title: Combined rpoB duplex PCR and hsp65 PCR restriction fragment length polymorphism with capillary electrophoresis as an effective algorithm for identification of mycobacterial species from clinical isolates.
abstract: BACKGROUND
  Mycobacteria can be quickly and simply identified by PCR restriction-enzyme analysis (PRA), but misidentification can occur because of similarities in band sizes that are critical for discriminating among species. Capillary electrophoresis can provide computer-aided band discrimination. The aim of this research was to develop an algorithm for identifying mycobacteria by combined rpoB duplex PRA (DPRA) and hsp65 PRA with capillary electrophoresis.
  
  
  RESULTS
  Three hundred and seventy-six acid-fast bacillus smear-positive BACTEC cultures, including 200 Mycobacterium tuberculosis complexes (MTC) and 176 non-tuberculous mycobacteria (NTM) were analyzed. With combined hsp65 and rpoB DPRA, the accuracy rate was 100% (200 isolates) for the MTC and 91.4% (161 isolates) for the NTM. Among the discordant results (8.6%) for the NTM, one isolate of Mycobacterial species and an isolate of M. flavescens were found as new sub-types in hsp65 PRA.
  
  
  CONCLUSIONS
  This effective and novel identification algorithm using combined rpoB DPRA and hsp65 PRA with capillary electrophoresis can rapidly identify mycobacteria and find new sub-types in hsp65 PRA. In addition, it is complementary to 16 S rDNA sequencing.
authors: Department of internal medicine, Executive Yuan Department of health, Division of Respiratory and Critical Care Medicine, Taichung Hospital, Taichung, Taiwan.
authors: Chen-Cheng
authors: CC
authors: Huang
authors: Jiann-Hwa
authors: JH
authors: Chen
authors: Shiau-Ting
authors: ST
authors: Hu
authors: Chien-Shun
authors: CS
authors: Chiou
authors: Wei-Chang
authors: WC
authors: Huang
authors: Jeng-Yuan
authors: JY
authors: Hsu
authors: Jang-Jih
authors: JJ
authors: Lu
authors: Gwan-Han
authors: GH
authors: Shen
mesh_terms: D001426:Bacterial Proteins / Q000235:genetics*; D001431:Bacteriological Techniques / Q000379:methods; D018834:Chaperonin 60 / Q000235:genetics*; D012321:DNA-Directed RNA Polymerases / Q000235:genetics*; D019075:Electrophoresis, Capillary / Q000379:methods*; D006801:Humans; D025202:Molecular Diagnostic Techniques / Q000379:methods; D060885:Multiplex Polymerase Chain Reaction / Q000379:methods*; D009161:Mycobacterium / Q000145:classification* / Q000235:genetics / Q000302:isolation & purification*; D012680:Sensitivity and Specificity; D014376:Tuberculosis / Q000175:diagnosis / Q000382:microbiology*
chemical_list: D001426:Bacterial Proteins; D018834:Chaperonin 60; C074119:heat-shock protein 65, Mycobacterium; D012321:DNA-Directed RNA Polymerases; C105078:RNA polymerase beta subunit