WO2006029014A2 - rRNA OLIGONUCLEOTIDE PROBES FOR SPECIFIC DETECTION OF MYCOBACTERIA AND METHODS OF USE THEREOF - Google Patents

Abstract

Disclosed are oligonucleotides (e.g., DNA, RNA, or peptide nucleic acids (PNA)) that are complementary to, or hybridize under stringent hybridization conditions to, ribosomal RNA (rRNA) sequences from microbial cells, and particularly, mycobacterial cells. These oligonucleotide probes have been designed to be species-specific or phylogenetic group-specific, and are intended to be used primarily as in situ hybridization probes or as PCR primers. Also disclosed are cell permeabilization and hybridization techniques that are useful for the detection of microbial cells in a sample.

Description

rRNA Oligonucleotide Probes for Specific Detection of Mycobacteria and Methods of Use Thereof

Field of the Invention The present invention relates to rRNA oligonucleotide probes for the detection of mycobacteria at the species-specific and phylogenetic group-specific level, and to a permeabilization protocol for use in hybridization methods for detecting mycobacteria and other Gram-positive bacteria.

Background of the Invention

The difficulty of rapidly identifying Mycobacteria beyond the genus level seriously compromises treatment of patients who present with putative mycobacterial infections. Currently, only two assays have been approved by the FDA for rapid molecular identification of Mycobacterium tuberculosis in sputum samples. Traditionally, identification of mycobacteria beyond the genus level has required, as a first step, isolation of microbes in pure culture. However, many pathogenic mycobacteria species are characterized by slow, fastidious growth that often requires weeks to months for their isolation. Clinical care is thus complicated, because acute mycobacterial infections require treatment long before positive identification of the causative species can be accomplished. In the case of pulmonary infections, rapid, FDA-approved diagnostic tests can be applied to sputum samples in order to determine the presence or absence of Mycobacterium tuberculosis. However, FDA-approved assays for other mycobacteria in sputum samples or for any mycobacterial species in tissue samples do not exist, to the best of the present inventors' knowledge.

Mycobacterium tuberculosis (Mtb) is the causative agent of tuberculosis. Mtb infections often result in pulmonary disease but also can cause skin infections, lymphandentis, meningitis and other manifestations (Ashford, 2001; Chao, 2002). Detection of Mtb generally relies on a combination of acid fast staining of bacilli (AFB) followed by in vitro cultivation (Kehinde, 2005). However, AFB staining is unable to speciate different mycobacteria, and at different growth stages some mycobacteria are reported to lose their "acid fastness" (Chandrasekhar, 1992; Wall, 1993; Wang, 2001). After cultivation, a variety of methods are used to identify Mtb including PCR of IS6110 or 16S rRNA genes (Watterson, 2000). Several commercial products that use 16S rRNA sequences as probes or amplification primers are approved for molecular identification of mycobacteria (Drobniewski, 2000; Tortoli, 1996; Woods, 2001). These assays either require cultivation of the organism or are approved only for sputum samples^'. The Mycobacterium avium complex (MAC) consists of genetically similar, slowly growing bacteria that includes Mycobacterium avium ssp. avium, Mycobacterium avium ssp. paratuberculosis, Mycobacterium avium ssp. silvaticum, Mycobacterium avium ssp. hominis, and Mycobacterium intracellular (De Groote, 2003). MAC organisms are opportunistic pathogens that can be isolated from multiple environmental sources including drinking water, soil, plants, dairy products, bioaerosols, and animals (Falkinham, 2003; Primm, 2004), and are implicated in several chronic, idiopathic diseases (De Groote, 2003; Eccles, 1995; Falkinham, 1996). Although human exposure to MAC is ubiquitous most individuals rarely develop infections. Immunocompromised persons, such as those with Acquired Immune Deficiency Syndrome (AIDS) or following transplant surgery, are at the greatest risk for MAC infection (Benson, 1994; Horsburgh, 1999). Before the emergence of AIDS, most MAC infections were pulmonary in nature and typically affected patients with preexisting lung diseases, such as emphysema or cystic fibrosis (De Groote, 2004, Pulmonary infection in non-HIV infected individuals; Kilby, 1992; Oliver, 2001). MAC also is the most frequent cause of pediatric cervical lymphadenitis (Chao, 2002; Fitzpatrick, 1996). In recent years an emergence of "hot tub lung" and "life guard lung" cases have been noted, in which disease occurs secondary to a hypersensitivity that develops due to aerosolized MAC bacteria in therapeutic pools, hot tubs, or indoor swimming pools (Falkinham, 2003; Iivanainen, 1999).

Although M. avium ssp. avium (MAA) and M. intracellular (MIN) are currently the main causative agents of human MAC diseases (De Groote, 2004, Skin, bone, and soft tissue infections), M. avium ssp. paratuberculosis (MAP) is suggested as an emerging human pathogen (Greenstein, 2004). In addition to the well-accepted MAC infections, members of MAC are implicated in causing human granulomatous diseases, such as sarcoidosis and Crohn's disease (CD) (McFadden, 1996). Sarcoidosis is an idiopathic, multisystem, disease that has many features in common with mycobacterial infections (Hance, 1998, Kon, 1997; Rutherford, 2004). Although acid-fast bacilli have not been detected in sarcoid granulomas, several studies have reported the detection of mycobacteria, including MAP, in granulomatous tissue by molecular techniques (Drake, 2002; el-Zaatari, 1996; Mangiapan, 1995; Mirmirani, 1999). Two recent medical case reports demonstrate that MAP is able to infect humans. In 2002, a German AIDS patient developed a disseminated mycobacterial infection from which MAP was isolated (Richter, 2002). Secondly, Hermon-Taylor reported a case of pediatric cervical lymphadenitis in which MAP was the primary infectious agent (Hermon-Taylor, 1998). It is perhaps noteworthy that this patient later developed Crohn's disease (CD). MAP has long been suspected as a trigger of CD, a chronic human inflammatory bowel disease of unknown etiology (Behr, 2004; Chiodini, 1989; el-Zaatari, 2001; Greenstein, 2003; Hermon-Taylor, 2002). CD clinically resembles Johne's disease (JD), which occurs in ruminants and non-human primates, and is characterized by severe gastroenteritis caused by MAP (Chacon, 2004; Greenstein, 2003; Van Kruiningen, 1999). CD is hypothesized to be a zoonotic infection that arises from exposure to dairy products contaminated with M. avium spp. paratuberculosis, the causative agent of bovine JD (Greenstein, 2004). The public health implications of a link between human Crohn's disease and bovine Johne's disease demand careful consideration, given the increasing incidence of Johne's disease in cattle herds (Chacon, 2004) and the presence of viable MAP in commercial milk supplies (Chiodini, 1993; Miller, 1996).

Detection of MAP has been reported by either culture or molecular techniques in a subset of CD cases (Bull, 2003; Collins, 2000; Hulten, 2001; Naser, 2002; Roholl, 2002; Ryan, 2002; Sechi, 2001). However, unambiguous identification of MAP as the etiologic agent of CD is hampered by the difficult and time-consuming methods necessary to detect the organism. MAP is extremely difficult to culture and requires prolonged incubation of weeks to months (Woods, 2001). Currently, most molecular analyses of JD and CD rely on detection of the MAP-specifϊc insertion element, IS900, by nested and/or quantitative PCR (Bull, 2000; Cousins, 1999; Englund, 2003; Vansnick, 2004). However, as is the case with culture studies, the application of these molecular techniques to CD has led to contradictory results (Chiodini, 1986; Kanazawa, 1999; Moss, 1991; Wall, 1993). An alternative approach, in situ hybridization (ISH), is an attractive technique for the detection of MAP and other mycobacteria. ISH not only allows for a more rapid diagnosis than can be achieved by culture methods, but fewer bacilli can be detected with ISH as opposed to current PCR methods. Furthermore, unlike PCR, ISH provides detailed, tissue-based morphological information.

One of the major obstacles to the use of ISH to identify mycobacteria is the tough, lipid-containing cell walls of these organisms, which renders cells impermeable to oligonucleotide probes (Lambert, 2002). However, Hulten et al. (2000, Vet Microbiol.; 2000, J. Microbiol. Methods) developed an ISH methodology to identify MAP by microscopic detection of IS900. The IS900 ISH procedure utilizes a digoxygenin (DIG) or biotin labeled double stranded DNA probe and enzymatic amplification of the signal with alkaline phosphatase (AP) conjugated antibodies. However, the IS900 ISH technique can detect only

"cell wall deficient" forms of MAP, which are hypothesized to play a role in colonization or _persistence in Crohn's disease (Hulten, 2000, Vet Microbiol.; Wall, 1993). By this method, both Hulten et al. (2001) and Sechi et al. (2001; 2004) reported detection of MAP in tissues obtained from Crohn's patients. However, permeabilization issues due to the large size of the double stranded DNA probe (241 base pairs) and the low copy number of IS900 targets (1-14 copies per MAP genome) render this technique and the results questionable. It seems unlikely that a few hybridization targets per cell, even including potential IS900 transcripts, would be able to sequester sufficient probe to visualize a single bacterial cell. Indeed, the inventors have now shown that IS900 ISH is non-specific and thus can not definitively rule in or out the presence of MAP in clinical specimens (St. Amand 2005; see also Examples 1-4). An alternative hybridization approach to IS900 ISH is the use of oligonucleotide probes that target ribosomal RNA (rRNA) (Amann, 1990; Giovannoni, 1988). Ribosomal RNA typically is present in cells at high copy number (thousands of copies/cell), and thus provides a far greater number of potential targets than do the IS900 genes. The abundance of rRNA in cells allows for ready visualization of individual organisms when fluorescent probes bind to their rRNA targets. Phylogenetic comparisons of rRNA gene sequences can be used to design oligonucleotide probes that can distinguish between very closely related organisms, such as species of mycobacteria (Lane, 1985). Several commercial products are approved for molecular detection of mycobacteria, and most use 16S rRNA sequences as probes (e.g. AccuProbe rRNA hybridization; Gen-Probe, Inc., San Diego, CA) or amplification primers (e.g. Amplified Mycobacterium Tuberculosis Direct Test (MTD); Gen-Probe, Inc., San Diego, CA) (Drobniewski, 2000; Tortoli, 1996; Watterson, 2000; Woods, 2001). However, all of these commercial assays either require cultivation of the organism or are approved only for the assay of sputum samples. Moreover, existing assays and probes are not available to rapidly (i.e., without cultivation of the microbe) detect any mycobacterial cell in a species- or phylogenetic group-specific manner, in any type of sample. Therefore, there is a clear need for rapid diagnostic methodologies that are applicable to many different tissue types in addition to sputum, that are applicable to the detection of any mycobacterial species or phylogenetic groups, and that do not require cultivation of the microbial cell prior to the assay.

