Development of an Oligonucleotide Array for Identification of Clinically Important Anaerobic Bacteria

• Main Authors: Yu-tzu Lin, 林佑姿
• Other Authors: Tsung-chain Chang
• Format: Others
• Language: zh-TW
• Published: 2009
• Online Access: http://ndltd.ncl.edu.tw/handle/12795715873654656982

碩士 === 國立成功大學 === 醫學檢驗生物技術學系碩博士班 === 97 === Anaerobic bacteria are clinically important pathogens that can cause a wide variety of infections. Because of some serious infections are caused by anaerobic bacteria and conventional identification of these bacteria is time-consuming, rapid and accurate identification of these microorganisms may have clinical importance. In this study, an oligonucleotide array was developed to identify clinically frequently isolated anaerobes. Species-specific oligonucleotide probes were designed from the 16S-23S ribosomal DNA intergenic spacer (ITS) regions and immobilized on nylon membrane. The ITS regions of anaerobic bacteria were amplified by PCR and hybridized to probes on the membrane for species identification. A total of 52 probes, including 46 species-specific probes and 6 group-specific probes, were used to fabricate an oligonucleotide array (0.9 cm × 0.8 cm) to identify 27 species of anaerobic bacteria and the genus Veillonella. A collection of 119 reference target strains (strains I aimed to identify) were tested by the array for species identification and four strains were found to produce discrepant identification by the array. The four discordant strains were further analyzed by sequencing of the 16S rRNA gene for species clarification, three strains were found to be correctly identified by the array, with the remaining one being an nontarget strains. In addition, a collection of 189 target clinical isolates (identified by Rapid ID 32A or RapID ANA II) were identified by the array and among them, 160 stains were correctly identified, with 29 strais producing discrepant identification. These 29 discrepant clinical isolates were retested with Rapid ID 32A and sequencing of the 16S rRNA gene. The results demonstrated that among the 189 clinical isolates, 181 were target strains. In addition, two additional strains that was originally identified as nontarget strains were found to be target strains by array hybridization and sequencing of the 16S rRNA gene. Of the 183 target clinical isolates, 169 were correctly identified by the array. Moreover, 105 nontarget strains (50 species, including 45 reference strains and 60 clinical isolates) were used for specificity test of the array. Among these nontarget strains, only one strain was misidentified by the array. In conclusion, The sensitivity and specificity of the array for identification of the 27 species of anaerobic bacteria and Veillonella were 95.3% (287/301) and 99.0% (104/105), respectively. The current array can be used as an accurate alternative of conventional methods for identification of clinically important anaerobic bacteria.