Abstract: The present invention provides primers which can be used for M. tuberculosis complex-specific detection of .alpha.-antigen DNA in a diagnostic assay performed on clinical specimens or in a culture-confirmation assay following growth of the organism in vitro. These primers and probes can also be employed in a reverse transcriptase-mediated amplification system for M. tuberculosis complex .alpha.-antigen mRNA. Such an assay provides a means by which to determine the viability of M. tuberculosis complex organisms either in clinical specimens or when grown in culture. The specific DNA or mRNA target region can be amplified using SDA, PCR, LCR, Nucleic Acid Sequence Based Amplification (NASBA), Self-sustained Sequence Replication (3SR) or Q.beta. Replicase-mediated systems. Also described are methods for the detection of the products of amplification with a radiolabeled probe by chemiluminescent assay or fluorescence polarization analysis.

Abstract: Fluorescence polarization methods for detection of nucleic acid amplification at thermophilic temperatures employ a fluorescently labeled oligonucleotide signal primer which is converted from single- to double-stranded form in a target amplification-dependent manner. This conformational change is accompanied by an increase in fluorescence polarization values. The decrease in FP typically observed for the duplex at elevated temperatures is overcome by double-stranded DNA binding proteins which are believed to stabilize the double-stranded structure by reducing the single-strandedness normally associated with higher temperatures. The inventive methods provide a closed, homogeneous system for amplification and detection of amplification in real-time or at an endpoint.

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.

Abstract: Methods for identification or detection of a species of an organism or a group of related species of an organism by species non-specific amplification of a target sequence followed by species- or group-specific detection of the amplification products. Also provided are a target sequence which is amplifiable in multiple species of mycobacteria using a single pair of amplification primers and species- and group-specific detector probes for hybridization to the assay regioin of the amplified target. Blocking oligonucleotides are employed to allow discrimination among species in which the amplified target sequences are sufficiently similar that they cross-hybridize to an assay probe.

Abstract: The present invention provides methods for detecting amplified or unamplified nucleic acid target sequences at increased temperatures by changes in fluorescence polarization. The decrease in fluorescence polarization associated with hybridization of oligonucleotides at higher, more stringent, temperatures is overcome by including a double-stranded DNA binding protein in the assay. At elevated temperatures, the double-stranded DNA binding protein restores, and often enhances, the magnitude of the change in fluorescence polarization associated with single- to double-stranded conversion of an oligonucleotide probe or primer.