Verma A, Rattan A, Tyagi J S
Department of Biotechnology, All India Institute of Medical Sciences, New Delhi.
Indian J Biochem Biophys. 1994 Aug;31(4):288-94.
The partial nucleotide sequence of a recombinant plasmid containing the 23S rRNA gene of Mycobacterium tuberculosis was determined and an assay was developed for amplifying 23S rRNA gene sequences of mycobacteria. The PCR-based non-radioactive test enabled us to distinguish Mycobacterium from other closely related genera and was sensitive enough to detect 2 bacterial genome equivalents. The assay was extended to the detection of mycobacterial DNA in uncultured clinical specimens; 23S rRNA sequences were detected in thirty four of forty eight (70.8%) sputum and cerebrospinal fluid (CSF) specimens by the PCR assay, whereas direct smear examination and culture methods demonstrated a positivity rate of 29.2% and 16.7% respectively for the same specimens. A RNA-based PCR assay with a detection limit of 1 genome equivalent was also developed. These PCR assays should prove useful for the early and rapid detection of mycobacterial infection in uncultured clinical specimens.
测定了含有结核分枝杆菌23S rRNA基因的重组质粒的部分核苷酸序列，并开发了一种用于扩增分枝杆菌23S rRNA基因序列的检测方法。基于PCR的非放射性检测使我们能够区分分枝杆菌与其他密切相关的属，并且灵敏度足以检测2个细菌基因组当量。该检测方法扩展到未培养临床标本中分枝杆菌DNA的检测；通过PCR检测，在48份痰液和脑脊液（CSF）标本中的34份（70.8%）中检测到了23S rRNA序列，而相同标本的直接涂片检查和培养方法的阳性率分别为29.2%和16.7%。还开发了一种检测限为1个基因组当量的基于RNA的PCR检测方法。这些PCR检测方法应证明对未培养临床标本中分枝杆菌感染的早期快速检测有用。
