Multiplexed pcr assay for detecting disseminated mycobacterium avium complex infection
AssigneeOrtho-Clinical Diagnostics, Inc.
InventorsKEVIN M. GORMAN, JOHN A. MCELVER, CHARLES P. CARTWRIGHT, DAVID R. PATTERSON
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Abstract
The present invention relates to nucleic acid primers and probes specific for organisms of the Mycobacterium avium complex (MAC) and to their use in nucleic acid amplification methods for the detection and differentiation of such organisms in biological samples. The invention also relates to diagnostic kits for detecting and differentiating the various organisms comprising the MAC.
Claims15
A method for amplifying nucleic acids from organisms of the Mycobacterium avium complex (MAC) comprising contacting a sample suspected of containing MAC nucleic acids with four different nucleoside triphosphates, a thermostable DNA polymerase, and a set of oligonucleotide primers specific for two or more of the following three gene regions:
- MacSequevar gene region, M. avium 19 kilodalton protein gene region, and M. intracellulare ribosomal protein sl gene region, under conditions such that said MAC nucleic acids are amplified.
A method for amplifying and detecting nucleic acids from organisms of the Mycobacterium avium complex (MAC) comprising:
- (i)contacting a sample suspected of containing MAC nucleic acids with four different nucleoside triphosphates, a thermostable DNA polymerase, and a set of oligonucleotide primers specific for two or more of the following three gene regions:
- MacSequevar gene region, M. avium 19 kilodalton protein gene region, and M. intracellulare ribosomal protein sl gene region, under conditions such that said MAC nucleic acids are amplified;
- (ii) denaturing said amplified nucleic acids to form single stranded nucleic acids;
- and
- (iii) detecting the presence or absence of said amplified MAC nucleic acids.
A diagnostic kit useful for amplification of organisms of the MAC, said kit comprising:
- (i) a primer set specific for MacSequevar gene region;
- and
- (ii) a primer set specific for M. avium 19 kilodalton protein gene regions;
- (iii) a primer set specific for M. intracellulare ribosomal protein sl gene regions;
- and
- (iv) a thermostable DNA polymerase.
A method for detecting and distinguishing M. avium from other organisms of the Mycobacterium avium complex (MAC) comprising contacting a sample suspected of containing nucleic acids from organisms of the MAC with an oligonucleotide probe comprising nucleotide sequence:
- 5′ CCC TGA GAC AAC ACT XGG TCC GTC C 3′, wherein X is any nucleotide other than C and detecting the presence or absence of a complex formed between said probe and said nucleic acids present in said sample.
A composition comprising one or more of the following oligonucleotides:
- Msqv F4;
- MSqv F2;
- MSqv R5;
- MSqv R2;
- MSqv-Av;
- MSqv P1.21c;
- MSqv-MAC;
- MSqv P1;
- MSqv 1.3c;
- MAV19K F1;
- MAV19K R1;
- MAV19K P1;
- rpsl F1;
- rpsl R1;
- and
- rspl P1.
An oligonucleotide probe capable of selectivity hybridizing to nucleic acids from Mycobacterium avium comprising nucleotide sequence:
- 5′ CCC TGA GAC AAC ACT XGG TCC GTC C 3′, wherein X is any nucleotide other than C.
Dependencies are inferred from the claim text.