Summary of the Invention

One embodiment of the present invention relates to an oligonucleotide probe consisting of a nucleic acid sequence that hybridizes under stringent conditions to the complement of an oligonucleotide sequence selected from the group consisting of any one of SϊsQ ID JNUs:2-iy, wnerein the oligonucleotide probe also hybridizes under stringent conditions to rRNA in a mycobacteria. In one aspect of the invention, at least one nucleotide in the oligonucleotide probe has been modified by a Locked Nucleic Acid (LNA) modification. For example, LNA-modifϊed oligonucleotide probes of the invention are represented by SEQ ID NOs: 13-19. The oligonucleotide probe can be any length, and is preferably between about 8 nucleotides and about 50 nucleotides in length, and in another preferred embodiment, is between about 8 nucleotides and about 25 nucleotides in length. In one aspect, the oligonucleotide consists of a nucleic acid sequence is at least 90% identical, or at least about 95% identical, to any one of SEQ ID NOs:2-19. In one aspect of this embodiment, the oligonucleotide probe hybridizes under stringent conditions to rRNA from the Mycobacterium avium complex, including, but not limited to, Mycobacterium avium ssp. avium, Mycobacterium avium ssp. paratuherculosis, Mycobacterium avium ssp. silvaticum, Mycobacterium avium ssp. hominis, and Mycobacterium intracellular. A preferred oligonucleotide probe comprises a nucleic acid sequence of SEQ ID NO :4.

In another aspect, the oligonucleotide probe hybridizes under stringent conditions to rRNA from Mycobacterium intracellular. A preferred oligonucleotide probe comprises a nucleic acid sequence selected from SEQ ID NO: 5 and SEQ ID NO: 6.

In another aspect, the oligonucleotide probe hybridizes under stringent conditions to rRNA from Mycobacterium avium ssp. avium and Mycobacterium avium ssp. paratuberculosis. A preferred oligonucleotide probe comprises a nucleic acid sequence selected from SEQ ID NO:2, SEQ ID NO:3, and SEQ ID NO: 16.

In yet another aspect, the oligonucleotide probe hybridizes under stringent conditions to rRNA from Mycobacterium tuberculosis. A preferred oligonucleotide probe comprises a nucleic acid sequence selected from SEQ ID NO: 7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:17, SEQ ID NO:18 and SEQ ID NO:19.

In yet another aspect, the oligonucleotide probe hybridizes under stringent conditions to rRNA from Mycobacterium abscessus. A preferred oligonucleotide probe comprises a nucleic acid sequence selected from the group consisting of SEQ ID NO: 10 and SEQ ID NO:11.

In another aspect, the oligonucleotide probe hybridizes under stringent conditions to rRNA from Mycobacterium kansasii. A preferred oligonucleotide probe comprises the nucleic acid sequence of SEQ ID NO: 12. in any or me above-described embodiments, the oligonucleotide probe can be operatively linked to a detectable label, including, but not limited to, a fluorescent label, a colorimetric label, biotin, an enzyme and a radiolabel.

Another embodiment of the present invention relates to a panel of oligonucleotide probes, comprising at least two of any of the oligonucleotide probes described herein. In one aspect, the probes are immobilized on a substrate in an array.

Another embodiment of the present invention relates to an assay kit for the detection of mycobacteria in a sample, comprising at least one oligonucleotide probe as described herein. Yet another embodiment of the present invention relates to a primer for polymerase chain reaction comprising an oligonucleotide probe as described herein.

Another embodiment of the present invention relates to a method to detect mycobacteria in a sample, comprising: (a) contacting a fixed sample suspected of containing mycobacterial cells with Xylene; (b) rehydrating the sample; (c) enzymatically digesting the sample with lysozyme and achromapeptidase; (d) washing and drying the sample; (e) contacting the permeabilized sample with at least one oligonucleotide probe as described herein in a hybridization buffer; (f) heating the sample in the buffer at a temperature sufficient to dissociate polynucleotide strands; (g) cooling the sample in the buffer at a temperature sufficient to allow polynucleotide strands to hybridize; (h) washing the sample; and (i) visualizing the sample to detect the presence or absence of mycobacteria in the sample. In one aspect, the sample is selected from: a microbial cell sample, a tissue sample, a sputum sample, and an environmental sample. In another aspect, the method is used to diagnose a mycobacterial disease in a subject. In another aspect, step (e) comprises contacting the sample with two or more different oligonucleotide probes. For example, in one aspect, the two or more different oligonucleotide probes hybridize to rRNA from the same species or phylogenetic group of mycobacteria. In another aspect, the two or more different oligonucleotide probes hybridize to rRNA from different species or phylogenetic groups of mycobacteria and are labeled such that the different species or phylogenetic groups can be differentiated. Yet another embodiment of the present invention relates to a method to permeabilize microbial cells for use in in situ hybridization protocols, comprising: (a) contacting fixed microbial cells with Xylene; (b) rehydrating the microbial cells; (c) enzymatically digesting the microbial cells with lysozyme and achromapeptidase; and (d) washing and drying the microbial cells. Another ^"embodiment of the present invention relates to a method for in situ hybridization of microbial cells, comprising: (a) contacting fixed microbial cells with Xylene; (b) rehydrating the microbial cells; (c) enzymatically digesting the microbial cells with lysozyme and achromapeptidase to produce permeabilized microbial cells; (d) washing and drying the permeabilized microbial cells; (e) contacting the permeabilized microbial cells with at least one oligonucleotide probe in a hybridization buffer; (f) heating the cells in the buffer at a temperature sufficient to dissociate polynucleotide strands; (g) cooling the cells in the buffer at a temperature sufficient to allow polynucleotide strands to hybridize; (h) washing the cells to remove excess hybridization buffer; and (i) visualizing the cells. In either above the methods described directly above, the microbial cells are preferably mycobacterial cells. The cells may be, for example, in culture or in a tissue. Preferably, in the above methods, the cells are not permeabilized using Proteinase K.

In one aspect of the above-methods, steps (a)-(d) comprise: (a) contacting fixed microbial cells with 100% Xylene for from about 1 to about 30 minutes; (b) rehydrating the microbial cells; (c) enzymatically digesting the microbial cells with from about 0.1 mg/ml to about 10 mg/ml lysozyme, and from about 3 units/ml to about 300 units/ml achromapeptidase, for between about 5 and about 60 minutes; and (d) washing and drying the microbial cells. In one aspect, step (a) comprises contacting fixed microbial cells with 100% Xylene for from about 5 to about 20 minutes. In another aspect, step (c) comprises enzymatically digesting the microbial cells with about 1 mg/ml lysozyme and from about 3 units/ml to about 300 units/ml achromapeptidase. In another aspect, step (c) comprises enzymatically digesting the microbial cells with from about 0.1 mg/ml to about 10 mg/ml lysozyme, and about 30 units/ml achromapeptidase. In yet another aspect, step (c) comprises enzymatically digesting the microbial cells with lysozyme and achromapeptidase for from about 15 to about 35 minutes.

In the above-methods, in one aspect, the oligonucleotide probe is a species-specific or phylogenetic group-specific oligonucleotide that hybridizes under stringent conditions to rRNA in microbial cells, such as microbial cells are mycobacterial cells. In another aspect, the oligonucleotide probe hybridizes under stringent conditions to rRNA from a mycobacterium species selected from the group consisting of: Mycobacterium avium ssp. avium, Mycobacterium avium ssp. paratuberculosis, Mycobacterium avium ssp. silvaticum, Mycobacterium avium ssp. hominis, Mycobacterium intracellular, Mycobacterium tuberculosis, Mycobacterium kansasii, and Mycobacterium abscessus. Preferably, the oligonucleotide probe does not cross-hybridize to rRNA from another mycobacterium species. In one aspect, the oligonucleotide probe hybridizes under stringent conditions to rRNA from Mycobacterium avium complex. Preferably, the oligonucleotide probe does not cross-hybridize to rRNA from a non-Mycobacteήum avium complex species. In another aspect, the oligonucleotide probe includes any of the oligonucleotide probes described herein. In another aspect of the above-described methods, steps (e)-(h) comprise: (e) contacting the permeabilized microbial cells with at least one oligonucleotide probe in a hybridization buffer comprising NaCl, Tris, SDS and formamide; (f) heating the cells in the buffer at a temperature of about 92°C to 98°C for from about 1 minute to about 5 minutes; (g) cooling the cells in the buffer at a temperature of between about 37°C and about 60⁰C for from about 30 minutes to about 12 hours; and (h) washing the cells in a buffer comprising NaCl, EDTA, SDS and Tris. In one aspect, step (f) comprises heating the cells in the buffer at a temperature of about 94°C for from about 1 minute to about 5 minutes. In another aspect, step (g) comprises cooling the cells in the buffer at a temperature of between about 38°C and about 42⁰C, from about 30 minutes to about 12 hours. In yet another aspect, steps (e)-(h) comprise: (e) contacting the permeabilized microbial cells with at least one oligonucleotide probe at a concentration of about 2 ng/μl in a hybridization buffer comprising about 90OmM NaCl, about 2OmM Tris pH 8, about 0.01% SDS and about 20% formamide; (f) heating the cells in the buffer at a temperature of about 94⁰C for about 3 minutes; (g) cooling the cells in the buffer at a temperature of about 4O⁰C for from about 6 hours to about 12 hours; and (h) washing the cells in a buffer comprising about 225mM NaCl, about 5mM EDTA, about 0.01% SDS and about 2OmM Tris pH 8.

In the methods described above, in one aspect, the cells are visualized by microscopy. In one aspect, the cells are visualized using direct fluorescent microscopy or enzymatic amplification of a colorimetric substrate. In one aspect, the cells are visualized by radiolabeling.

Another embodiment of the present invention relates to method to identify oligonucleotide probes for the species-specific or phylogenetic group-specific detection of mycobacteria in a sample, comprising: (a) identifying or determining an rRNA sequence from a mycobacterial species or phylogenetic group; (b) designing an oligonucleotide probe that is complementary to the rRNA sequence; (c) contacting rRNA from mycobacterial cells of the mycobacterial species or phylogenetic group of (a) with the oligonucleotide probe of (b) using the method of in situ hybridization described above; (d) contacting rRNA from mycobacterial cells of a different mycobacterial species or phylogenetic group than the mycobacterial species or phylogenetic group of (a) with the oligonucleotide probe of (b) using the method of in situ hybridization described above; and (e) selecting an oligonucleotide probe for the species-specific or phylogenetic group-specific detection of mycobacteria that hybridizes to the rRNA from (c) but does not cross-hybridize to the rRNA from (d).

Brief Description of the Drawings of the Invention

Fig. IA is a digitized image showing a specificity test using M. avium ssp. avium culture hybridized with MAVP187ssu- and MAVP5151su-Cy3 probes.

Fig. IB is a digitized image showing a specificity test using MAP culture hybridized with MAVP 187ssu- and MAVP5151su-Cy3 probes.

Figs. 1C and ID are digitized images showing a specificity test using M. intracellular* dual probed with EUB338-6' FAM (Fig. 1C) and MAVP187ssu- and MAVP5151su-Cy3 probes (Fig. ID) in the same field.

