Detection of mycobacteria by multiplex nucleic acid amplificatio

Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Preparing compound containing saccharide radical

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435 6, 536 2432, 536 2433, 935 17, 935 77, 935 78, C12Q 168, C12P 1934, C07H 1512, C07H 1700

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054707234

ABSTRACT:
Primers and methods for adapter-mediated multiplex amplification of the IS6110 insertion element of Mycobacterium tuberculosis (M.tb) and the 16S ribosomal gene of Mycobacterium tuberculosis, useful for simultaneously detecting and/or identifying species of the M. tuberculosis complex and other clinically relevant Mycobacterium species. Multiplex Strand Displacement Amplification (SDA) is used in a single amplification reaction which is capable of simultaneously identifying M. tuberculosis and providing a screen for substantially all of the clinically relevant species of Mycobacteria. Also disclosed are methods for adapter-mediated multiplex amplification of multiple target sequences and a single internal control sequence for determination of sample efficacy or quantitation of the targets. In a preferred embodiment, an internal control sequence is included in the amplification reaction and coamplified with the IS6110 ant 16S target sequences as an indication of sample amplification activity or to quantitate the initial amount of target sequences in the sample.

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G. T. Walker, et al. "Strand displacement amplification--an isothermal in vitro DNA amplification technique" Nuc. Acids Res. 20:1691-1696 (1992).
G. T. Walker, et al. "Isothermal in vitro amplification of DNA by a restriction enzyme/DNA polymerase system" Proc. Natl. Acad. Sci. USA 89:392-396 (1992).
P. R. Mueller, et al. "In Vivo Footprinting of a Muscle Specific Enhancer by Ligation Mediated PCR" Science 246:780-246 (1989).

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