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首页> 外文期刊>Journal of Clinical Microbiology >PCR-DNA probe assays for identification and detection of Prevotella intermedia sensu stricto and Prevotella nigrescens.
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PCR-DNA probe assays for identification and detection of Prevotella intermedia sensu stricto and Prevotella nigrescens.

机译：PCR-DNA探针测定法用于鉴定和检测中型普氏杆菌和黑氏普氏杆菌。

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The purpose of this study was to construct PCR-DNA probe assays specific for Prevotella intermedia sensu stricto and Prevotella nigrescens based on the ability of randomly amplified polymorphic DNA (RAPD) fingerprinting to generate species-specific markers. The strategy included four steps: (i) construction of first-generation DNA probes from a 850-bp RAPD marker for P. intermedia sensu stricto and a 1,300-bp RAPD marker for P. nigrescens, (ii) cloning and sequencing of each RAPD marker, (iii) designing of primer pairs flanking specific internal sequences of 754 bp for P. intermedia sensu stricto and of ca. 1,100 bp for P. nigrescens, and (iv) synthesis (by PCR amplification) and digoxigenin labeling of quantities of DNA probes 754 and ca. 1,100 bp in size. The PCR-DNA probe assays combine either PCR amplification of a 754-bp specific sequence in the genomic DNA of strains of P. intermedia sensu stricto and hybridization with the 754-bp digoxigenin-labeled probe or amplification of a ca. 1,100-bp sequence of P. nigrescens and hybridization with the ca. 1,100-bp probe. Specific hybridization was observed with the amplified DNAs from 25 strains of P. intermedia and 24 strains of P. nigrescens, and no reaction was observed with the PCR products from 20 foreign species. The PCR-DNA probe assays described here should allow a highly specific and sensitive detection of P. intermedia sensu stricto and P. nigrescens in mixed infections.
机译：这项研究的目的是基于随机扩增的多态性DNA（RAPD）指纹图谱产生物种特异性标记的能力，构建对中间小肠细小夜蛾和黑短肠小夜蛾特异性的PCR-DNA探针测定。该策略包括四个步骤：（i）从中度假单胞菌的850-bp RAPD标记和黑僵菌的1300-bp RAPD标记构建第一代DNA探针，（ii）每个RAPD的克隆和测序标记，（iii）设计用于中间致密体育的约754bp的内部特异性序列的引物对，以及约200bp的引物对。黑僵菌的1,100 bp，以及（iv）合成（通过PCR扩增）和洋地黄毒苷标记的DNA探针754和ca的数量。大小为1100 bp。 PCR-DNA探针测定法结合了中间致病假单胞菌菌株基因组DNA中754bp特异性序列的PCR扩增和与754bp地高辛配基标记的探针的杂交或ca.的扩增。黑僵菌1,100 bp序列并与ca. 1,100-bp探针。观察到来自25个中间P. intermedia和24个Nigrescens的扩增DNA的特异性杂交，并且未观察到来自20个外来物种的PCR产物的反应。此处描述的PCR-DNA探针检测应能够在混合感染中高度特异性和灵敏地检测中间感假单胞菌和黑斑假单胞菌。

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