title: Rapid and specific detection of Mycobacterium tuberculosis from acid-fast bacillus smear-positive respiratory specimens and BacT/ALERT MP culture bottles by using fluorogenic probes and real-time PCR.
abstract: A real-time PCR assay using the LightCycler (LC) instrument for the specific identification of Mycobacterium tuberculosis complex (MTB) was employed to detect organisms in 135 acid-fast bacillus (AFB) smear-positive respiratory specimens and in 232 BacT/ALERT MP (MP) culture bottles of respiratory specimens. The LC PCR assay was directed at the amplification of the internal transcribed spacer region of the Mycobacterium genome with real-time detection using fluorescence resonance energy transfer probes specific for MTB. The results from the respiratory specimens were compared to those from the Amplicor M. tuberculosis PCR test. Specimens from MP culture bottles were analyzed by Accuprobe and conventional identification methods. MTB was cultured from 105 (77.7%) respiratory AFB smear-positive specimens; 103 of these samples were positive by LC PCR and Amplicor PCR. Two samples negative in the LC assay contained rare numbers of organisms; both were positive in the Amplicor assay. Two separate samples negative by Amplicor PCR contained low and moderate numbers of AFB, respectively, and both of these were positive in the LC assay. There were 30 AFB smear-positive respiratory specimens that grew mycobacteria other than tuberculosis (MOTT), and all tested negative in both assays. Of the 231 MP culture bottles, 114 cultures were positive for MTB and all were positive by the LC assay. The remaining 117 culture bottles were negative in the LC assay and grew various MOTT. This real-time MTB assay is sensitive and specific; a result was available within 1 h of having a DNA sample available for testing.
authors.affiliation: Department of Pathology. Department of Microbiology and Immunology, University of Miami School of Medicine, Miami, Florida 33136, USA. nmiller@med.miami.edu
authors.forename: Nancimae
authors.initials: N
authors.lastname: Miller
authors.forename: Tim
authors.initials: T
authors.lastname: Cleary
authors.forename: Günter
authors.initials: G
authors.lastname: Kraus
authors.forename: Andrea K
authors.initials: AK
authors.lastname: Young
authors.forename: Gina
authors.initials: G
authors.lastname: Spruill
authors.forename: H James
authors.initials: HJ
authors.lastname: Hnatyszyn
mesh_terms: D015373:Bacterial Typing Techniques; D003470:Culture Media; D031541:Fluorescence Resonance Energy Transfer; D005456:Fluorescent Dyes; D006801:Humans; D009169:Mycobacterium tuberculosis / Q000235:genetics / Q000254:growth & development / Q000302:isolation & purification*; D016133:Polymerase Chain Reaction / Q000379:methods*; D011933:Reagent Kits, Diagnostic; D012137:Respiratory System / Q000382:microbiology*; D012680:Sensitivity and Specificity; D013048:Specimen Handling / Q000379:methods*; D013696:Temperature; D013997:Time Factors; D014397:Tuberculosis, Pulmonary / Q000382:microbiology
chemical_list: D003470:Culture Media; D005456:Fluorescent Dyes; D011933:Reagent Kits, Diagnostic