title: Novel wide-range quantitative nested real-time PCR assay for Mycobacterium tuberculosis DNA: development and methodology.
abstract: 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.
authors.affiliation: Advanced Research Institute for the Sciences and Humanities, Nihon University, Tokyo, Japan. teruyuk@med.nihon-u.ac.jp
authors.forename: Teruyuki
authors.initials: T
authors.lastname: Takahashi
authors.forename: Masato
authors.initials: M
authors.lastname: Tamura
authors.forename: Yukihiro
authors.initials: Y
authors.lastname: Asami
authors.forename: Eiko
authors.initials: E
authors.lastname: Kitamura
authors.forename: Kosuke
authors.initials: K
authors.lastname: Saito
authors.forename: Tsukasa
authors.initials: T
authors.lastname: Suzuki
authors.forename: Sachiko Nonaka
authors.initials: SN
authors.lastname: Takahashi
authors.forename: Koichi
authors.initials: K
authors.lastname: Matsumoto
authors.forename: Shigemasa
authors.initials: S
authors.lastname: Sawada
authors.forename: Eise
authors.initials: E
authors.lastname: Yokoyama
authors.forename: Toshiaki
authors.initials: T
authors.lastname: Takasu
mesh_terms: D015169:Colony Count, Microbial / Q000379:methods; D004269:DNA, Bacterial / Q000235:genetics / Q000302:isolation & purification*; D006801:Humans; D009169:Mycobacterium tuberculosis / Q000235:genetics / Q000302:isolation & purification*; D010957:Plasmids; D016133:Polymerase Chain Reaction / Q000379:methods* / Q000592:standards; D012015:Reference Standards; D012680:Sensitivity and Specificity; D014390:Tuberculosis, Meningeal / Q000175:diagnosis* / Q000382:microbiology
chemical_list: D004269:DNA, Bacterial