[Rapid identification of mycobacteria by combined method of polymerase chain reaction and the gen-probe DNA hybridization system].
We developed the rapid detection and identification method of mycobacteria, involving amplification of mycobacterial 16S rRNA gene by nested PCR and identification of M. tuberculosis complex or M. avium-intracellulare complex (MAC) by hybridization protection assay (HPA) using the acridinium-ester (AE) labeled DNA probe. The specificity of the nested PCR combined with DNA probe test was excellent in terms of detection of mycobacterial organisms and identification of M. tuberculosis or MAC. The detection limits of the present method were 10 fg DNA for M. tuberculosis, and 100 fg DNA for MAC, respectively. We further investigated on the optimum temperature for hybridization in HPA with AE labeled DNA probe because there was the difference in the mode of DNA-RNA hybridization from that of DNA-DNA hybridization. In our method, the optimum temperature of hybridization was estimated as 55 +/- 1 degrees C. In preliminary experiments on two clinical cases, we practically detected and identified M. tuberculosis and MAC in clinical specimens, such as sputa, by using this newly devised method. We concluded that this method is useful for rapid detection and identification of M. tuberculosis and MAC in clinical specimens.
Second Department of Internal Medicine, Nagasaki University School of Medicine, Japan.
A
A
Hashimoto
H
H
Koga
S
S
Kohno
Y
Y
Miyazaki
H
H
Ohno
K
K
Ogawa
Y
Y
Higashiyama
K
K
Tomono
M
M
Kaku
K
K
Hara
D000328:Adult; D000368:Aged; D000369:Aged, 80 and over; D015342:DNA Probes; D004269:DNA, Bacterial / Q000032:analysis*; D005260:Female; D006801:Humans; D008297:Male; D009161:Mycobacterium / Q000235:genetics / Q000302:isolation & purification*; D015269:Mycobacterium avium Complex / Q000235:genetics / Q000302:isolation & purification; D009169:Mycobacterium tuberculosis / Q000235:genetics / Q000302:isolation & purification; D009693:Nucleic Acid Hybridization; D016133:Polymerase Chain Reaction
D015342:DNA Probes; D004269:DNA, Bacterial