Becton Dickinson and Company Limited

Other

Patricia A Spears

Daryl D Shank

James G Nadeau

Colleen M Nycz

James L Schram

George T Walker

Stewart R Jurgensen

G Terrance Walker

Carl Preston Linn

Michael C Little

Kathleen D Eisenach

Lucy Ellen DesJardin

Mac Donald Cave

Tobin James Hellyer

Daryl Dee Shank

Patricia Brinkley Scott

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.

Detection of M. tuberculosis complex via reverse transcriptase SDA

Amplification primers useful in assays for species-specific detection of a target sequence in the cryptic plasmid of C. trachomatis are described. The primers of the invention amplify a target in the region of nucleotides 2219-2366 of the cryptic plasmid sequence, and the target binding sequences disclosed may be adapted for use in amplification primers for a variety of amplification reactions.

Amplification and detection of chlamydia trachomatis nucleic acids

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.

Method for detection of nucleic acid targets by amplification and

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 and 16S target sequences as an indication of sample amplification activity or to quantitate the initial amount of target sequences in the sample.

Multiplex nucleic acid amplification

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.

Amplification and detection of mycobacterial DNA K nucleic acids

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.

Fluorescence polarization detection of nucleic acids

Detection of mycobacteria by multiplex strand displacement nucleic acid

Oligonucleotide probes and primers which exhibit M. kansasii-specificity in nucleic acid hybridization assays and in nucleic acid amplification reactions are provided. The full-length M. kansasii-specific sequence, identified herein as clone MK7, is 493 base pairs in length and has a GC content of 63%. Several M. kansasii-specific subsequences of MK7 are also provided. The probes and primers are useful in assays for species-specific detection and identification of M. kansasii.

Nucleic acid sequences specific for mycbacterium kansasii

M. kansasii-specific oligonucleotide amplification primers and methods for detecting and identifying M. kansasii nucleic acids using the amplification primers. One hundred percent of the clinical and environmental M. kanasii isolates tested were positive in amplification assays using the inventive amplification primers, with no cross-reactivity in other species of mycobacteria or closely related non-mycobacteria.

Species-specific detection of Mycobacterium kansasii

Genus- and species-specific oligonucleotide probes derived from the M. paratuberculosis 70 kD heat shock protein gene sequence. The probes are useful for detecting Mycobacteria and for identifying specific species of Mycobacteria.

Detection and identification of mycobacteria

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.

Detection of mycobacteria by multiplex nucleic acid amplification

Oligonucleotide probes derived from Mycobacteria Kansasii capable of selectively hybridizing to Mycobacteria nucleic acid are disclosed. The oligonucleotide probe is selected from the group consisting of: (a) an oligonucleotide probe consisting essentially of the DNA sequence given herein as SEQ ID NO:1 (MK14); (b) oligonucleotide probes comprising fragments of MK14 which retain the capability of MK14 of selectively hybridizing to Mycobacteria nucleic acid; (c) oligonucleotide probes which hybridize to MK14 and are capable of selectively hybridizing to Mycobacteria nucleic acid; and (d) oligonucleotide probes which are complementary to any of the foregoing and are capable of selectively hybridizing to Mycobacteria nucleic acid. The probes are useful in nucleic acid amplification and hybridization assays for genus-specific detection of the mycobacteria, specific detection of M. kansasii and specific detection of the slow-growing mycobacteria. Methods of using the probes to detect and amplify Mycobacteria nucleic acid and kits containing the same are also disclosed.

Growing community of inventors

Raleigh, NC, United States of America

Patricia A Spears