Organic compounds -- part of the class 532-570 series – Organic compounds – Carbohydrates or derivatives
Reexamination Certificate
2000-12-15
2004-06-08
Fredman, Jeffrey (Department: 1637)
Organic compounds -- part of the class 532-570 series
Organic compounds
Carbohydrates or derivatives
C435S006120, C435S091200, C536S023100, C536S063000
Reexamination Certificate
active
06747141
ABSTRACT:
FIELD OF THE INVENTION
This invention relates to in vitro diagnostic detection of pathogenic bacteria, and specifically relates to compositions and assays for amplifying nucleic acid of
Mycobacterium avium
complex (MAC) organisms (e.g.,
M. avium, M. intracellulare
) by using in vitro nucleic acid amplification.
BACKGROUND OF THE INVENTION
Detection of Mycobacterium species of the
Mycobacterium avium
complex (MAC) in clinical samples is important as a diagnostic tool.
M. avium
complex organisms include
M. avium, M. intracellulare
and other species that are difficult to differentiate from these, such as
M. paratuberculosis
. MAC organisms are frequently found in clinical samples and are common causative agents of opportunistic infections in immunocompromisied individuals, such as HIV-infected individuals or individuals undergoing chemotheraphy or using immunosuppressive drugs (Good et al., 1982, J. Infect. Dis. 146: 829-833; Gill et al., 1985, J. Clin. Microbiol. 22: 543-546). Therefore, assays that can detect MAC species and distinguish them from other species are important for clinical diagnosis.
Clinical diagnostic assays for Mycobacterium species often rely on time-consuming methods that analyze bacterial physical characteristics (e.g., staining and microscopic detection), physiological characteristics (e.g., growth on defined media) and/or biochemical characteristics (e.g., membrane lipid composition). Such methods often require relatively high bacterial concentrations in the sample and may require a high degree of experience and expertise to properly determine the infective species. Diagnostic assays that require in vitro growth of the bacteria are costly both in terms of delayed or inappropriate early treatment of the patient and in terms of the amount of laboratory equipment and space required to culture Mycobacterium, which is often difficult to grow in vitro.
Assays that use molecular biology techniques to detect the presence Mycobacterium nucleic acid in the sample have been introduced to increase the sensitivity and relative speed of diagnosis (U.S. Pat. Nos. 5,030,557, 5,567,587, 5,595,874, 5,601,984 and 5,677,128; PCT No. WO/95/06755). These assays may directly detect the nucleic acid sequences present in the sample or may rely on in vitro nucleic acid amplification of nucleic acids present in the sample before the detection step (U.S. Pat. Nos. 5,554,516, 5,766,849, 5,906,917, 5,908,744; European Patent Nos. EP 0528306 and EP 0818465; and PCT Nos. WO 9636733 and WO 9723618). Many in vitro nucleic acid amplification reactions require amplification oligonucleotides that serve as primers for a polymerase reaction that uses the nucleic acid present in the sample as a template. Detection of the amplified nucleic acid often requires use of specific nucleic acid probes that hybridize to the amplified sequences to produce a detectable signal or complex.
The present invention provides compositions and in vitro nucleic acid amplification methods that produce relatively long amplified nucleic acid sequences to allow detection of MAC species present in a biological sample.
