van der Vliet G M, Schepers P, Schukkink R A, van Gemen B, Klatser P R
Department of Biomedical Research, Royal Tropical Institute, Amsterdam, The Netherlands.
Antimicrob Agents Chemother. 1994 Sep;38(9):1959-65. doi: 10.1128/AAC.38.9.1959.
We investigated whether the presence of intact RNA is a valuable indicator of viability of mycobacteria with Mycobacterium smegmatis. M. smegmatis was exposed to various concentrations of rifampin and ofloxacin suspended in broth for different periods of time. The NASBA nucleic acid amplification system was used because of its rapid, sensitive, and specific detection of 16S rRNA. During drug exposure, the viability of the mycobacteria, expressed by the number of CFU, was compared with the presence of 16S rRNA as determined by NASBA and with the presence of DNA coding for 16S rRNA as determined by PCR. Both NASBA and PCR were shown to have a detection limit of approximately 5 x 10(2) CFU/ml. The intensity of the NASBA signal corresponded well with the number of CFU, and the lack of NASBA signal coincided with a loss of viability, which was reached after 3 days of exposure to bactericidal concentrations of both drugs. The presence of mycobacterial DNA, as determined by the intensity of the PCR signal, and the viability of M. smegmatis were not related, but an increase in the number of cells and intensity of PCR signal correlated well. Bacterial viability may thus be assessed by a rapid, sensitive, and specific, and semiquantitative technique by using NASBA. This system of viability testing provides the potential for rapid evaluation of drug susceptibility testing.
我们研究了完整RNA的存在是否是耻垢分枝杆菌分枝杆菌活力的重要指标。将耻垢分枝杆菌暴露于悬浮在肉汤中的不同浓度的利福平和氧氟沙星中不同时间。使用NASBA核酸扩增系统是因为它能快速、灵敏且特异地检测16S rRNA。在药物暴露期间，将以CFU数量表示的分枝杆菌活力与通过NASBA测定的16S rRNA的存在以及通过PCR测定的编码16S rRNA的DNA的存在进行比较。结果表明，NASBA和PCR的检测限均约为5×10(2) CFU/ml。NASBA信号强度与CFU数量良好对应，NASBA信号缺失与活力丧失一致，在暴露于两种药物的杀菌浓度3天后达到这种情况。通过PCR信号强度测定的分枝杆菌DNA的存在与耻垢分枝杆菌的活力无关，但细胞数量增加和PCR信号强度良好对应。因此，可以通过使用NASBA的快速、灵敏、特异且半定量技术来评估细菌活力。这种活力测试系统为药物敏感性测试的快速评估提供了潜力。
