在鏈球菌屬(Streptococcus)、腸球菌屬(Enterococcus)、和營養需求鏈球菌(nutritionally variant streptococci, 包含Abiotrophia和Granulicatella)中，有許多菌種為人類的致病菌。本研究欲利用16S和23S rRNA基因的內轉錄區域(intergenic spacer, ITS)的序列及DNA晶片來鑑定這四個菌屬的菌株。本研究定序62標準菌株(type strain) (55種)的ITS序列，並結合由GenBank取得的12個ITS序列構成一個ITS序列資料庫。不同菌種的ITS長度介於189-602個鹼基，不同種間(interspecies) ITS序列變化大，其序列相似值(sequence similarity)介於0.31到0.996之間，而種內(intraspecies)不同菌株間有相當高的相似性(Granulicatella elegans除外)，其序列相似值介於0.98到1之間。以此資料庫鑑定227菌株[包含89參考菌株(reference strain)和138臨床分離株]，鑑定結果不一致的臨床菌株再次利用Rapid ID 32 STREP system (bioMrieux Vitek, Marcy l’Etoile, France)鑑定，最後仍有11.9% (27株/227株)的菌株不相符合。以ITS序列和生化鑑定結果不同的菌株，再以16S rRNA基因序列做確認，結果顯示ITS序列鑑定的正確率高達98.2% (223株/227株)，其中未能鑑定的4株菌是因為資料庫中未含有該菌種的ITS序列。因此利用ITS序列鑑定Abiotrophia, Enterococcus, Granulicatella和Streptococcus具有很高的正確性。本研究進一步利用ITS當作目標基因，設計一個寡核酸晶片並利用雜合反應(hybridization)來鑑定這些菌種。一共測試了391菌株，包含336目標菌株(target strain) (160參考菌株和176臨床菌株)和55非目標菌株(nontarget strain)，晶片鑑定之靈敏度(sensitivity)為100% (336株/336株)，特異性(specificity)為96.4% (53株/55株)。本研究的結論是利用ITS序列或晶片雜合法來鑑定Abiotrophia, Enterococcus, Granulicatella和Streptococcus具有很高的正確性。

　Many species belonging to Enterococcus, Streptococcus, and nutritionally variant streptococci (NVS) including Abiotrophia and Granulicatella, are human pathogens. The feasibility of sequence analysis of the 16S-23S ribosomal DNA intergenic spacer (ITS) for identification of strains belonging to the four genera was evaluated. In this study, ITS sequences of 62 type strains (55 species) and 12 ITS sequences obtained from the GenBank database were used to construct an ITS database for identicication of strains belonging to Enterococcus, Streptococcus, and NVS. The ITS lengths of different species varied from 189 to 602 bp, with interspecies sequence similarities ranging from 0.31 to 0.996. ITS sequences were highly conserved among strains within the same species, with intraspecies similarity ranged from 0.98 to 1.0, except for strains of Granulicatella elegans. A total of 227 strains, including 89 reference strains and 138 clinical isolates, were identified by ITS sequencing. Cinical isolates producing discrepant species names by ITS sequencingwere reconfirmed by the Rapid ID 32 STREP system (bioMe’rieux Vitek, Marcy l’Etoile, France). A total of 27 strains (11.9%, 27/227), including reference strains and clinical isolates, were found to produce discrepant identification results between by ITS sequencing and. The sequences of 16S rRNA genes were further determined for species confirmation of strains producing discrepant identification. The sensitivity of ITS sequence analysis for species identification was 98.2% (226/230). Four strains were not identified by ITS sequencing because their corresponding ITS sequences were not available in the ITS sequence database. Based on ITS sequence, an oligonucleotide array was designed to identify these bacteria. A total of 391 strains, including 336 target strains (160 reference strains and 176 clinical isolates) and 55 nontarget strains were identified by the array. The sensitivity and specificity of the array were 100% (336/336) and 96.4% (53/55), respectively. In conclusion, both ITS sequence analysis and the array hybridization method could be used as accurate alternatives for species identification of Abiotrophia, Enterococcus, Granulicatella, and Streptococcus.

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