Nucleic acid amplification and detection of mycobacterium...

Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Preparing compound containing saccharide radical

Reexamination Certificate

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C435S091100, C536S024320, C536S024300

Reexamination Certificate

active

06664081

ABSTRACT:

FIELD OF THE INVENTION
This invention relates to in vitro diagnostic detection of pathogenic bacteria, and specifically relates to compositions and assays for detecting many species of Mycobacterium by using in vitro nucleic acid amplification and detection of amplified products.
BACKGROUND OF THE INVENTION
Detection of Mycobacterium species in clinical species is important as a clinical diagnostic tool. Historically,
M. tuberculosis
was thought to be the only clinically significant pathogen in this genus. A rise in the incidence of drug-resistant strains of
M. tuberculosis
has further emphasized the need to detect this species. Other Mycobacterium species, however, are also clinically important. These are sometimes referred to as “MOTT” for Mycobacterium other than
tuberculosis
, commonly including
M. avium/intracellulare
complex organisms (
M. avium, M. intracellulare, M. paratuberculosis
, commonly referred to as MAIC),
M. gordonae, M. fortuitum, M. chelonae, M. mucogenicum
and mixtures of Mycobacterium species in a clinical specimen. For example, fast-growing opportunistic infections by
M. avium
complex (MAC) bacteria have been shown to occur frequently in AIDS and other immunocompromised individuals. In such infected individuals, at least 10
6
MAC cells/ml of sputum sediment have been found. Therefore, detection assays that can detect, and optimally distinguish between, many species of Mycobacterium are clinically important.
Many clinical methods for detecting and identifying Mycobacterium species in samples require analysis of the bacteria's physical characteristics (e.g., acid-fast staining and microscopic detection of bacilli), physiological characteristics (e.g., growth on defined media) or biochemical characteristics (e.g., membrane lipid composition). These methods require relatively high concentrations of bacteria in the sample to be detected, may be subjective depending on the clinical technician's experience and expertise, and are time-consuming. Because Mycobacterium species are often difficult to grow in vitro and may take several weeks to reach a useful density in culture, these methods can also result in delayed patient treatment and costs associated with isolating an infected individual until the diagnosis is completed.
More recently, assays that detect the presence of nucleic acid derived from bacteria in the sample have been preferred because of the sensitivity and relative speed of the assays. In particular, assays that use in vitro nucleic acid amplification of nucleic acids present in a clinical sample can provide increased sensitivity and specificity of detection. Such assays, however, can be limited to detecting one or a few Mycobacterium species depending on the sequences amplified and/or detected.
Assays and reagents for detecting Mycobacterium nucleic acid sequences have been previously disclosed, for example, in 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 published PCT Patent Applications WO 9636733 and WO 9723618.
The present invention provides compositions and relatively simple diagnostic methods that detect a wide spectrum of Mycobacterium species that may be present in a clinical sample.
SUMMARY OF THE INVENTION
According to one aspect of the invention, there is provided a method of detecting Mycobacterium species present in a biological sample, comprising the steps of providing a biological sample containing nucleic acid from at least one Mycobacterium species comprising a Mycobacterium 16S ribosomal RNA (rRNA) or DNA encoding 16S rRNA; amplifying the Mycobacterium 16S rRNA or DNA in an in vitro nucleic acid amplification mixture comprising at least one polymerase activity, and at least two primers having sequences selected from the group consisting of SEQ ID NO:1 to SEQ ID NO:34, SEQ ID NO:37 and SEQ ID NO:38 to produce amplified Mycobacterium nucleic acid; and detecting the amplified Mycobacterium nucleic acid by detecting a label associated with the amplified Mycobacterium nucleic acid. One embodiment of the method further comprises the steps of adding to the biological sample at least one capture oligonucleotide that specifically hybridizes to the Mycobacterium 16S rRNA and an immobilized nucleic acid that hybridizes to the capture oligonucleotide under hybridizing conditions to produce a hybridization complex; and separating the hybridization complex from other components of the biological sample before the amplifying step. In one embodiment, the amplifying step amplifies 16S rRNA or DNA encoding 16S rRNA from
M. tuberculosis
or a Mycobacterium other than
tuberculosis
(MOTT) species. In another embodiment, the amplifying step amplifies 16S rRNA or DNA encoding 16S rRNA from
M. abscessus, M. africanum, M. asiaticum, M. avium, M. bovis, M. celatum, M. chelonae, M. flavescens, M. fortuitum, M. gastri, M. gordonae, M. haemophilum, M. intracellulare, M. interjectum, M. intermedium, M. kansasii, M. malmoense, M. marinum, M. non
-
chromogenicum, M. paratuberculosis, M. phlei, M. scrofulaceum, M. shimodei, M. simiae, M. smegmatis, M. szulgai, M. terrae, M. triviale, M. tuberculosis, M. ulcerans or M. xenopi
. One embodiment, in the detecting step, uses at least one probe that hybridizes specifically to the amplified Mycobacterium nucleic acid. In a preferred embodiment, the detecting step uses at least one labeled probe that hybridizes specifically to the amplified Mycobacterium nucleic acid. In another preferred embodiment, the detecting step uses a plurality of probes that hybridize specifically to the amplified Mycobacterium nucleic acid. Embodiments of the method, in the amplifying step, use a combination of at least a first primer and a second primer, wherein the first primer is selected from the group consisting of SEQ ID NO:1 to SEQ ID NO:12, and the second primer is selected from the group consisting of SEQ ID NO:13 to SEQ ID NO:34, SEQ ID NO:37 and SEQ ID NO:38. Some embodiments, in the amplifying step, use a combination of at least a first primer and a second primer, wherein the first primer is selected from the group consisting of SEQ ID NO:7 to SEQ ID NO:12, and the second primer is selected from the group consisting of SEQ ID NO:13 to SEQ ID NO:34, SEQ ID NO:37 and SEQ ID NO:38. Preferred embodiments of the method use, in the amplifying step, a combination of at least a first primer and a second primer selected from the group consisting of: the first primer having the sequence of SEQ ID NO:7, and the second primer having the sequence of SEQ ID NO:13; the first primer having the sequence of SEQ ID NO:7, and the second primer having the sequence of SEQ ID NO:14; the first primer having the sequence of SEQ ID NO:7, and the second primer having the sequence of SEQ ID NO:15; the first primer having the sequence of SEQ ID NO:7, and the second primer having the sequence of SEQ ID NO:16; the first primer having the sequence of SEQ ID NO:8, and the second primer having the sequence of SEQ ID NO:13; the first primer having the sequence of SEQ ID NO:8, and the second primer having the sequence of SEQ ID NO:14; the first primer having the sequence of SEQ ID NO:8, and the second primer having the sequence of SEQ ID NO:15; the first primer having the sequence of SEQ ID NO:9, and the second primer having the sequence of SEQ ID NO:13; the first primer having the sequence of SEQ ID NO:9, and the second primer having the sequence of SEQ ID NO:14; the first primer having the sequence of SEQ ID NO:9, and the second primer having the sequence of SEQ ID NO:15; the first primer having the sequence of SEQ ID NO:19, and the second primer having the sequence of SEQ ID NO:16; the first primer having the sequence of SEQ ID NO:11, and the second primer having the sequence of SEQ ID NO:13; the first primer having the sequence of SEQ ID NO:11, and the second primer having the sequence of SEQ ID NO:16; the first primer having the sequence of SEQ ID NO:11, and the second primer having the sequence of SEQ ID NO:17; the first primer having the sequence of SEQ ID NO:11, and

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