Details

Autor(en) / Beteiligte

• Mantha, Stacey Lynn

Titel

Direct detection and rapid identification of clinically significant mycobacteria from Bactec 12B medium and acid-fast bacilli positive sputum specimens

Ort / Verlag

ProQuest Dissertations & Theses

Erscheinungsjahr

2000

Quelle

ProQuest Dissertations & Theses A&I

Beschreibungen/Notizen

• With the advent of the AIDS epidemic, infections due to Mycobacterium tuberculosis and pathogenic non-tuberculous mycobacteria have become increasingly common. Present identification of mycobacteria from clinical specimens can take up to two months and be inconclusive. The introduction of nucleic acid probes has greatly decreased the time required for identification although these methods are expensive and not available for all clinically significant mycobacterial species. We evaluated the use of a PCR-RFLP (PCR/REA) method involving PCR amplification of a 439bp fragment of the 65kDa heat shock protein gene, ubiquitous to all mycobacteria, followed by restriction enzyme analysis of the product with HaeIII and BstEII. In brief, the DNA from 47 acid fast bacilli (AFB) smear positive concentrated sputum specimens, and 106 AFB smear positive cultures grown in Bactec 12B medium, were amplified using primers Th11 and Th12. The amplicons, were then digested with restriction enzymes and analyzed using agarose gel electrophoresis. This method allows for speciation in 6 hours following a positive PCR result (11 hours in total) and is a vast improvement over the standard method presently in use in diagnostic laboratories with respect to both cost and timeliness.