Widjojoatmodjo M N, Fluit A C, Verhoef J
p4p4man Winkler Institute for Medical and Clinical Microbiology, University Hospital Utrecht, The Netherlands.
J Clin Microbiol. 1995 Oct;33(10):2601-6. doi: 10.1128/jcm.33.10.2601-2606.1995.
PCR-single-strand conformation polymorphism (PCR-SSCP) analysis is a rapid and convenient technique for the detection of mutations and allelic variants. We have adapted this technique for the identification of bacteria by PCR with fluorescein-labeled primers chosen from the conserved regions of the 16S rRNA gene flanking a variable region. The PCR product was denatured, separated on a nondenaturing gel, and detected by an automated DNA sequencer. The mobility of the single-stranded DNA is sequence dependent and allows the identification of a broad panel of bacteria. A single nucleotide difference in the amplified region was sufficient to obtain different PCR-SSCP patterns. The simultaneous amplification of multiple polymorphic regions by multiplex PCR with subsequent multiplex SSCP increased the discriminatory power of PCR-SSCP. A broad range of gram-negative and gram-positive bacteria were tested by PCR-SSCP, including, e.g., Escherichia coli, Enterobacter spp., Klebsiella spp., Haemophilus spp., Neisseria spp., Staphylococcus spp, Streptococcus spp., Enterococcus spp., and Bacillus spp. In total, a panel of 178 strains of bacteria representing 51 species in 21 genera was examined. Although a limited number of strains from each species were tested, the strains tested gave species-specific patterns, with only one exception: Shigella species were indistinguishable from E. coli. PCR is a sensitive technique; as few as 10 CFU of E. coli was sufficient to produce PCR-SSCP patterns suitable for identification. The whole fluorescence PCR-SSCP procedure takes approximately 8 h for the detection and identification of low numbers of bacteria.2+ fluorescence PCR-SSCP seems to be a promising method for the differentiation of a broad range of pathogens found in usually sterile clinical sites, such as blood and cerebrospinal fluid.
聚合酶链反应-单链构象多态性（PCR-SSCP）分析是一种用于检测突变和等位基因变异的快速便捷技术。我们已将该技术进行改进，通过使用从16S rRNA基因可变区侧翼保守区域选取的荧光素标记引物进行PCR来鉴定细菌。PCR产物经变性后，在非变性凝胶上进行分离，然后通过自动DNA测序仪进行检测。单链DNA的迁移率取决于序列，可用于鉴定多种细菌。扩增区域内的单个核苷酸差异足以产生不同的PCR-SSCP图谱。通过多重PCR同时扩增多个多态性区域并随后进行多重SSCP，提高了PCR-SSCP的鉴别能力。我们用PCR-SSCP对多种革兰氏阴性菌和革兰氏阳性菌进行了检测。例如，包括大肠杆菌、肠杆菌属、克雷伯菌属、嗜血杆菌属、奈瑟菌属、葡萄球菌属、链球菌属、肠球菌属和芽孢杆菌属。总共检测了代表21个属51个物种的178株细菌。尽管每个物种检测的菌株数量有限，但所检测的菌株呈现出物种特异性图谱，只有一个例外：志贺菌属与大肠杆菌无法区分。PCR是一种灵敏的技术；仅10个大肠杆菌菌落形成单位就足以产生适用于鉴定的PCR-SSCP图谱。整个荧光PCR-SSCP程序检测和鉴定少量细菌大约需要8小时。荧光PCR-SSCP似乎是一种很有前景的方法，可用于区分通常无菌临床部位（如血液和脑脊液）中发现的多种病原体。
