title: Rapid detection and identification of clinically important bacteria by high-resolution melting analysis after broad-range ribosomal RNA real-time PCR.
abstract: BACKGROUND
  Broad-range PCR provides valuable information for detecting bacterial infections. This study assesses the combined use of broad-range real-time PCR and high-resolution melting analysis for rapid detection and identification of clinically important bacteria.
  
  
  METHODS
  We subjected 46 bacterial culture colonies representing 25 clinically important bacterial species to LightCycler real-time PCR amplification of the 16S rRNA gene in the presence of LCGreen I fluorescent dye. We performed high-resolution melting analysis of the PCR products with the HR-1 instrument and used melting profiles as molecular fingerprints for bacterial species identification. We validated this method via assessment of 54 consecutive bacteria culture colonies obtained from a clinical microbiology laboratory.
  
  
  RESULTS
  The 16S rRNA gene of all 25 bacterial species was amplifiable by this method, with PCR product lengths of 216 or 217 bp. Of the 25 bacterial species, we identified 11 via a 1-step post-PCR high-resolution melting analysis. The remaining bacterial species were identified via the high-resolution melting plots obtained by heteroduplex formation between the PCR products of the tested and reference bacterial species or by a 2nd real-time PCR targeting a different region of the 16S rRNA gene. A high-resolution melting database and a working protocol were established for identifying these 25 bacterial species. In the validation assay, a 94% accuracy rate was achieved when the bacterial species were in the high-resolution melting database.
  
  
  CONCLUSIONS
  This assay requires no multiplexing or hybridization probes and provides a new approach for bacterial species identification in a molecular diagnostic laboratory.
authors.affiliation: School of Medical Laboratory Science and Biotechnology, China Medical University, Taichung, and Department of Pathology and Laboratory Medicine, Li Shin Hospital, Tao-Yuan, Taiwan, Republic of China.
authors.forename: Ju-Chien
authors.initials: JC
authors.lastname: Cheng
authors.forename: Chien-Ling
authors.initials: CL
authors.lastname: Huang
authors.forename: Chung-Ching
authors.initials: CC
authors.lastname: Lin
authors.forename: Chi-Ching
authors.initials: CC
authors.lastname: Chen
authors.forename: Yi-Chih
authors.initials: YC
authors.lastname: Chang
authors.forename: Shy-Shin
authors.initials: SS
authors.lastname: Chang
authors.forename: Ching-Ping
authors.initials: CP
authors.lastname: Tseng
mesh_terms: D001419:Bacteria / Q000145:classification* / Q000235:genetics / Q000302:isolation & purification; D001424:Bacterial Infections / Q000175:diagnosis*; D004269:DNA, Bacterial / Q000737:chemistry* / Q000235:genetics*; D019937:Diagnostic Techniques and Procedures; D009691:Nucleic Acid Denaturation; D012336:RNA, Ribosomal, 16S / Q000235:genetics*; D015203:Reproducibility of Results; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D013696:Temperature
chemical_list: D004269:DNA, Bacterial; D012336:RNA, Ribosomal, 16S