Figs. IE and IF are digitized images showing a specificity test using M. tuberculosis dual probed with EUB338-6¹ FAM (Fig. IE) and MAVP187ssu- and MAVP5151su-Cy3 probes (Fig. IF) in the same field.

Figs. IG and IH are digitized images showing a specificity test using M. avium culture dual probed with EUB338-6¹ FAM (Fig. IG) and MAC2543-Cy3 probe (Fig. IH) in the same field. Figs. II and IJ are digitized images showing a specificity test using M. intracellulare culture dual hybridized with EUB338-6¹ FAM (Fig. II) plus MAC2543-Cy3 (Fig. IJ) probe in the same field.

Figs. IK and IL are digitized images showing a specificity test using M. tuberculosis dual probed with EUB338-6¹ FAM (Fig. IK) and MAC2543-Cy3 probe (Fig. IL) in the same field.

Figs. IM and IN are digitized images showing a specificity test using M. avium ssp. avium culture dual probed with EUB338-6' FAM (Fig. IM) and MIN3511su- and MIN15861su-Cy3 probes (Fig. IN) in the same field.

Figs. IO and IP are digitized images showing a specificity test using M. intracellulare culture dual hybridized with EUB338-6' FAM (Fig. 10) and MIN3511su- and MIN15861su-Cy3 (Fig. IP) in the same field.

Figs. IQ and IR are digitized images showing a specificity test using M. tuberculosis dual probed with EUB338-6¹ FAM (Fig. IQ) and MIN3511su- and MIN15861su-Cy3 probe (Fig. IR) same field. Fϊgs^""2Α and 2B are digitized images showing a representative application of the

MAVP187ssu- and MAVP5151su-Cy3 probes to bovine tissue (Fig. 2A=negative control

MLN probed with MAVP187ssu- and MAVP5151su-Cy3; Fig. 2B=positive control MLN probed with MAVP187ssu- and MAVP5151su-Cy3; Scale Bar=l micron; original magnification 100OX; arrows indicate bacilli in expected morphology).

Fig. 2C is a digitized image showing a laser scanning confocal picture of positive control MLN stained with DAPI and probed with MAVP187ssu-Cy3 (Scale Bar=l micron; Original magnification 630X; arrows indicate bacilli in expected morphology).

Fig 2D is a digitized image showing a Brightfield picture of positive control ileum hybridized with MAVP187ssu- and MAVP5151su-6' FAM probes and visualized with anti- fluorescein-AP antibodies and INT/BCIP substrate (Scale Bar=l micron; Original magnification 100Ox; arrows indicate bacilli in expected morphology).

Fig. 3A is a digitized image showing a resected lung from a patient with a M. avium ssp. avium infection probed with MAVP187ssu- and MAVP5151su-6' FAM; signal was visualized using anti-fluorescein AP Fab fragments and INT/BCIP; arrows indicate bacilli in expected morphology.

Fig. 3B is a digitized image showing that MAVP187ssu- and MAVP5151su-Cy3 probes detect red fluorescent M. avium ssp. avium bacilli in a tissue section from a hand of a patient with tenosynovitis; arrows indicate bacilli in expected morphology. Fig. 3C is a digitized image showing that MAVP187ssu- and MAVP5151su-Cy3 probes detect red fluorescent bacilli in a tissue section from a pediatric CD patient's duodenum; arrows indicate bacilli in expected morphology.

Fig. 3D is a digitized image showing a small bowel biopsy from the pediatric CD patient in Fig. 3C that was probed with MAVP187ssu- and MAVP5151su-6' FAM; signal was visualized using anti-fluorescein AP Fab fragments fluorescein and INT/BCIP; arrows indicate bacilli in expected morphology.

Fig. 3E is a digitized image showing a lung tissue section from patient with an M. intracellular pulmonary infection probed with MIN3511su- and MTN15861su-6' FAM and visualized with anti-fluorescein AP antibodies and INT/BCIP substrate; arrows indicate bacilli in expected morphology.

Fig. 3F is a digitized image showing sputum (stained with DAPI) from a chronic smoker with possible mycobacterial infection probed with MAC25431su-6' FAM; signal was visualized with anti-fluorescein AP antibodies and INT/BCIP substrate. Micrograph represents a merged image ot bright iield and epiiluorescence (Scale Bar=l micron; Original magnification 1000X;Arrows indicate bacilli in expected morphology).

Fig. 4A is a digitized image showing a negative control MLN stained with the Ziehl- Neelsen acid-fast procedure; (Scale Bar=l micron; Original magnification 1000X). Fig. 4B is a digitized image showing a positive control MLN stained with the Ziehl-

Neelsen acid-fast procedure; (Scale Bar=l micron; Original magnification 1000X).

Fig. 4C is a digitized image showing a negative control MLN hybridized with MAVP187SSU-6¹ FAM ISH; (Scale Bar=l micron; Original magnification 1000X).

Fig. 4D is a digitized image showing a positive control MLN hybridized with MAVP187SSU-6' FAM ISH; (Scale Bar=l micron; Original magnification 1000X).

Fig. 4E is a digitized image showing a negative control tissue hybridized with IS900- biotin; (Scale Bar=l micron; Original magnification 1000X).

Fig. 4F is a digitized image showing a positive control Johne's tissue hybridized with IS900-biotin; (Scale Bar=l micron; Original magnification 1000X). Fig. 4G is a digitized image showing a negative control tissue hybridized with IS900- fluoroscein; (Scale Bar=l micron; Original magnification 1000X).

Fig. 4H is a digitized image showing a positive control Johne's tissue hybridized with IS900-fluoroscein; (Scale Bar=l micron; Original magnification 1000X).

Fig. 5A is a digitized image showing B. subtilis dual probed with IS900-fluorescein (lighter, punctuate structures) and EUB338-Cy3 (darker, rod-like structures) Scale Bar = 1 micron; Original magnification 100OX; Arrows indicate amorphous nonspecific signal.

Fig. 5B is a digitized image showing Rhodococcus sp. probed with IS900-biotin ISH Scale Bar = 1 micron; Original magnification 100OX; Arrows indicate amorphous nonspecific signal. Fig. 5C is a digitized image showing M. kansasii probed with IS900-biotin ISH Scale

Bar = 1 micron; Original magnification 100OX; Arrows indicate amorphous nonspecific signal.

Fig. 5D is a digitized image showing M. avium ssp. avium hybridized with EUB338-6' FAM ISH; Scale Bar = 1 micron; Original magnification 100OX; Arrows indicate signal positive bacilli.

Figs. 6A and 6E are digitized images showing M. tuberculosis dual hybridized with EUB338-FAM (Fig. 6A) and MTB-Cy3 probes (Fig. 6E) Original Magnification 100Ox.

Figs. 6B and 6F are digitized images showing M. intracellulare dual hybridized with

EUB338-FAM (Fig. 6B) and MTB-Cy3 probes (Fig. 6F) Original Magnification 100Ox. Figs. 6(J and 6ϋ are digitized images showing M. avium dual hybridized with EUB338-FAM (Fig. 6C) and MTB-Cy3 probes (Fig. 6G) Original Magnification 100Ox.

Figs. 6D and 6H are digitized images showing M. kansasii dual hybridized with EUB338-FAM (Fig. 6D) and MTB-Cy3 probes (Fig. 6H); Original Magnification 100Ox. Fig. 61 is a digitized image showing fluorescent in situ hybridization utilizing three oligonucleotide probes: MTB-FAM probes, MAVP187-Cy3, and EUB338-Cy5, hybridized with a mixed culture which contains M. tuberculosis, M. avium, M. intracellular e, and Corynebacterium sp (EUB338-Cy5 hybridized with all the bacteria; long M. tuberculosis rods are stained; short M. avium rods are stained; long M. intracellular rods and short Corynebacterium sp. rods are detected by the EUB338-Cy5 probe but not by the MTB-FAM probe or the MAVP187-Cy3 probe; Original Magnification 630X.

Fig. 61 is a digitized image showing sputum from a patient with a history of tuberculosis hybridized with MTB-FAM probes; arrow indicates a positive Mtb bacillus.

Fig. 6N is a digitized image showing a phase contrast micrograph of the same field as Fig. 6 J; arrow indicates the same bacilli as in Fig. 6 J.

Figs. 6K and 6L are digitized images showing Brightfield micrographs from AFB staining of Mtb H37rv infected guinea pig lung and mouse lung sections, respectively, counterstained with methylene blue (Brightfield micrograph taken with Nikon Eclipse E600 microscope; Original Magnification 1000X). Fig. 6M is a digitized image showing a Brightfield micrograph from AFB staining of

MAP infected bovine lymph node, counterstained with methylene blue (Brightfield micrograph taken with Nikon Eclipse E600 microscope; Original Magnification 1000X).

Fig. 60 is a digitized image showing that MTB-FAM probes hybridize with multiple Mtb bacilli in a representative granuloma of the guinea pig lung; nuclei were counterstained with DAPI (Epifluorescent micrograph taken with Leica Laser Scanning Confocal microscope; Original magnification 630X).

Fig. 6P is a digitized image showing a representative micrograph of a mouse lung specimen probed with the MTB-FAM oligos and visualized with enzymatic amplification of a colorimetric dye INT/BCIP (bacilli), counterstained with methylene blue (Brightfield micrograph taken with Nikon Eclipse E600 microscope; Original Magnification 1000X).

Fig. 6Q is a digitized image showing MTB-FAM probes did not hybridize with the bacilli in the MAP infected bovine lymph node (fuzzy background). MAVP187-Cy3 did hybridize to the MAP within the tissue (bacilli) (Epifluorescent micrograph taken with Leica

Laser Scanning Confocal microscope; Original magnification 630X). figs"^, .^ ^^ ^g ^ digitized images showing tissue from a patient with bronchiectasis and lung destruction probed with the M. avium probe, MAVPl 87 ssu.

Figs. 8A and 8B are digitized images showing the use of M. abscessus cy3 probe to probe expectorated sputum from a chronically infected patient with M. abscessus (Fig. 8A) and control sputum (Fig. 8B).

Fig. 8C is a digitized image showing heavily differentiated Carbol fuchsin acid-fast stain; arrow demonstrates the only positive rod in entire specimen (>300 high power (1000X) fields).

Figs. 8D and 8E are digitized images showing the use of M. abscessus probe (Fig. 8D) and M. avium probe (Fig. 8E) to probe a specimen from a patient with known M. avium pulmonary disease.

Figs. 9A-9C are digitized images showing the hybridization of BAC338LNA1 Cy3 (SEQ ID NO:13; Fig. 9A), BAC338LNA2 Cy3 probe (SEQ ID NO:14; Fig. 9B), and BAC338 (SEQ ID NO: 1) to fixed E. coli cultures.

