Tung Sheng Kai, Teng Lee Jene, Vaneechoutte Mario, Chen Hung Mo, Chang Tsung Chain
Department of Medical Laboratory Science and Biotechnology, School of Medicine, National Cheng Kung University, Tainan, Taiwan, Republic of China, and Department of Clinical Chemistry, Microbiology and Immunology, Ghent University Hospital, Belgium.
J Clin Microbiol. 2006 Dec;44(12):4414-24. doi: 10.1128/JCM.01712-06. Epub 2006 Oct 25.
Some species of enterococci and streptococci are difficult to differentiate by phenotypic traits. The feasibility of using an oligonucleotide array for identification of 11 viridans group streptococci was previously established. The aim of this study was to expand the array to identify species of Abiotrophia (1 species), Enterococcus (18 species), Granulicatella (3 species), and Streptococcus (31 species and 6 subspecies). The method consisted of PCR amplification of the ribosomal DNA intergenic spacer (ITS) regions, followed by hybridization of the digoxigenin-labeled PCR products to a panel of oligonucleotide probes (16- to 30-mers) immobilized on a nylon membrane. Probes could be divided into three categories: species specific, group specific, and supplemental probes. All probes were designed either from the ITS regions or from the 3' ends of the 16S rRNA genes. A collection of 312 target strains (162 reference strains and 150 clinical isolates) and 73 nontarget strains was identified by the array. Most clinical isolates were isolated from blood cultures or deep abscesses, and only those strains having excellent species identification with the Rapid ID 32 STREP system (bioMérieux Vitek, Taipei, Taiwan) were used for array testing. The test sensitivity and specificity of the array were 100% (312/312) and 98.6% (72/73), respectively. The whole procedure of array hybridization took about 8 h, starting from isolated colonies, and the hybridization patterns could be read by the naked eye. The oligonucleotide array is accurate for identification of the above microorganisms and could be used as a reliable alternative to phenotypic identification methods.
某些肠球菌和链球菌的种别很难通过表型特征加以区分。此前已证实使用寡核苷酸芯片鉴定11种草绿色链球菌群的可行性。本研究的目的是扩展该芯片，以鉴定阿比奥罗菲亚菌属（1个种）、肠球菌属（18个种）、颗粒链菌属（3个种）以及链球菌属（31个种和6个亚种）。该方法包括核糖体DNA基因间隔区（ITS）的PCR扩增，随后将地高辛标记的PCR产物与固定在尼龙膜上的一组寡核苷酸探针（16至30聚体）进行杂交。探针可分为三类：种特异性探针、群特异性探针和补充探针。所有探针均从ITS区域或16S rRNA基因的3'端设计。用该芯片鉴定了312株靶菌株（162株参考菌株和150株临床分离株）以及73株非靶菌株。大多数临床分离株取自血培养物或深部脓肿，只有那些在快速ID 32 STREP系统（生物梅里埃公司，台湾台北）中能被准确鉴定种别的菌株才用于芯片检测。该芯片检测的敏感性和特异性分别为100%（312/312）和98.6%（72/73）。从分离的菌落开始，芯片杂交全过程约需8小时，杂交图谱肉眼即可读取。该寡核苷酸芯片对上述微生物的鉴定准确，可作为表型鉴定方法的可靠替代方法使用。
