This Article
Right arrow Full Text
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowReprints and Permissions
Right arrow Copyright Information
Right arrow Books from ASM Press
Right arrow MicrobeWorld
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Takahashi, T.
Right arrow Articles by Takasu, T.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Takahashi, T.
Right arrow Articles by Takasu, T.

 Previous Article  |  Next Article 

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{triangledown}

Teruyuki Takahashi,1* Masato Tamura,2 Yukihiro Asami,1 Eiko Kitamura,1 Kosuke Saito,1 Tsukasa Suzuki,1 Sachiko Nonaka Takahashi,3 Koichi Matsumoto,3 Shigemasa Sawada,4 Eise Yokoyama,5 and Toshiaki Takasu1,2

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.

* Corresponding author. Mailing address: Advanced Research Institute for the Sciences and Humanities, Nihon University School of Medicine, Research Center 2F, Ooyaguchi-kamimachi, 30-1 Itabashi-ku, Tokyo 173-8610, Japan. Phone: 81 3-3972-8337. Fax: 81 3-5964-0464. E-mail: teruyuk{at}med.nihon-u.ac.jp

{triangledown} Published ahead of print on 12 March 2008.

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.



This article has been cited by other articles:

  • 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]  


Copyright © 2009 by the American Society for Microbiology.   For an alternate route to Journals.ASM.org, visit: http://intl-journals.asm.org | More Info»