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Journal of Clinical Microbiology, May 2008, p. 1708-1715, Vol. 46, No. 5
0095-1137/08/$08.00+0     doi:10.1128/JCM.01200-07
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

Novel Wide-Range Quantitative Nested Real-Time PCR Assay for Mycobacterium tuberculosis DNA: Development and Methodology

Advanced Research Institute for the Sciences and Humanities, Nihon University, Tokyo, Japan,¹ Division of Neurology, Department of Medicine, Nihon University School of Medicine, Tokyo, Japan,² Division of Nephrology and Endocrinology, Department of Medicine, Nihon University School of Medicine, Tokyo, Japan,³ Department of Internal Medicine, Nihon University Nerima-Hikarigaoka Hospital, Tokyo, Japan,⁴ Department of Public Health, Nihon University School of Medicine, Tokyo, Japan⁵

Received 14 June 2007/ Returned for modification 11 October 2007/ Accepted 27 February 2008

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 10⁵) 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 ahead of print on 12 March 2008.

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• Takahashi, T., Tamura, M., Asami, Y., Kitamura, E., Saito, K., Suzuki, T., Takahashi, S. N., Matsumoto, K., Sawada, S., Yokoyama, E., Takasu, T. (2008). Novel Wide-Range Quantitative Nested Real-Time PCR Assay for Mycobacterium tuberculosis DNA: Clinical Application for Diagnosis of Tuberculous Meningitis. J. Clin. Microbiol. 46: 1698-1707 [Abstract] [Full Text]