的扩增曲线;根据所述样品的Ct值和扩增曲线判断检测结果,当Ct值小于32且出现“S”型曲线则为阳性,即检测样品为含有苦荞麦叶斑病菌木樨黑孢霉;反之,则为阴性,
将所述样品的Ct值代入标准曲线方程计算出样品中目标病原菌的DNA量或拷贝数。
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