Organic compounds -- part of the class 532-570 series – Organic compounds – Carbohydrates or derivatives
Reexamination Certificate
1998-06-23
2002-10-15
Jones, W. Gary (Department: 1655)
Organic compounds -- part of the class 532-570 series
Organic compounds
Carbohydrates or derivatives
C536S024300, C536S023100, C435S006120, C435S091200
Reexamination Certificate
active
06465638
ABSTRACT:
FIELD OF THE INVENTION
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.
BACKGROUND INFORMATION
The
Mycobacterium avium
complex (MAC) is composed of a large number of organisms, many of which are classified as
M. avium
or
M. intracellulare.
In addition, there are a number of organisms within the MAC that cannot be properly classified because they have the characteristics of both
M. avium
and
M. intracellulare,
or because they have combined characteristics of another mycobacterium with either
M. avium
or
M. intracellulare
(Wayne, et al., International J. Systematic Bacteriol., 43(3):482-489 (1993)).
The
Mycobacterium avium
complex consists of at least 26 serovars. These organisms were originally defined by their agglutination in the presence of specific agglutinating antisera (through immune reaction with their cell wall surface antigens).
M. avium
is considered to include serovars 1 through 6, 8 through 11, and 21, while
M. intracellulare
is considered to include serovars 7, 12-17, 19, 20, 25 (Wayne, et al., Clin. Microbiol. Rev. 5:1-25 (1992) and H. Saito, et al., J. Clin. Microbiol., 28:1694-1697 (1990)).
Frothingham et al. (J. Bacteriol., 175(10):2818-2825 (1993)) further classified these organisms into sequevars. The sequevar classification was derived by sequencing the 16s-23s rRNA internal transcribed spacer region of reference strains of organisms representing the MAC. This classification system, based on the genetic sequence, revealed a wide range of genetic diversity among non-
M. avium,
non-
M. intracellulare
MAC strains. Frothingham et al. classified these reference strains as Mav-A through Mav-D, for
M. avium
organisms, Min-A, for
M. intracellulare
organisms, and MAC-A through MAC-H, for all
M. avium
complex strains that fit in neither of the avium or intracellulare categories (see also, Frothingham, et al., J. Infect. Diseases, 169:305-312 (1994)).
Infections caused by members of the MAC have become a major clinical problem, particularly in individuals who have AIDS (especially those individuals with extremely low CD4 counts). Yakrus et al. (J. Clin. Microbiol., 28:926-929, (1990)) identified MAC organisms that were most frequently associated with disseminated disease:
M. avium
serovar 4 (40%),
M. avium
serovar 8 (17%), non-typeable MAC (13%), and
M. avium
serovar 1 (9%).
Laboratory diagnosis of disseminated MAC traditionally has been based on culture methodology. MAC culture methods are labor, material, and resource intensive, and require relatively long periods of time for definitive diagnosis. Because of this, a polymerase chain reaction (PCR) test for the detection of MAC infection would be advantageous.
PCR-based amplification of target nucleic acids allows rapid and sensitive detection of target DNA sequences. Amplified sequences accumulate to concentrations that are easily detected using non-isotopic detection methods. PCR technology theoretically allows the practitioner to identify a specific target nucleic acid in samples which may contain just a single target.
Kulski et al. (J. Clin. Microbiol., 33:668, (1995)) investigated the use of multiplex PCR to detect members of the Mycobacterium genus and to detect and differentiate
M. tuberculosis, M. avium,
and
M. intracellulare.
These investigators coamplified regions of the 16s rRNA gene with a region of the MPB70 gene to detect and differentiate
M. avium, M. intracellulare,
and
M. tuberculosis.
Abed et al. (Res. Microbiol., 146:405, 1995) amplified the entire 16s to 23s rRNA spacer region and used a secondary technique of RAPD fingerprinting to differentiate 56 strains belonging to 11 Mycobacterium species. Their PCR primers resided outside the intergenic region, with their forward and reverse primers targeted to the 16s rRNA gene and the 23s rRNA gene, respectively.
Barry et al. (EP Publication No. 0395292), and Rossau et al. (EP Publication No. 0525095), describe amplification of the entire 16s to 23s intergenic region, and diagnostic tests for bacterial organisms using probes targeted for sequences within the 16s to 23s intergenic region. As in Abed et al., the primers used by Barry et al. and Rossau et al. were targeted for sequences located within the genes encoding 16s or 23s rRNA.
Booth et al. (Infection and Immunity, 61(4):1509, (1993)) found a high degree of similarity between the 19 kd protein genes of
M. avium, M. tuberculosis,
and
M. intracellulare.
This high degree of similarity at the level of the gene persists at the protein level. Nair et al. (Molecular Microbiology, 6(11):1431, (1992)) demonstrated that the
M. intracellulare
gene encodes a seroactive lipoprotein. This lipoprotein was considered to be the
M. intracellulare
homologue of the serologically active 19 kd
M. tuberculosis
protein.
In designing assays to detect the presence of the MAC, the skilled artisan is faced with a significant challenge in selecting primer and probe combinations that detect all of the organisms that comprise the MAC and that do not cross react with non-MAC organisms. The present invention overcomes these problems by providing primers and probes specific for organisms of the MAC that can be used to co-amplify at least two, and preferably three, gene regions of these organisms.
SUMMARY OF THE INVENTION
In one embodiment, the present invention relates to a method for amplifying nucleic acids from MAC organisms. The method involves contacting a sample suspected of containing nucleic acids from MAC organisms 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 the nucleic acids are amplified.
In another embodiment, the present invention relates to a method for amplifying and detecting nucleic acids from MAC organisms. The method involves contacting a sample suspected of containing nucleic acids from MAC organisms 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 the nucleic acids are amplified. The amplified product is then denatured and detected.
In a further embodiment, the present invention relates to a method for detecting and distinguishing
M. avium
from other organisms of the
Mycobacterium avium
complex 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 DGG TCC GTC C 3′ (SEQ ID NO:1), wherein D is any nucleotide other than C and detecting the presence or absence of a complex formed between the probe and nucleic acids present in the sample.
Various other objects and advantages of the present invention will be apparent from the detailed description of the invention.
All publications mentioned herein are hereby incorporated by reference.
DETAILED DESCRIPTION OF THE INVENTION
The development of a nucleic acid co-amplification system that detects all MAC organisms without crossreacting with non-MAC mycobacteria is very challenging. Applicants have overcome several obstacles to arrive at the present invention relating to the amplification and/or detection of nucleic acids from MAC organisms.
In the present invention, three gene regions have been identified that are compatible with each other in a multiplexed system and can, therefore, be use
Cartwright Charles P.
Gorman Kevin M.
McElver John A.
Patterson David R.
Einsmann Juliet C.
Gowen Catherine K.
Jones W. Gary
Ortho-Clinical Diagnostics, Inc.
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