Detailed Description of the Invention

The present invention generally relates to oligonucleotides (e.g, DNA, RNA, or peptide nucleic acids (PNA)) that are complementary to, or hybridize under stringent hybridization conditions to, ribosomal RNA (rRNA) sequences from microbial cells, and particularly, mycobacterial cells. These oligonucleotide probes have been designed to be species-specific or phylogenetic group-specific, and are intended to be used primarily as in situ hybridization probes. Specifically, the present inventors have developed and validated a set of oligonucleotide probes that permit the detection and differentiation of several species, or closely related groups, of the genus Mycobacteria. The oligonucleotides have been designed to specifically detect and differentiate from other microbes the following species or phylogenetic groups: 1) Mycobacterium abscessus, 2) Mycobacterium tuberculosis complex members, 3) Mycobacterium avium spp. avium and Mycobacterium avium spp. paratuberculosis, 4) Mycobacterium intracellular and 5) Mycobacterium kansasii. These species are of great clinical importance because they can cause a variety of tuberculous and non-tuberculous diseases. These oligonucleotides bind to the cellular ribosomes of fixed samples in a sequence-dependent manner. Modification of the oligonucleotides by addition of fluorescent dyes, or other small moieties or labels (e.g., biotin) suitable for detection procedures, allows for the detection of hybridized probes by either fluorescence microscopy or indirect colorimetric assay. Under the proper conditions, such as by using the novel cell peπheabϊϊizatiόh^'aήd hybridization techniques described herein, detection ot tne Hybridized oligonucleotides provides proof that the targeted microbial cells are physically present in a particular sample.

The development of suites of phylogenetically informative, single-cell probes is of considerable utility to the clinical microbiology community. Traditionally, identification of mycobacteria beyond the genus level has required, as a first step, isolation of microbes in pure culture. However, many pathogenic mycobacteria species are characterized by slow, fastidious growth that often requires weeks to months for their isolation. Clinical care is thus complicated, because acute mycobacterial infections require treatment long before positive identification of the causative species can be accomplished. There is a clear need in the art for rapid, diagnostic tests and probes for the detection of the presence or absence of mycobacterial species that are applicable to many different tissue types in addition to sputum, that does not require cultivation of the microbial sample prior to detection, and that can distinguish mycobacterial species or phylogenetic groups from one another. Moreover, even with regard to the testing of sputum, there is a need in the art to be able to detect any species of Mycobacteria, or at least species other than Mycobacterium tuberculosis, in sputum samples, so that appropriate treatment can be prescribed for the patient. The present invention provides the solution to these needs.

The inventors report herein the development of several different rRNA probes and techniques for reproducible and efficient permeabilization of mycobacteria in tissue, sputum, and culture. Furthermore, using bovine Johne's disease as a model system, the inventors have compared their methods to previously published IS900 protocols, and have shown that the method and the probes of the present invention are superior to these prior protocols. Finally, the present inventors have demonstrated the general applicability of rRNA probes to a variety of proven and suspected human diseases.

First, the inventors describe rRNA-based oligonucleotide probes, referred to collectively herein as Mycobacterium avium complex (MAC) probes, that specifically detect M. intracellular (probes MIN351ssu (SEQ ID NO:5) and MIN15861su (SEQ ID NO:6)), the two M. avium subspecies (MAA and MAP; probes MAVPl 87ssu (SEQ ID NO:2) and MAVP5151su (SEQ ID NO:3)) and all members of the M. avium complex (probe MAC25431SU (SEQ ID NO:4)). To assess the specificities and sensitivities of the MAC probes, the inventors applied these probes to four types of samples: 1) axenic bacterial cultures, M. kansasii and M. tuberculosis; pathogens closely related to MAC; 2) bovine

Johne's disease tissue infected with MAP; 3) human clinical samples with a variety of confirmed mycobacterial infections; and 4) human and bovine samples not infected with MAC. With the methodology of the present invention, the ISH probes specifically labeled the mycobacterial species against which they were designed, in both tissues and culture samples. The specificity of these MAC probes has been further demonstrated by their successful hybridization to pure cultures of MAA and MAP; under the permeabilization and hybridization conditions of the present invention, these probes fail to detect other mycobacterial species (M. intracellular, M. tuberculosis, M. kansasii, M. abscessus, M. phlei, and M. parafortuitum) as well as other, more distantly related, microbial species (Corynebacterium spp., Rhodoccus spp., Bacillus subtilis, and Escherichia coli). Second, the inventors also describe herein rRNA-based oligonucleotide probes, referred to herein collectively as Mycobacterium tuberculosis complex (MTB) probes, that specifically detect Mycobacterium tuberculosis (probes MTB770 (SEQ ID NO:7), MTB226 (SEQ ID NO:8), and MTB187 (SEQ ID NO:9)). To assess the specificities and sensitivities of the MTB probes, the inventors applied these probes to five types of samples: 1) axenic bacterial cultures of M. tuberculosis, M. avium, M. intracellular, M. kansasii, M. parafortuitium, M. phlei, Escherichia coli, Bacillus subtilis, Rhodococcus sp. and Corynebacterium sp.; 2) mixed bacterial cultures of M. tuberculosis, M. avium, M. intracellular and Corynebacterium sp.; (3) clinical sputa samples that were acid-fast bacillus (AFB) positive but culture negative; (4) M. tuberculosis-infected guinea pig lung, mouse lung, and bovine lymph node; and (5) non-infected guinea pig lung, mouse lung, and bovine lymph node. With the methodology of the present invention, the ISH probes specifically labeled the mycobacterial species against which they were designed, in infected sputum, tissues and culture samples, but not control samples.

Third, the inventors describe herein Locked Nucleic Acid (LNA)-modified versions of the rRNA oligonucleotides described herein. LNA is a novel type of nucleic acid modification that contains a 2'-O, 4'-C methylene bridge. This bridge restricts the flexibility of the ribofuranose ring and locks the sugar into the N-type conformation of the A-form DNA. LNA monomers also induce the adjacent DNA or RNA nucleotides to adopt the N type conformation. Addition of LNA monomers to specific oligonucleotides leads to increases in specificity and thermal stability when hybridized to the DNA or RNA target. The inventors have demonstrated that incorporation of LNA bases into the oligonucleotide probes enhanced hybridization efficiency.

In addition to the development of a variety of species-specific or phylogenetic group- specific oligonucleotide probes as discussed above, the present inventors have developed an assay that can identify a variety of mycobacterial species without prior cultivation. This assay can be applied to clinical (e.g., tissue, sputum) samples and/or environmental (e.g., water, biofϊlm) samples to detect mycobacteria in pure or mixed cultures. Application of this invention will be of substantial utility to clinicians and clinical or environmental microbiologists, and/or veterinarians who treat tuberculous and non-tuberculous mycobacterial diseases. A presumptive diagnosis of the species or species-complex (e.g., M. tuberculosis complex group) of mycobacteria present in a clinical specimen, made on the basis of this assay and/or using the probes of the present invention, would allow definitive treatment decisions to be made weeks or even months before cultivation of the causative agent has succeeded.

More specifically, the present inventors report herein a rapid, highly reliable technique for the permeabilization and identification of specific species or phylogenetic groups of mycobacteria in tissues and cultures by in situ hybridization (ISH). The novel ISH technique of the present invention allows for direct observation of bacterial distribution and morphology in the context of the histopathology of the tissue. Additional specific oligonucleotide probes that distinguish between closely related organisms, other than those described herein, can be developed based on comparisons of rRNA gene sequences (Giovannoni, 1988; Lane, 1985) and used in the present methods. The present method provides a rapid means for evaluating additional candidate rRNA-hybridizing probes, which probes must be tested for utility after design, due to the complex three-dimensional structure of the ribosome which precludes many candidate probes from productively binding to their complementary sequences.

Because of their waxy, mycolic acid-laden cell walls, mycobacteria normally are recalcitrant to the typical permeabilization steps used for in situ hybridizations with bacteria. When performing ISH with mycobacteria, the present inventors have discovered that results were reproducible only when cells were permeabilized with a combination of xylene, lysozyme, and achromopeptidase treatments. Omission of any one of these steps either diminished or abolished signals from the probes. The permeabilization protocol of the present invention also works well with other Gram-positive bacteria, such as Corynebacterium sp., Rhodococcus sp. and B. subtilis, but in these instances, the xylene step is not critical.

Proteinase K often is used to permeabilize mycobacteria in culture and tissue, in preparation for in situ hybridization (Hulten, 2001; Sechi, 2001). As shown herein, however

(see Examples 1-4), the use of proteinase K for mycobacterial ISH resulted either in inadequate ^'"peimeabilizaSon (i.e. lack of signal) or in significant tissue damage and non¬ specific binding of probes to tissue (i.e. excessive background signal). Overdigestion with proteinase K is therefore likely to increase the occurrence of false positive results, as were observed for IS900 ISH (discussed below). In contrast, lysozyme and achromapeptidase digest only bacterial cell walls and therefore do not affect the surrounding mammalian tissue. The rRNA probes of the invention were visualized in method of the present invention by two techniques: 1) direct fluorescence and 2) enzymatic amplification of a colorimetric substrate. One important benefit of the fluorescent method is its applicability to confocal laser microscopy, which allows for high-resolution imaging of microbes in tissues. For example, in both bovine and human tissues, MAC cells were observed to be embedded in tissue sections, often within the cytoplasm of eukaryotic cells, thus eliminating the possibility of cross-contamination of specimens during the preparation of tissue sections (see Examples 1-4). In the present inventors' experience, it was important to use high-resolution microscopy, either under IOOOX oil-immersion or by confocal microscopy, in order to confirm by morphology the staining of microbial cells, although one of skill in the art will appreciate that in other detection techniques than the ones described in the Examples, visualization can be achieved by other suitable methods (e.g., radiolabeling techniques). In addition to direct visualization by fluorescence, ISH probes also were indirectly visualized by bright-field microscopy following reaction of a colorimetric dye (INT/BCIP) with antibody- conjugated alkaline phosphatase. This method is not influenced by tissue autofluorescence, which can obscure the true signals of fluorescently labeled probes. Although autofluorescence typically is not a problem, tissues that are densely packed with red blood cells and/or mast cells can be prone to high autofluorescent backgrounds. Because this indirect method of visualization can be performed on tissue sections following direct fluorescent detection, the two approaches can be used to corroborate results and thereby diminish the possibility of false positive results.

