A real-time fluorescent PCR method for detection of Botryosphaeria stevensii from apple using TaqMan probe
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摘要:目的
针对我国检疫性植物病原真菌苹果壳色单隔孢溃疡病菌Botryosphaeria stevensii，建立实时荧光PCR检测方法。
方法根据苹果壳色单隔孢溃疡病菌及其近似种的β微管蛋白基因(β-tubulin基因)保守序列设计1对特异性引物和1条TaqMan MGB探针，分别以病菌DNA和β-tubulin基因靶标序列重组质粒DNA为阳性标准品检验探针的特异性和灵敏度。
结果探针BsP267对苹果壳色单隔孢溃疡病菌菌株表现出特异性阳性扩增，且与近缘种及其他常见的果腐病菌无交叉反应，扩增效率为105.858%，探针检测DNA的灵敏度达到1 fg/μL。
结论本研究建立的苹果壳色单隔孢溃疡病菌实时荧光PCR检测法具有高特异性和灵敏度，可用于该病害的防控检测和检疫工作。
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关键词:
- 苹果壳色单隔孢溃疡病菌 /
- 实时荧光PCR /
- TaqMan探针 /
- 快速检测 /
- β微管蛋白基因
Abstract:ObjectiveTo establish a real-time fluorescent PCR assay for detecting Botryosphaeria stevensii, which is the causal agent of Diplodia canker on apple and is presently subjected to phytosanitary legislation in China.
MethodA pair of specific primers and a TaqMan MGB probe were designed based on the conserved sequences of β-tubulin genes of B. stevensii and related species. The specificity and sensitivity of the probe were evaluated using DNAs of B. stevensii strains and recombinant plasmid of β-tubulin gene sequence as positive standard, respectively.
ResultThe probe of BsP267 displayed specificity to B. stevensii strains with positive amplification, while there was no crossing reaction with related species and other common fruit rot pathogens. The PCR amplification efficiency was 105.858% and the detection sensitivity of DNA reached 1 fg/μL.
ConclusionThe real-time PCR method developed in this study can detect B. stevensii from apple with strong specificity and high sensitivity, thus can be used for the prevention, control, detection and quarantine of this disease.
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图 1 靶标基因阳性质粒克隆序列
阴影部分从左到右依次为BsF1(绿色)、BsP267(黄色)和BsR1(蓝色)
Figure 1. Cloning sequence of positive plasmid of target gene
The shaded parts with different colours from left to right are BsF1(green), BsP267(yellow) and BsR1(blue), respectively
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图 2 探针BsP267对苹果壳色单隔孢溃疡病菌的特异性检测
1~3为Botryosphaeria stevensii基因组DNA(CBS 112553、CBS 120834、CQ01)；4~27为其他无扩增曲线的样品(CBS112555、CBS112876、PP5.3-2、CFCC51611、CFCC50629、CFCC86567、CFCC84207、PP5.3-6、CFCC86251、CFCC86538、PP5.12-2、ACCC37263、CBS121886、CBS121887、CBS120835、CBS121104、CBS124253、CBS124130、CBS124462、CBS115176、11-40460、PG5.31、16-4966-2、15-128331)，28为空白对照(无菌水)
Figure 2. Specificity idendification of Botryosphaeria stevensii using probe BsP267
1−3: Cultured mycelial DNA of Botryosphaeria stevensii(CBS 112553, CBS 120834, CQ01)；4−27: Negative control (CBS112555, CBS112876, PP5.3-2, CFCC51611, CFCC50629, CFCC86567, CFCC84207, PP5.3-6, CFCC86251, CFCC86538, PP5.12-2, ACCC37263, CBS121886, CBS121887, CBS120835, CBS121104, CBS124253, CBS124130, CBS124462, CBS115176, 11-40460, PG5.31, 16-4966-2, 15-128331), 28: Blank control (ddH2O)
