Tuberculosis Research Today is a free monthly online journal that collates and summarizes the latest research about Tuberculosis, including details on symptoms, causes, treatment, pulmonary, mycobacterium.

Novel wide-range quantitative nested real-time PCR assay for Mycobacterium tuberculosis DNA: development and methodology.

Takahashi T, Tamura M, Asami Y, Kitamura E, Saito K, Suzuki T, Takahashi SN, Matsumoto K, Sawada S, Yokoyama E, Takasu T

Advanced Research Institute for the Sciences and Humanities, Nihon University, Tokyo, Japan. teruyuk@med.nihon-u.ac.jp

Previously, we designed an internally controlled quantitative nested real-time (QNRT) PCR assay for Mycobacterium tuberculosis DNA in order to rapidly diagnose tuberculous meningitis. This technique combined the high sensitivity of nested PCR with the accurate quantification of real-time PCR. In this study, we attempted to improve the original QNRT-PCR assay and newly developed the wide-range QNRT-PCR (WR-QNRT-PCR) assay, which is more accurate and has a wider detection range. For use as an internal-control "calibrator" to measure the copy number of M. tuberculosis DNA, an original new-mutation plasmid (NM-plasmid) was developed. It had artificial random nucleotides in five regions annealing specific primers and probes. The NM-plasmid demonstrated statistically uniform amplifications (F = 1.086, P = 0.774) against a range (1 to 10(5)) of copy numbers of mimic M. tuberculosis DNA and was regarded as appropriate for use as a new internal control in the WR-QNRT-PSR assay. In addition, by the optimization of assay conditions in WR-QNRT-PCR, two-step amplification of target DNA was completely consistent with the standard curve of this assay. Due to the development of the NM-plasmid as the new internal control, significantly improved quantitative accuracy and a wider detection range were realized with the WR-QNRT-PCR assay. In the next study, we will try to use this novel assay method with actual clinical samples and examine its clinical usefulness.

Published 7 May 2008 in J Clin Microbiol, 46(5): 1708-15.
Full-text of this article is available online (may require subscription).

© 2004-2008 Tuberculosis Research Today. All Rights Reserved.