SUMMARY OF THE INVENTION
According to one aspect of the invention, there is provided a method of detecting
Mycobacterium avium
complex (MAC) species present in a biological sample. The method includes the steps of: providing a biological sample containing nucleic acid from at least one MAC species selected from the group consisting of
M. tuberculosis, M. avium, M. intracellulare
, and
M. paratuberculosis
, the nucleic acid comprising 16S ribosomal RNA (rRNA) or DNA encoding 16S rRNA; amplifying the 16S rRNA or DNA in an in vitro nucleic acid amplification mixture comprising at least one polymerase activity, and at least one first primer having a sequence selected from the group consisting of SEQ ID NO:1 to SEQ ID NO:6 and at least one second primer having a sequence selected from the group consisting of SEQ ID NO:7, SEQ ID NO:8 and SEQ ID NO:9, to produce an amplified nucleic acid; and detecting the amplified nucleic acid. In one embodiment, the detecting step further comprises hybridizing the amplified nucleic acid to at least one probe and detecting a signal resulting from the amplified nucleic acid that is hybridized to the probe. In another embodiment, the detecting step uses at least one labeled probe comprising sequence complementary to a portion of the amplified nucleic acid. Another embodiment of the method further includes the step of using at least one capture oligonucleotide that specifically hybridizes to nucleic acid from at least one MAC species to bind the nucleic acid from the MAC species to an immobilized nucleic acid, to purify the nucleic acid from the MAC species from other components in the sample before the amplifying step. In another embodiment, the amplifying step amplifies 16S rRNA from
M tuberculosis, M. avium, M. intracellulare, M. paratuberculosis
or any combination thereof. In some embodiments of the method, the amplifying step uses a combination selected from the group consisting of: the first primer having the sequence of SEQ ID NO:1, and the second primer having the sequence of SEQ ID NO:7; the first primer having the sequence of SEQ ID NO:1, and the second primer having the sequence of SEQ ID NO:8; the first primer having the sequence of SEQ ID NO:1, and the second primer having the sequence of SEQ ID NO:9; the first primer having the sequence of SEQ ID NO:2, and the second primer having the sequence of SEQ ID NO:7; the first primer having the sequence of SEQ ID NO:2, and the second primer having the sequence of SEQ ID NO:8; the first primer having the sequence of SEQ ID NO:2, and the second primer having the sequence of SEQ ID NO:9; the first primer having the sequence of SEQ ID NO:3, and the second primer having the sequence of SEQ ID NO:7; the first primer having the sequence of SEQ ID NO:3, and the second primer having the sequence of SEQ ID NO:8; the first primer having the sequence of SEQ ID NO:3, and the second primer having the sequence of SEQ ID NO:9; the first primer having the sequence of SEQ ID NO:4, and the second primer having the sequence of SEQ ID NO:7; the first primer having the sequence of SEQ ID NO:4, and the second primer having the sequence of SEQ ID NO:8; the first primer having the sequence of SEQ ID NO:4, and the second primer having the sequence of SEQ ID NO:9; the first primer having the sequence of SEQ ID NO:5, and the second primer having the sequence of SEQ ID NO:7; the first primer having the sequence of SEQ ID NO:5, and the second primer having the sequence of SEQ ID NO:8; the first primer having the sequence of SEQ ID NO:5, and the second primer having the sequence of SEQ ID NO:9; the first primer having the sequence of SEQ ID NO:6, and the second primer having the sequence of SEQ ID NO:7; the first primer having the sequence of SEQ ID NO:6, and the second primer having the sequence of SEQ ID NO:8; and the first primer having the sequence of SEQ ID NO:6, and the second primer having the sequence of SEQ ID NO:9. In other embodiments, the amplifying step uses a combination of at least one first primer having a sequence selected from the group consisting of SEQ ID NO:1 to SEQ ID NO:3 and at least one second primer having a sequence selected from the group consisting of SEQ ID NO:7, SEQ ID NO:8 and SEQ ID NO:9. Some preferred embodiments use a combination selected from the group consisting of: the first primer having the sequence of SEQ ID NO:1, and the second primer having the sequence of SEQ ID NO:7; the first primer having the sequence of SEQ ID NO:1, and the second primer having the sequence of SEQ ID NO:8; the first primer having the sequence of SEQ ID NO:1, and the second primer having the sequence of SEQ ID NO:9; the first primer having the sequence of SEQ ID NO:2, and the second primer having the sequence of SEQ ID NO:7; the first primer having the sequence of SEQ ID NO:2, and the second primer having the sequence of SEQ ID NO:8; the first primer having the sequence of SEQ ID NO:2, and the second primer having the sequenc
Brentano Steven T.
Lankford Roger L.
Fredman Jeffrey
Gen-Probe Incorporated
Gritzmacher Christine A.
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