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图 3 探针BsP267对阳性质粒DNA的灵敏度检测
1~7分别为100 000、10 000、1 000、100、10、1、0.1 fg质粒DNA
Figure 3. Sensitivity idendification of positive plasmid DNA using probe BsP267
1−7 are 100 000、10 000、1 000、100、10、1、0.1 fg plasmid DNA, respectively
表 1 本研究供试菌株的相关信息
Table 1 Information of strains used in the study
菌株编号
Strain No.来源
Origin学名
Scientific name异名
Synonym寄主
Host地理来源
Geographic originCBS112553 荷兰微生物菌种保藏中心 CBS Botryosphaeria stevensii Diplodia mutila Vitis 葡萄牙 Portugal CBS120834 荷兰微生物菌种保藏中心 CBS Botryosphaeria stevensii Diplodia mutila Prunus 南非 South Africa CQ01 重庆海关 Chongqing Customs Botryosphaeria stevensii Diplodia mutila Glycine 阿根廷 Argentina CBS112555 荷兰微生物菌种保藏中心 CBS Botryosphaeria obtusa Diplodia seriata Vitis 葡萄牙 Portugal CBS112876 荷兰微生物菌种保藏中心 CBS Botryosphaeria obtusa Diplodia seriata Vitis 南非 South Africa PP5.3-2 黄埔海关 Huangpu Customs Botryosphaeria rhodina Lasiodiplodia theobromae Eriobotrya 中国 China CFCC51611 中国林业微生物保藏管理中心 CFCC Botryosphaeria rhodina Lasiodiplodia theobromae Rubia 中国 China CFCC50629 中国林业微生物保藏管理中心 CFCC Botryosphaeria rhodina Lasiodiplodia theobromae Vitis 中国 China CFCC86567 中国林业微生物保藏管理中心 CFCC Botryosphaeria parva Neofusicoccum parvum Prunus 中国 China CFCC84207 中国林业微生物保藏管理中心 CFCC Botryosphaeria parva Neofusicoccum parvum Vitis 中国 China PP5.3-6 黄埔海关 Huangpu Customs Botryosphaeria parva Neofusicoccum parvum Eriobotrya 中国 China CFCC86251 中国林业微生物保藏管理中心 CFCC Botryosphaeria dothidea Malus 中国 China CFCC86538 中国林业微生物保藏管理中心 CFCC Botryosphaeria dothidea Malus 中国 China PP5.12-2 黄埔海关 Huangpu Customs Botryosphaeria dothidea Eriobotrya 中国 China ACCC37263 中国农业微生物菌种保藏管理中心 Agricultural Culture Collection of China Botryosphaeria dothidea Lilium 中国 China CBS121886 荷兰微生物菌种保藏中心 CBS Diplodia olivarum Olea 意大利 Italy CBS121887 荷兰微生物菌种保藏中心 CBS Diplodia olivarum Olea 意大利 Italy CBS120835 荷兰微生物菌种保藏中心 CBS Diplodia africana Prunus 南非 South Africa CBS121104 荷兰微生物菌种保藏中心 CBS Diplodia africana Prunus 南非 South Africa CBS124253 荷兰微生物菌种保藏中心 CBS Diplodia malorum Malus 葡萄牙 Portugal CBS124130 荷兰微生物菌种保藏中心 CBS Diplodia malorum Malus 葡萄牙 Portugal CBS124462 荷兰微生物菌种保藏中心 CBS Diplodia intermedia Malus 葡萄牙 Portugal CBS115176 荷兰微生物菌种保藏中心 CBS Sphaeropsis pyriputrescens Pyrus 美国 USA 11-40460 黄埔海关 Huangpu Customs Monilinia fructicola Malus 智利 Chile PG5.31 黄埔海关 Huangpu Customs Alternaria alternata Malus 中国 China 16-4966−2 黄埔海关 Huangpu Customs Botrytis cinerea Vitis 智利 Chile 15-128331 黄埔海关 Huangpu Customs Botryotinia fuckeliana Malus 南非 South Africa 
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