The 16S and 23 S rRNA sequences of MAP and MAA are identical and so rRNA probes cannot distinguish between the two organisms. PCR amplification of the IS900 gene, which is present in the genome of MAP, but not MAA, is an effective means of typing the two subspecies, and could be used subsequent to the method of the present invention, if desired, to distinguish between the two sub-species. However, although theoretically possible, the detection of MAP by IS900-based in situ hybridization presents several technical challenges. First, as demonstrated by rRNA-specific oligonucleotide probes (see

Examples 1-4), proper permeabilization is absolutely essential for gaining access to hybridization targets within a cell. Large hybridization probes, such as those produced by PCR amplification or nick-translation of the IS900 gene, should require even more careful control of permeabilization conditions than is necessary for smaller probes. Second, MAP genomes are reported to carry only 1-14 copies of the IS900 gene, which necessitates an extremely sensitive assay to detect so few targets. Bacterial mRNAs are very difficult to detect by ISH, so IS900 mRNA is unlikely to be detectable in these assays. Nevertheless, IS900-based ISH has been suggested as a means of detecting MAP in clinical tissues. Using IS900 ISH, both Sechi et al. (2001) and Hulten et al. (2001) have reported the presence of MAP in Crohn's disease specimens. In the present inventors' experience, however, in situ hybridization with IS900 probes resulted in false positive-signals, especially if the colorimetric dye (INT/BCIP) was allowed to react with alkaline-phosphatase for prolonged time periods (2 hours to overnight). When reaction times were limited to less than two hours, no signal was seen in positive control tissues (data published in St. Amand, 2005).

Bacterial cell morphology should be used as a benchmark for the validation of a positive signal with any kind of ISH because of the possibility of non-specific signals (Figs. 4 and 5 and Examples 1-4 below) (Roholl, 2002). Often this morphology can only be visualized by examination at IOOOX under oil immersion because single, one-micron rods (the typical size of MAP cells) are extremely difficult to visualize at 400X, the magnification typically used in clinical analyses. When visualized under oil immersion, the structures labeled with IS900 probes and INT/BCIP had large indistinct boundaries, as compared to the clearly defined morphology of cells labeled with rRNA probes of the present invention and INT/BCIP.

IS900 false positive results observed by the present inventors were not due solely to the colorimetric labeling and detection method. Under high magnification, fluorescently labeled IS900 probes were observed to bind the outer cell walls of a variety of bacterial species. In contrast, rRNA probes of the present invention were observed to label the interiors of cells uniformly. Thus, false positive results with the IS900 probes most likely arise from non-specific binding of DNA probes to cells, coupled with non-specific precipitation and aggregation of dye. Overall, the lack of specificity and apparently artifactual nature of the IS900 probes makes their use questionable for assay of mammalian tissues, such as Crohn's disease specimens, for the presence or absence of MAP bacteria.

Furthermore, the inventors demonstrate herein the ability of the mycobacteria- specific rRNA probes to detect and identify mycobacterial organisms in a variety of animal and human tissues, including sputum. The probes of the present invention offer researchers and clinicians^' an incisive method for identifying members of mycobacterial complexes, such as members of the MAC-complex or the MTB complex, in tissue and in culture. The present inventors have demonstrated that in several cases, as exemplified by probing an M. intracellulare-infected lung and M. avium tenosynovitis, the rRNA probes of the present invention detected more bacilli than were evident by standard acid fast staining procedures. Mycobacteria could not be detected by PCR in many of these specimens, possibly due to the low abundance of organisms or the presence of PCR inhibitors in the tissue. On the other hand, ISH can detect single cells and thus provide greater sensitivity than PCR. ISH probes hold the promise of more rapid detection and differentiation of mycobacterial species than traditional clinical microbiological techniques. Oligonucleotide Probes of the Invention

Accordingly, one embodiment of the present invention relates to an oligonucleotide probe that is useful for the detection of mycobacteria in a sample. The oligonucleotide probes useful in the present invention are oligonucleotides that are complementary to, or hybridize under stringent hybridization conditions to, ribosomal RNA (rRNA) sequences from mycobacterial cells. The oligonucleotide probes are species-specific or phylogenetic group-specific, and are suitable for use as in situ hybridization (ISH) probes. According to the present invention, a "phylogenetic group" refers to a higher-order evolutionary group of related organisms, such as several species within a genus that are evolutionarily related. For example, the Mycobacterium avium complex (MAC) is a phylogenetic group that consists of genetically similar, slowly growing bacteria that includes Mycobacterium avium ssp. avium, Mycobacterium avium ssp. paratuberculosis, Mycobacterium avium ssp. silvaticum, Mycobacterium avium ssp. hominis, and Mycobacterium intracellular (De Groote, 2003). As such, the oligonucleotide probes of the invention do not cross-hybridize (cross-react, substantially bind to) rRNA from mycobacterial species or phylogenetic groups other than the species or groups to which the probe is designed to hybridize. Such an oligonucleotide probe according to the invention can be produced by: (a) determining or obtaining the nucleotide sequence of rRNA (e.g., 16S or 23S), or the gene encoding the rRNA, from a specific mycobacterial species or phylogenetic group; (b) designing an oligonucleotide probe that is complementary to a fragment of the rRNA sequence of the target rRNA, but is not expected to hybridize to rRNA from a different mycobacterial species or phylogenetic group; and (c) validating the sensitivity and selectivity of the designed oligonucleotide probe. The sequences of the 16S and 23 S rRNA for many bacterial and mycobacterial species are known in the art or can be determined using methodology well-known in the art. Hybridization efficiency of these probes is dependent on the ability to choose a sterically accessible binding site(s) on the three-dimensional surface of the ribosome, and therefore, this evaluation is included in the design of the oligonucleotide probe. Step (c) is performed by determining whether the probe selectively binds to the target rRNA {i.e., from mycobacterial cells of the selected mycobacterial species or phylogenetic group) but does not substantially bind to rRNA from different mycobacterial species {i.e., the probe differentiates among mycobacterial species). Step (c) is also used to determine whether the probe is sensitive enough to be used in ISH, alone or in combination with other probes. The validation procedure is preferably performed using the novel permeabilization and/or ISH protocol described herein (described in detail below). The present inventors have determined the parameters for preparing samples for ISH so that they can be rapidly and successfully probed using specific oligonucleotide probes useful in the present invention.

The present inventors have designed, produced and validated several specific oligonucleotide sequences that can detect and differentiate between certain species and phylogenetic groups of mycobacteria, including Mycobacterium abscessus, Mycobacterium tuberculosis complex members, Mycobacterium avium spp. avium and Mycobacterium avium spp. paratuberculosis, Mycobacterium intercellulare, and Mycobacterium kansasii, and the sequences of these oligonucleotide probes are described herein. However, it will be apparent to those of skill in the art that these sequences can be modified to produce related variants that will retain specificity and sufficient sensitivity to be used in an ISH assay described herein. Therefore, one embodiment of the present invention relates to an oligonucleotide probe consisting of a nucleic acid sequence that hybridizes under stringent conditions to the complement of an oligonucleotide sequence selected from any one of SEQ ID NOs:2-19. The oligonucleotide probe also hybridizes under stringent conditions to rRNA in a mycobacteria.

In accordance with the present invention, an isolated polynucleotide, or an isolated nucleic acid molecule, which includes an oligonucleotide, is a nucleic acid molecule that has been removed from its natural milieu (i.e., that has been subject to human manipulation), its natural milieu being the genome or chromosome in which the nucleic acid molecule is found in nature. As such, "isolated" does not necessarily reflect the extent to which the nucleic acid molecule has been purified, but indicates that the molecule does not include an entire genome or an entire chromosome in which the nucleic acid molecule is found in nature. Polynucleotides such as those used in a method of the present invention to detect rRNAs

(e.g., by hybridization to a gene or RNA transcript of a gene) are complementary to a portion of the target rIUSTA and are suitable for use as a hybridization probe for the identification of the target rRNA in a given sample (e.g., a cell sample, a tissue sample, a sputum sample, etc.).

Reference to any isolated nucleic acid molecule can include reference to a gene or a

5 portion of a gene (e.g., the regulatory region or promoter). An isolated nucleic acid molecule that includes a gene is not a fragment of a chromosome that includes such gene, but rather includes the coding region and regulatory regions associated with the gene, but no additional genes naturally found on the same chromosome. An isolated nucleic acid molecule, including an oligonucleotide as described herein, can also include a specified nucleic acid

0 sequence flanked by (i.e., at the 5' and/or the 3' end of the sequence) additional nucleic acids that do not normally flank the specified nucleic acid sequence in nature (i.e., heterologous sequences). Isolated nucleic acid molecules can include DNA, RNA (e.g., rRNA, mRNA), or derivatives of either DNA or RNA (e.g., cDNA). Although the phrase "nucleic acid molecule" primarily refers to the physical nucleic acid molecule and the phrase "nucleic acid

5 sequence" primarily refers to the sequence of nucleotides on the nucleic acid molecule, the two phrases can be used interchangeably. Preferably, an isolated nucleic acid molecule of the present invention is produced using chemical synthesis or recombinant DNA technology (e.g., polymerase chain reaction (PCR) amplification, cloning). Methods to deduce a complementary sequence are known to those skilled in the art.

0 According to the present invention, a probe (oligonucleotide probe) is a nucleic acid molecule which typically ranges in size from about 8 to about 100 nucleotides, to several hundred nucleotides, to several thousand nucleotides in length. Typically, probes useful in the ISH methods according to the present invention are of a length from about 8 nucleotides to about 50 nucleotides, and more preferably from about 8 nucleotides to about 25

5 nucleotides. Therefore, a probe can be any suitable length for use in an assay described herein, including any length in the range of about 8 nucleotides to 50 nucleotides, in whole number increments (e.g., 8, 9, 10...22, 23, 24...49, 50). The probes are used to identify a target nucleic acid sequence (i.e., an rRNA sequence in the present invention) in a sample by hybridizing to such target nucleic acid sequence under stringent hybridization conditions.

) Hybridization conditions are described in detail below.

General reference to an oligonucleotide described herein is to be interpreted to include peptide nucleic acids (PNA). PNAs are analogues of DNA in which the backbone of the molecule is a pseudopeptide rather than a sugar. More specifically, a PNA monomer is 2- aminoethyl glycine linked by a methylenecarbonyl linkage to one of the four bases (adenine, guanine, thymine, or cytosine) found in DNA. Like amino acids, PNA monomers have amino and carboxyl termini. Unlike nucleotides, PNA's lack pentose sugar phosphate groups. PNA monomers are linked by peptide bonds into a single chain oligomer. PNA mimics the behavior of DNA and binds complementary nucleic acid strands, similar to a conventional nucleotide-based oligonucleotide. The neutral backbone of PNA results in stronger binding and greater specificity than is normally achieved using DNA-based oligonucleotides. PNA monomers are easily synthesized into oligomers as long as 20 bases using protocols for standard peptide synthesis (e.g., using commercially available L monomers supplied as Boc- benzyloxycarbonyl-protected derivatives that are coupled in DMF/pyridine containing HBTU and a tertiary amine, with completion by TFMSA cleavage of the oligomer from the resin, and purification by reverse phase HPLC).

