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首页> 外文期刊>Plant Disease >Quantitative Real-Time Polymerase Chain Reaction Assay for Detection of Pectobacterium wasabiae Using YD Repeat Protein Gene-Based Primers
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Quantitative Real-Time Polymerase Chain Reaction Assay for Detection of Pectobacterium wasabiae Using YD Repeat Protein Gene-Based Primers

机译：基于YD重复蛋白基因的引物定量实时定量聚合酶链反应检测芥末杆菌

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Kim, M. H., Cho, M. S., Kim, B. K., Choi, H. J., Hahn, J. H., Kim, C., Kang, M. J., Kim, S. H., and Park, D. S. 2012. Quantitative real-time polymerase chain reaction assay for detection of Pectobacterium wasabiae using YD repeat protein gene-based primers. Plant Dis. 96:253-257. The aim of this study was to develop a quantitative polymerase chain reaction (qPCR) assay for specific detection of Pectobacterium wasabiae using a primer pair based on the YD repeat protein gene for amplification of a 140-bp DNA fragment from infected wasabi (Wasabia japonica), a member of the crucifer family. The soft rot caused by P wasabiae is an emerging disease that is present in many wasabi-producing areas. However, specific and reliable methods for identifying the pathogen are not available. Therefore, a qPCR primer set for accurate diagnosis of P wasabiae was developed from publically available genome sequences. A SYBR Green qPCR primer set was designed based on a YD repeat protein gene of P. wasabiae WPP163 because it is known that this gene is structurally diverse among species, pathovars, or subspecies. The specificity of the primer set was evaluated using genomic DNA from 5 isolates of P wasabiae, 5 different species of Pectobacterium, and 16 other pathogenic reference bacteria. The primer set used in the PCR assay successfully amplified a 140-bp amplicon for all five P. wasabiae strains. No amplification was obtained from 29 other pathogenic bacteria. The assay was also able to detect at least two genomic DNA, or 3 CFU per reaction, when using calibrated cell suspension.
机译：Kim，MH，Cho，MS，Kim，BK，Choi，HJ，Hahn，JH，Kim，C.，Kang，MJ，Kim，SH和Park，DS2012。定量实时聚合酶链反应检测试剂盒使用基于YD重复蛋白基因的引物对山葵油杆菌进行鉴定。植物病96：253-257。这项研究的目的是使用基于YD重复蛋白基因的引物对开发一种定量聚合酶链反应（qPCR）分析法，用于特异性检测芥末分形，以扩增被感染芥末（Wasabia japonica）的140 bp DNA片段。 ，十字花科家族的成员。由芥末引起的软腐病是一种新兴疾病，在许多芥末产区都存在。但是，尚无用于鉴定病原体的特定且可靠的方法。因此，从公众可获得的基因组序列开发了一种用于准确诊断芥末的qPCR引物。基于芥末假单胞菌WPP163的YD重复蛋白基因设计了SYBR Green qPCR引物集，因为已知该基因在物种，病原体或亚种之间在结构上是不同的。使用来自芥末假单胞菌的5种分离株，5种不同种类的油杆菌和16种其他病原参照细菌的基因组DNA评估了引物组的特异性。 PCR分析中使用的引物组成功扩增了所有五个芥末假单胞菌菌株的140 bp扩增子。没有从其他29种致病细菌中扩增。当使用校准的细胞悬液时，该测定还能够检测至少两个基因组DNA，或每个反应3 CFU。

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