多重实时定量PCR检测马铃薯主要的真细菌病原菌
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摘要
我国马铃薯的种植面积与产量均居世界首位,但是马铃薯的主要病害一直威胁着我国马铃薯产业的发展。其中主要的马铃薯真细菌病害有：马铃薯晚疫病(Phytophthora infestans (Mont.)deBary),马铃薯干腐病(Fusarium spp.),马铃薯黑痣病(Rhizoctonia solani Kuhn),马铃薯软腐病(Erwinia spp.),马铃薯青枯病(Ralstonia solanacearum)等。提高种薯的质量,检测去除含有潜在病原危害的不合格种薯,是目前防控的主要方法。这就需要发展一种快速高效的病害检测方法用以严格监控种薯生产各环节以保证种薯质量。最新发展的Real Time PCR由于其具有操作简单、快速便捷、灵敏度高、重复性好、污染率低等优点,被广泛应用于病原体检测。
本实验根据以上几种重要的马铃薯真细菌病原菌保守序列设计了Taqman探针及引物。马铃薯晚疫病(Pi)、黑痣病(Rhs)和软腐病(Ec)探针用JOE荧光标记,干腐病(Fz)、青枯病(Rs)以及马铃薯内参基因(细胞色素氧化酶Cox)探针用FAM标记。马铃薯内参COX的引入在检测过程中可以有效地避免因DNA提取失败或PCR抑制从而导致的假阴性结果出现。实验中分别用荧光染料SYRB GREEN I和上述标记的Taqman探针建立了实时荧光定量PCR,并根据探针荧光标记不同建立了双重探针Real time PCR。双重定量PCR的组合为：Rhs与Cox,Fz与Ec,Pi与Rs；Pi与Fz完全不能组合在一起。比较荧光定量检测方法与普通PCR检测的灵敏度,荧光定量PCR灵敏度比普通PCR高10-100倍。本方法兼具多重PCR与实时定量PCR的优点,灵敏度高、特异性强、降低成本、节省时间等,可为脱毒种薯的生产质量提供可靠的保证。
China is the biggest potato producer in the world and is ranked #1 in both total yield and acerage. Potato production in China is threatened by a number of diseases. Major potato diseases in China include late blight caused by oomycete pathogen Phytophthora infestans, dry rot caused by fungal pathogens Fusarium spp., and soft rot and bacterial wilt caused by bacterial pothgens Fusarium spp. and Ralstonia solanacearum, respectively. Currently, the primary approach for controlling these diseases in China is by application of disease-free seed potatoes. Thus, development of efficient and accurate methods for detecting various potato disease pathogens is essential for production and application of quality seed potatoes. Various techniques have been developed to detect the pathogens that cause different potato diseases. However, the recently developed real-time polymerase chain reaction (PCR) methodology is widely used in detecting different potato pathogens. The real-time PCR methodology is simple, fast, highly sensitive, and more reproducible than most of the traditional techniques.
To establish real-time PCR procedures for detecting pathogens associated with several potato diseases, DNA primers were designed from conserved DNA sequences associated with pathogens that are responsibe for potato late blight (Pi), potato black scurf (Rhs), potato soft rot (Ec), potato dry rot (Fz), and potato bacterial wilt (Rs). The potato gene Cox was used as a internal control to eliminate false negatives in the detection procedure. In our study, we establish Real time PCR using DNA binding dye SYRB GREEN I and labelled fluorescence Taqman probe. In addition, we developed a double Real time PCR to detect the diseases according to their different fluorescence:Rhs and Cox, Fz and Ec, Pi and Rs. But Pi and Fz can not be combined together. Sensitivity test showed to us Real time PCR is 10～100 times more than conventional PCR. The new detection procedure is simple, rapid and reliable. It can be used for control the seed production areas strictly so that ensure the quality of seed.
本实验根据以上几种重要的马铃薯真细菌病原菌保守序列设计了Taqman探针及引物。马铃薯晚疫病(Pi)、黑痣病(Rhs)和软腐病(Ec)探针用JOE荧光标记,干腐病(Fz)、青枯病(Rs)以及马铃薯内参基因(细胞色素氧化酶Cox)探针用FAM标记。马铃薯内参COX的引入在检测过程中可以有效地避免因DNA提取失败或PCR抑制从而导致的假阴性结果出现。实验中分别用荧光染料SYRB GREEN I和上述标记的Taqman探针建立了实时荧光定量PCR,并根据探针荧光标记不同建立了双重探针Real time PCR。双重定量PCR的组合为：Rhs与Cox,Fz与Ec,Pi与Rs；Pi与Fz完全不能组合在一起。比较荧光定量检测方法与普通PCR检测的灵敏度,荧光定量PCR灵敏度比普通PCR高10-100倍。本方法兼具多重PCR与实时定量PCR的优点,灵敏度高、特异性强、降低成本、节省时间等,可为脱毒种薯的生产质量提供可靠的保证。
China is the biggest potato producer in the world and is ranked #1 in both total yield and acerage. Potato production in China is threatened by a number of diseases. Major potato diseases in China include late blight caused by oomycete pathogen Phytophthora infestans, dry rot caused by fungal pathogens Fusarium spp., and soft rot and bacterial wilt caused by bacterial pothgens Fusarium spp. and Ralstonia solanacearum, respectively. Currently, the primary approach for controlling these diseases in China is by application of disease-free seed potatoes. Thus, development of efficient and accurate methods for detecting various potato disease pathogens is essential for production and application of quality seed potatoes. Various techniques have been developed to detect the pathogens that cause different potato diseases. However, the recently developed real-time polymerase chain reaction (PCR) methodology is widely used in detecting different potato pathogens. The real-time PCR methodology is simple, fast, highly sensitive, and more reproducible than most of the traditional techniques.
To establish real-time PCR procedures for detecting pathogens associated with several potato diseases, DNA primers were designed from conserved DNA sequences associated with pathogens that are responsibe for potato late blight (Pi), potato black scurf (Rhs), potato soft rot (Ec), potato dry rot (Fz), and potato bacterial wilt (Rs). The potato gene Cox was used as a internal control to eliminate false negatives in the detection procedure. In our study, we establish Real time PCR using DNA binding dye SYRB GREEN I and labelled fluorescence Taqman probe. In addition, we developed a double Real time PCR to detect the diseases according to their different fluorescence:Rhs and Cox, Fz and Ec, Pi and Rs. But Pi and Fz can not be combined together. Sensitivity test showed to us Real time PCR is 10～100 times more than conventional PCR. The new detection procedure is simple, rapid and reliable. It can be used for control the seed production areas strictly so that ensure the quality of seed.
引文
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