The oligonucleotides of the present invention can also be used as primers for polymerase chain reaction (PCR). PCR primers are also nucleic acid sequences, although PCR primers are typically oligonucleotides of fairly short length as compared to probes (e.g., 8-30 nucleotides, although probe and primer lengths can overlap, particularly with regard to the present invention) that are used in polymerase chain reactions. PCR primers, as well as and hybridization probes, can readily be developed and produced by those of skill in the art, using sequence information from the target sequence. (See, for example, Sambrook et al., supra or Glick et al., supra). Nucleic acid hybridization (and PNA hybridization) involves contacting a probe and target nucleic acid under conditions where the probe and its complementary target can form stable hybrid duplexes through complementary base pairing. As used herein, hybridization conditions refer to standard hybridization conditions under which nucleic acid molecules are used to identify similar nucleic acid molecules. Such standard conditions are disclosed, for example, in Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Labs Press, 1989. Sambrook et al., ibid., is incorporated by reference herein in its entirety (see specifically, pages 9.31-9.62). In addition, formulae to calculate the appropriate hybridization and wash conditions to achieve hybridization permitting varying degrees of mismatch of nucleotides are disclosed, for example, in Meinkoth et al., 1984, Anal. Biochem. 138, 267-284; Meinkoth et al., ibid., is incorporated by reference herein in its entirety. Nucleic acids that do not form hybrid duplexes are washed away from the hybridized nucleic acids and the hybridized nucleic acids can then be detected, typically through detection of an attached detectable label. It is generally recognized that nucleic acids are denatured by increasing the temperature or decreasing the salt concentration of the buffer containing the nucleic acids. Under low stringency conditions (e.g., low temperature and/or high salt) hybrid duplexes (e.g., DNA:DNA, RNA:RNA, or RNA:DNA) will form even where the annealed sequences are not perfectly complementary. Thus, the specificity of hybridization is reduced at lower stringency. Conversely, at higher stringency (e.g., higher temperature or lower salt) successful hybridization requires fewer mismatches.

The methods of the present invention typically utilize high stringency hybridization conditions, so that the sensitivity and specificity of the assay is maximized. In one embodiment, stringent hybridization and washing conditions, as used herein, refer to conditions which permit the detection of nucleic acid molecules having at least about 90% nucleic acid sequence identity with the nucleic acid molecule or PNA being used as the probe in the hybridization reaction (i.e., conditions permitting about 10% or less mismatch of nucleotides). In a more preferred embodiment, hybridization conditions are sufficiently stringent that nucleic acid probes bind only to the target rRNA to which they were designed and so only the desired mycobacterial species are detected. Functionally, stringent hybridization conditions should enhance the specificity of hybridization with regard to species or groups of species to which the oligonucleotide probes were designed.

Most preferably, stringent conditions, as used herein, refer to the hybridization conditions described below, including the specifically exemplified conditions and the provided suitable ranges for the conditions, for the novel permeabilization and ISH method of the invention. Also, as discussed above, one of skill in the art can use the formulae in Meinkoth et al., ibid, to calculate the appropriate hybridization and wash conditions to achieve these particular levels of nucleotide mismatch. Such conditions may vary, depending on whether DNA:RNA, RNA:RNA, or PNA:RNA hybrids are being formed, as well as on whether PNA and DNA oligonucleotides have been further modified, such as by Locked Nucleic Acid (LNA) modification described below. For example, the calculated melting temperatures for DNA:DNA hybrids are 10°C less than for DNA:RNA hybrids. Suitable hybridization conditions for use in the present invention are described in detail in the Examples section, although it will be apparent to those of skill in the art that variation in the exact conditions is possible, while maintaining the ability to detect a target rRNA molecule according to the present invention.

The hybridized nucleic acids are detected by detecting one or more labels attached to the sample nucleic acids. The labels may be incorporated by any of a number of means well known to those of skill in the art. Detectable labels suitable for use in the present invention include any composition detectable by microscopic, spectroscopic, photochemical, biochemical, immunochemical, electrical, optical or chemical means. Useful labels in the present invention include fluorescent dyes (e.g., fluorescein, texas red, rhodamine, green fluorescent protein, yellow fluorescent protein and the like), biotin for staining with labeled streptavidin conjugate, radiolabels (e.g., ³H, ¹²⁵1, ³⁵S, ¹⁴C, or ³²P), enzymes (e.g., horse radish peroxidase, alkaline phosphatase and others commonly used in an ELISA), various colorimetric labels, colloidal gold, colored glass or plastic (e.g., polystyrene, polypropylene, latex, etc.) beads, or magnetic beads (e.g., Dynabeads.TM.). Means of detecting such labels are well known to those of skill in the art.

According to the present invention, an oligonucleotide probe useful in the present invention selectively or specifically hybridizes to a target rRNA sequence, without cross- hybridizing (cross-reacting, substantially binding to) an rRNA sequence from a different microbial species or phylogenetic group. Hybridization and stringent hybridization conditions are described above. As used herein, the term "selectively binds to" or "specifically binds to" refers to the specific binding of one molecule to another (e.g., an oligonucleotide to a target rRNA sequence), wherein the level of binding, as measured by any standard assay (e.g., hybridization, and particularly ISH), is statistically significantly higher than the background control for the assay. Statistical significance is typically defined as p<0.05. For example, when performing ISH assays, controls typically include a negative control, wherein an amount of hybridization (e.g., non-specific binding) by the oligonucleotide probe in the absence of the target rRNA is considered to be background.

One can readily determine whether a given oligonucleotide hybridizes with rRNA other than the target rRNA (i.e., whether the probe "cross-hybridizes", "cross-reacts", substantially binds to) an rRNA other than the rRNA against which the oligonucleotide probe was designed or produced using an assay which determines the hybridization (or lack thereof) of the probe to non-target rRNAs, such as by using the ISH assay described by the present invention. If the level of hybridization of a given probe to a non-target rRNA is statistically significantly higher than the background control for the assay, such that the hybridization is indicated to be other than a non-specific hybridization, then one may conclude that the given probe lacks specificity or selectivity for the target rRNA. Such a probe may still be useful in the present invention, for example, as a positive control for hybridization conditions or to detect morphology. The level of hybridization of a non-specific oligonucleotide probe to the non-target rRNA can be less than the level of binding of the probe to the target rRNA. A weakly cross-hybridizing probe can be defined herein as a probe that hybridizes to a non- target rRNA at a level that is about 20% or less (but statistically significantly higher than background) than the level of hybridization of the probe to the target rRNA. However, one of skill in the art will be able to determine an appropriate standard or limit for determining specific or non-specific hybridization based on the assay conditions, probes, sample types, and standards or controls used. In one embodiment of the invention, an oligonucleotide probe of the invention comprises, consists essentially of, or consists of, a nucleic acid sequence (or equivalent thereof, in the case of PNA) that is at least about 70% identical, and more preferably at least about 75% identical, and more preferably at least about 80% identical, and even more preferably at least about 85% identical, and even more preferably at least about 90% identical and even more preferably at least about 95% identical, and even more preferably at least about 96% identical, and even more preferably at least about 97% identical, and even more preferably at least about 98% identical, and even more preferably at least about 99% identical (or any percentage between 70% and 99%, in whole single percentage increments) to the reference nucleic acid sequence represented by any one of SEQ ID NOs:2-19, calculated over the full length of the reference nucleic acid sequence.

For all variants of the reference oligonucleotide probes that have been designed, produced and validated by the present inventors, in addition to hybridizing to the complement of the reference sequence under stringent conditions, also hybridizes to the target mycobacterial rRNA to which the reference sequence hybridizes, under the stringent conditions that are required to perfonn ISH according to the invention using the reference oligonucleotide probe. In some embodiments, the variant of the reference oligonucleotide can act as a competitive inhibitor of the reference oligonucleotide probe by inhibiting the hybridization of the reference oligonucleotide probe to its target rRNA (the rRNA that was used to design the reference oligonucleotide or against which the oligonucleotide has been validated).

As used herein, unless otherwise specified, reference to a percent (%) identity refers to an evaluation of homology which is performed using a BLAST homology search. BLAST homology searches can be performed using the BLAST database and software, which offers search programs including: (1) a BLAST 2.0 Basic BLAST homology search using blastn for nucleic acid searches with standard default parameters, wherein the query sequence is filtered for low complexity regions by default (described in Altschul, S.F., Madden, T.L., Schaaffer, A.A., Zhang, J., Zhang, Z., Miller, W. & Lipman, DJ. (1997) "Gapped BLAST and PSI- BLAST: a new generation of protein database search programs." Nucleic Acids Res.

25:3389-3402, incorporated herein by reference in its entirety); (2) a BLAST 2 alignment (using the "parameters described below); (3) and/or PSI-BLAST with the standard default parameters (Position-Specific Iterated BLAST. It is noted that due to some differences in the standard parameters between BLAST 2.0 Basic BLAST and BLAST 2, two specific sequences might be recognized as having significant homology using the BLAST 2 program, whereas a search performed in BLAST 2.0 Basic BLAST using one of the sequences as the query sequence may not identify the second sequence in the top matches.

Two specific sequences can be aligned to one another using BLAST 2 sequence as described in Tatusova and Madden, (1999), "Blast 2 sequences - a new tool for comparing protein and nucleotide sequences", FEMS Microbiol Lett. 174:247-250, incorporated herein by reference in its entirety. BLAST 2 sequence alignment is performed in blastn using the BLAST 2.0 algorithm to perform a Gapped BLAST search (BLAST 2.0) between the two sequences allowing for the introduction of gaps (deletions and insertions) in the resulting alignment. For purposes of clarity herein, a BLAST 2 sequence alignment between nucleic acid sequences is performed using the standard default parameters as follows. For blastn, using 0 BLOSUM62 matrix:

Reward for match = 1 Penalty for mismatch = -2 Open gap (5) and extension gap (2) penalties gap x_dropoff (50) expect (10) word size (11) filter (on) In another embodiment of the invention, the oligonucleotide probes of the invention can be modified. For example, the probes can be modified to improve the stability and/or the specificity of an oligonucleotide probe for its target. In one aspect of the invention, oligonucleotide probes can be modified to include one or more Locked Nucleic Acid (LNA) monomers in the sequence. As described above, LNA is a novel type of nucleic acid modification that contains a 2'-O, 4'-C methylene bridge. This bridge restricts the flexibility of the ribofuranose ring and locks the sugar into the N-type conformation of the A-form DNA. LNA monomers also induce the adjacent DNA or RNA nucleotides to adopt the N type conformation. Addition of LNA monomers to specific oligonucleotides leads to increases in specificity and thermal stability (melting temperature) when hybridized to the DNA or RNA target. The inventors have demonstrated that incorporation of LNA bases into the oligonucleotide probes enhanced hybridization efficiency. Preferably, oligonucleotides of the invention can be modified with 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more LNA monomers, wherein up to about 100% of the nucleic acid sequence of the oligonucleotide can be modified in this manner. In a preferred embodiment, probes of between about 8 and about 15 Bases are' modified up to about ^'lTJO% of positions; probes of between about 16 and about 25 bases are modified up to about 60% of positions; probes of between about 26 and about 40 bases are modified up to about 40% of positions; and longer probes are modified up to about 20% of positions (see, e.g., Proligo Inc. Technical Resources). Methods for optimizing LNA design of oligonucleotides are known in the art (e.g., see Tolstrup et al., Nucleic Acids Res. 2003 JuI l;31(13):3758-62). In addition, methods of producing LNA-modified sequences are known in the art, including by automated synthesis and purification (e.g., see Pfundheller et al., Methods MoI Biol. 2005 ;288: 127-46).

In one aspect of the present invention, the oligonucleotide probe preferably hybridizes under stringent conditions to rRNA from the Mycobacterium avium complex, but does not substantially hybridize (e.g., does not cross-react) under the novel ISH conditions described herein with rRNA from other species or phylogenetic groups of mycobacteria or other bacteria or microbes. In one embodiment, such an oligonucleotide probe hybridizes under stringent conditions to rRNA from mycobacterium selected from: Mycobacterium avium ssp. avium, Mycobacterium avium ssp. paratuberculosis, Mycobacterium avium ssp. silvaticum, Mycobacterium avium ssp. hominis, and Mycobacterium intracellular. The present inventors have provided and validated an oligonucleotide probe that is specific for the Mycobacterium avium complex rRNA, and that can be used alone or in combination with other probes described herein in any of the methods described herein. This oligonucleotide probe consists of the nucleic acid sequence of SEQ ID NO:4 (also identified herein as MAC25431su)

In one aspect of the present invention, the oligonucleotide probe preferably hybridizes under stringent conditions to rRNA from Mycobacterium intracellular, but does not substantially hybridize (e.g., does not cross-react) under the novel ISH conditions described herein with rRNA from other species or phylogenetic groups of mycobacteria or other bacteria or microbes. The present inventors have provided and validated two oligonucleotide probes that are specific for Mycobacterium intracellular rRNA and that can be used alone or in combination with each other or other probes described herein in any of the methods described herein. The oligonucleotide probes consist of a nucleic acid sequence selected from SEQ ID NO:5 and SEQ ID NO:6 (also identified herein as MIN351ssu and MIN15861su, respectively).

In another aspect of the present invention, the oligonucleotide probe preferably hybridizes under stringent conditions to rRNA from Mycobacterium avium ssp. avium and

Mycobacterium avium ssp. paratuberculosis, but does not substantially hybridize (e.g., does ^'hbt" crosS'-r'δ¹aet)""unclef" ffie novel^" ISH conditions described herein with rRNA from other species or phylogenetic groups of mycobacteria or other bacteria or microbes. The present inventors have provided and validated three oligonucleotide probes that are specific for Mycobacterium avium ssp. avium and Mycobacterium avium ssp. paratuberculosis rRNA (which are identical) and that can be used alone or in combination with each other or other probes described herein in any of the methods described herein. The oligonucleotide probes consist of a nucleic acid sequence selected from SEQ ID NO:2, SEQ ID NO:3, and SEQ ID NO: 16 (also identified herein as MAVP187ssu, MAVP5151su, and MAPVP 187LNA, respectively). It is noted that SEQ ID NO: 16 is an LNA-modified version of SEQ ID NO:2. In another aspect of the present invention, the oligonucleotide probe preferably hybridizes under stringent conditions to rRNA from Mycobacterium tuberculosis, but does not substantially hybridize (e.g., does not cross-react) under the novel ISH conditions described herein with rRNA from other species or phylogenetic groups of mycobacteria or other bacteria or microbes. The present inventors have provided and validated six oligonucleotide probes that are specific for Mycobacterium tuberculosis rRNA and that can be used alone or in combination with each other or other probes described herein in any of the methods described herein. The oligonucleotide probes consist of a nucleic acid sequence selected from SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:17, SEQ ID NO:18 and SEQ ID NO: 19 (also identified herein as MTB770, MTB226, MTB 187, MTB187LNA, MTB226LNA, and MTB770LNA, respectively). It is noted that SEQ ID NO: 17 is an LNA- modified version of SEQ ID NO:9; SEQ ID NO: 18 is an LNA-modified version of SEQ ID NO:8; and SEQ ID NO: 19 is an LNA-modified version of SEQ ID NO:7.

In another aspect of the present invention, the oligonucleotide probe preferably hybridizes under stringent conditions to rRNA from Mycobacterium abscessus, but does not substantially hybridize (e.g., does not cross-react) under the novel ISH conditions described herein with rRNA from other species or phylogenetic groups of mycobacteria or other bacteria or microbes. The present inventors have provided and validated two oligonucleotide probes that are specific for Mycobacterium abscessus rRNA and that can be used alone or in combination with each other or other probes described herein in any of the methods described herein. The oligonucleotide probes consist of a nucleic acid sequence selected from SEQ ID NO: 10 and SEQ ID NO: 11 (also identified herein as Mabsc-1# and Mabsc-2#, respectively).

In another aspect of the present invention, the oligonucleotide probe preferably hybridizes under stringent conditions to rRNA from Mycobacterium kansasii, but does not substantially hybridize (e.g., does not cross-react) under the novel ISH conditions described 'Herein wlftT fRNA from other species or phylogenetic groups of mycobacteria or other bacteria or microbes. The present inventors have provided and validated one oligonucleotide probe that is specific for Mycobacterium kansasii rRNA and that can be used alone or in combination with other probes described herein in any of the methods described herein. The oligonucleotide probe consists of a nucleic acid sequence of SEQ ID NO: 12 (also identified herein as M kan).

Finally, the present inventors have produced three LNA-modified versions of the previously known general bacterial probe, EUB338ssu (SEQ ID NO:1). These LNA- modified oligonucleotide probes consist of a nucleic acid sequence of SEQ ID NO: 13, SEQ ID NO: 14 and SEQ ID NO: 15 (also identified herein as BAC338LNA1, BAC338LNA2 and BAC338LNA3, respectively). These probes can be used as positive controls to identify bacteria in a sample and to test hybridization and/or permeabilization conditions in an assay.

Any of the oligonucleotide probes described herein can be operatively linked (conjugated, linked, ligated, etc.) to a detectable label. Detectable labels suitable for use in the present invention include any composition detectable by microscopic, spectroscopic, photochemical, biochemical, immunochemical, electrical, optical or chemical means. Detectable labels are well-known in the art and examples of suitable labels are described in detail above.

Another embodiment of the invention relates to a panel, suite or array (i.e., more than one or a plurality) of oligonucleotide probes as described herein. Preferably, the panel includes one, two, three, four, five, six, seven, eight, nine, ten, or more (up to all) of the oligonucleotide probes specifically described herein, including variants of such probes as described herein. Additional probes that are designed, produced and/or validated using the methods described herein may also be included in the panel. The probes of the invention may be used, for example, in combination to analyze complex samples, or to rapidly diagnose a particular mycobacterial infection in a given sample, so that appropriate treatment can commence immediately.

One embodiment of the present invention relates to the provision of two, a few, several, or multiple oligonucleotide probes of the present invention, alone, or in combination with other oligonucleotide probes, in an array format. Nucleic acid arrays are useful for detecting rRNA or cDNA in a method of the present invention. In general, in an array, an oligonucleotide occupies a known location on a substrate (e.g., in the methods of the present invention, a suitable substrate includes, but is not limited to, a slide). A nucleic acid target sample (e.g., labeled rRNA or cDNA) is hybridized with an array of such oligonucleotides and then tJae amount of target^'nucleic acids hybridized to each probe in the array is quantified or characterized. One preferred quantifying method is to use confocal microscope and fluorescent labels. Array techniques are well known in the art, and it will be apparent to those of skill in the art that any suitable detection methods can be used. One embodiment of the invention relates to an assay kit that includes one or more

(i.e., 2, 3, 4, 5,...up to all) of the oligonucleotide probes specifically described herein, including variants of such probes as described herein, in any combination. In one aspect, the oligonucleotide probes can be provided operatively linked to a detectable label, or reagents can be provided with the kit for the user to operatively link the probes to a particular label or any of a variety of labels. An assay kit can also include other components, including, but not limited to, control rRNA samples, positive and/or negative control oligonucleotide probes, reagents (e.g., sample fixing reagents and buffers, permeabilization reagents and buffers, hybridization reagents and buffers, microscopy reagents and buffers, reagents for visualizing a detectable label, etc.), tools (e.g. slides, assay plates, etc.), and/or directions for using the kit.

Methods of the Invention

Another embodiment of the present invention relates to a method to detect the presence or absence of microbial cells, and particularly, mycobacterial cells, in a sample. Related embodiments of the present invention include a method to permeabilize microbial cells for use in an in situ hybridization (ISH) protocol, and a method to detect microbial cells using in situ hybridization. These methods are not limited to the permeabilization and/or detection of mycobacterial cells, as these methods can also be used with other microbial cells, and particularly bacterial cells, and more particularly, Gram-positive bacterial cells. In preferred embodiments, however, the methods are used with the goal of detecting mycobacterial cells in a sample.

The methods of the present invention can be used to detect the presence or absence of such microbial cells in any sample, including, but not limited to, laboratory samples, clinical veterinary samples, and environmental samples. In particular, the methods can be used to detect the presence or absence of such microbial cells in cell culture samples, tissue samples and sputum samples, although other sample types may also be tested (e.g., blood, mucous, seminal fluid, saliva, bronchial lavage, breast milk, bile, urine, water (e.g., drinking water), soil, plants, dairy products, meats, bioaerosols, biofϊlms, etc.). The samples can be obtained from complex organisms, such as any member of the Vertebrate class, Mammalia, including, without limitation, primates, rodents, livestock and domestic pets. Livestock include mammals to b"e cόnsύmeS or mat produce useful products (e.g., sheep for wool production or cattle for milk or meat production). The methods can be used to diagnose a mycobacterial infection of a sample or of an organism or subject, such as a human, domestic pet, laboratory pet, livestock, etc. The methods of the invention are suitable for use in both clinical (human) and veterinary or livestock applications.

First, the inventors have developed a novel method for the permeabilization of microbial cells for use in in situ hybridization (ISH) and specifically, for the detection of microbial cells in a sample using ISH. As discussed above, the present inventors have significantly improved upon the state of the art by inventing and validating the specific rRNA oligonucleotide probe sequences described herein and by devising procedures by which ISH techniques can be applied to Mycobacteria and other Gram-positive bacteria. Specifically, it is necessary to permeabilize microbial cells such as Mycobacteria in order to use ISH and access the rRNA. The inventors have determined that only when cells are permeabilized with a combination of xylene, lysozyme, and achromopeptidase treatments (which can be expanded to functionally equivalent treatments thereof by those of skill in the art, such as by using enzymes and agents that perform substantially the same reaction, including derivatives or analogs of such enzymes and agents), would the ISH hybridization protocol be effective. Omission of any one of these steps either diminished or abolished signals from the probes. The permeabilization protocol of the present invention also works well with other Gram- positive bacteria, such as Corynebacterium sp., Rhodococcus sp. and B. subtilis, but in these instances, the xylene step is not critical. In addition, the present inventors have determined that the conventional use of Proteinase K in the permeabilization protocol is not required or desired, as it can lead to inadequate permeabilization (i.e. lack of signal) or significant tissue damage and non-specific binding of probes to tissue (i.e. excessive background signal), as well as false-positive results.

The permeabilization protocol of the invention generally includes the following steps: (a) contacting fixed microbial cells (or sample that is being tested for the presence or absence of the microbial cells) with xylene; (b) rehydrating the microbial cells or sample; (c) enzymatically digesting the microbial cells or sample with lysozyme and achromapeptidase; and (d) washing and drying the microbial cells or sample.

Prior to contact of the cells or sample with xylene, the sample is fixed. Fixation, or cell, tissue or sample fixing, is well-known in the art and is generally used to preserve (fix) the structure of freshly killed material (e.g., cells, tissue) in a state that most closely resembles the structure and/or composition of the original living state. Living material samples are typicalϊyTixed using ariy^" suitable reagent (fixative) including, but not limited to, formalin, formaldehyde, glutaraldehyde, etc. In a preferred embodiment, cell cultures or tissue samples (e.g., obtained by biopsy, cutting, slicing, or a punch) are fixed with formalin for a few to several hours, and then washed in a buffer and stored until use. For example, tissues may be fixed in 10% formalin for 24 hours, washed with a buffer such as phosphate- buffered saline (PBS), and stored in 70% ethanol/30%PBS at -2O⁰C. Cell cultures may be fixed in 10% formalin for between about 4 and about 10 hours, washed with PBS, and stored in 70% ethanol/30%PBS at -2O⁰C. The cultures or tissue samples can be placed onto (applied to) any suitable substrate for the permeabilization and hybridization protocols of the invention, including, but not limited, to slides, plates, dishes, and the like.

Contact of the sample with xylene (preferably 100% xylene) is performed by any suitable technique that allows any microbial cells in the sample to contact the xylene, such as dewaxing, dipping, immersing, pouring, covering, etc. According to the present invention, any suitable functional equivalent, derivative, or analog of xylene may also be used in this step, and therefore, reference to xylene treatment is not necessarily limited to a single type of xylene and is intended to encompass reference to the use of functional equivalents, derivatives and/or analogs thereof. The contact period with the xylene should be sufficient to allow the xylene to contact and interact with the sample and typically requires between about 1 and about 30 minutes. In a preferred embodiment, cell samples immobilized on a slide are dipped into 100% xylene for about 5 minutes, and tissue samples immobilized on a slide are dewaxed in 100% xylene for about 20 minutes. It is noted that this step can be omitted when the detection of non-Mycobacterial, Gram-positive microbes is performed.

After the xylene treatment, the sample is rehydrated, such as by exposing the sample to a series of increasing dilutions of ethanol (from 100% to 0%) in a suitable buffer (e.g., Tris). In a preferred embodiment, the sample is rehydrated using an ethanol series of 100%, 70%, 30% and 0% ethanol in Tris buffer, pH 7.5.

After rehydration, the sample is enzymatically digested with a combination of lysozyme and achromapeptidase in a suitable buffer (e.g., Tris). The enzymes can be obtained from any suitable source and can include any functionally equivalent enzymes (enzymes that catalyze substantially the same enzymatic reaction and/or that achieve substantially the same result as the reference enzymes) or derivatives or analogs of the reference enzymes. Therefore, reference herein to a lysozyme treatment or an achromapeptidase treatment is not necessarily limited to a single type of lysozyme or achromapeptidase, respectively, and is intended to encompass reference to the use of ^" functional equivalents, deπvatives and/or analogs thereof. The enzymes are typically diluted in a buffer (e.g., the same buffer used to rehydrate the sample) and are applied to (contacted with) the sample at a temperature and for a time suitable to allow the enzyme to contact the sample and catalyze the enzymatic reaction. Suitable temperatures are typically between about 30°C and about 4O⁰C, with 37⁰C being preferred. The enzymatic reaction can be performed for between about 5 minutes and about 60 minutes, which between about 15 minutes and about 35 minutes being preferred, and with about 25 minutes being particularly preferred. The concentration of the enzymes is a concentration suitable to permeabilize cells in the sample during the given time period, and can be determined by those of skill in the art. In a preferred embodiment, lysozyme is used at a concentration of about 0.1 mg/ml to about 10 mg/ml of buffer, including any concentration between about 0.1 mg/ml and 10 mg/ml, in increments of 0.1 mg/ml, with about 1 mg/ml being particularly preferred. In another preferred embodiment, achromapeptidase is used at a concentration of from about 3 to about 300 units/ml of buffer, including any concentration between about 3 units/ml and about 300 units/ml, in increments of 1 unit/ml, with about 30 units/ml being particularly preferred. Finally, the samples are washed to remove excess enzymes, preferably in the same buffer used in the rehydration and enzyme digestion steps (e.g., by washing in Tris pH 7.5 for 5 minutes). The samples are then dehydrated (e.g., by decreasing dilutions of ethanol) and allowed to dry prior to using the samples in a hybridization protocol. In one preferred embodiment, a permeabilization method of the present invention is performed as follows: Permeabilization of Microbe-Containing Cultures

• Cultures are fixed in 10% formalin for 4-10 hours, washed with phosphate buffered saline (PBS), and stored in 70% Ethanol/30% PBS at -20 C. • Fixed cultures are applied to Silane Prepped slides

• Slides are dipped in 100% Xylene for 5 minutes

• Cultures are rehydrated in a Ethanol series 100, 70, 30, 0% in Tris pH 7.5

• Cultures are enzymatically digested with lmg/mL of lysozyme and achromapeptidase (30 units/mL) in Tris pH 7.5 at 37°C for 25 minutes (this step was adapted from Amman et al.)

• Cultures are washed for 5 minutes in Tris pH 7.5 and dehydrated in an Ethanol series 30, 70, 100%

• Slides and cultures are allowed to air dry before hybridization Permeabilization of Tissue Samples

• Tissues are fixed in 10% formalin for 24 hours, washed with PBS, and stored in 70% Ethanol/30%PBS at -20 C.

• Tissue is embedded in paraffin wax and 4 micron sections are created using a microtome.

• Fixed sections are placed on Silane prepped slides.

• Tissue is dewaxed in 100% Xylene for 20 minutes

• Tissues are rehydrated in a Ethanol series 100, 70, 30, 0% in Tris pH 7.5

• Tissue slides are enzymatically digested with lmg/mL of lysozyme and achromapeptidase (30 units/mL) in Tris pH 7.5 at 37°C for 25 minutes (this step was adapted from Amman et al.)

• Tissues are washed for 5 minutes in Tris pH 7.5 and dehydrated in an Ethanol series 30, 70, 100%

• Tissue slides are allowed to air dry before hybridization The present inventors have also devised a novel method for in situ hybridization

(ISH) which uses samples that have been permeabilized as described above. This method is used, in conjunction with the rRNA-specific oligonucleotide probes of the present invention, to identify microbial cells in a sample. In addition, the inventors have incorporated and modified steps from a prior hybridization protocol for non-Mycobacterial hybridization (Amman et al.), and have also devised particular novel steps, such as the heating and cooling steps, that enable the rapid detection of microbial cells in a variety of samples. The ISH method of the present invention generally includes the steps of: (a) contacting the sample that has undergone the permeabilization procedure described above with at least one oligonucleotide probe that specifically hybridizes to rRNA, in a hybridization buffer; (b) heating the sample in the buffer at a temperature sufficient to dissociate polynucleotide strands; (c) cooling the sample in the buffer at a temperature sufficient to allow polynucleotide strands to hybridize; (d) washing the sample to remove excess hybridization buffer; and (e) visualizing the cells by microscopy.

First, the sample that has undergone the permeabilization procedure described above is contacted with at least one oligonucleotide probe that specifically hybridizes to rRNA, in a hybridization buffer. The oligonucleotide probe can include any oligonucleotide probe(s), or combination thereof, that hybridizes to rRNA and has been described herein, including variants of the specifically probes herein or probes designed and validated using the methods described' n'βreiri.^"' in tins emooαiment, the probes are operatively linked to a detectable label.

Control probes, such as the bacteria group-specific probe, EU338 (SEQ ID NO:1), can be applied simultaneously with the species-specific and/or phylogenetic group-specific probes of the present invention, in order to assess the extent of permeabilization and/or hybridization conditions.

The hybridization buffer can include any suitable hybridization buffer, but is preferably selected to optimize the specific contact of the oligonucleotide probe(s) with the sample to which it was designed, and in a preferred embodiment, includes a mixture of NaCl, Tris, SDS and formamide. In a particularly preferred embodiment, the hybridization buffer comprises 90OmM NaCl, 2OmM Tris pH 8, 0.01% sodium dodecyl sulfate (SDS) and 10-50% formamide, with 20% being a preferred embodiment.

The probe(s) are added to the hybridization buffer and sample at a final concentration of about 0.1 ng/μl to 10 ng/μl, with a concentration of about 2 ng/μl being preferred.

The hybridization reactions are then heated to from about 92°C to about 98⁰C, and preferably, to about 94°C, for from about 1 minute to about 5 minutes, with 3 minutes being preferred. This step, and the following cooling step, are preferably performed using a thermal cycler, although this is not absolutely required.

Following the heating step, the samples are rapidly cooled to a temperature from about 37°C to about 6O⁰C, and preferably, from about 37⁰C to about 45⁰C, and more preferably from about 38⁰C to about 42⁰C, and more preferably to about 4O⁰C, depending on the oligonucleotide probe(s) used. The samples are held at this temperature for from about 30 minutes to about 12 hours, and preferably from about 6 hours to about 12 hours. In a preferred embodiment, LNA probes are hybridized for from about 30 to about 90 minutes.

Following the hybridization steps, the samples are washed with a lower salt buffer that is similar to the hybridization buffer, at a temperature that is from about 1-5⁰C higher, and preferably about 1° higher, than the cooling (hybridization) step above. In a preferred embodiment, the wash buffer contains NaCl, EDTA, SDS and Tris. In a particularly preferred embodiment, the wash buffer contains 225mM NaCl, 5mM EDTA, 0.01% SDS and

2OmM Tris pH 8. The washing step is performed from about 5 minutes to about 60 minutes, with between about 10 minutes and about 30 minutes being preferred, and about 20 minutes being more preferred. Finally, the samples are dipped briefly in cold buffer (e.g., Tris pH 8) to remove excess salt, and the samples are mounted for microscopic visualization, preferably with an anti-fading agent. TM^saniplόs a^'K^" visualized by any suitable technology that can detect hybridization reactions, such technologies being well known in the art. In one preferred embodiment, the samples are visualized using high resolution microscopy, such as under IOOOX oil-immersion or by confocal microscopy, in order to confirm by morphology the staining of microbial cells. The samples can be visualized using direct fluorescent microscopy or enzymatic amplification of a colorimetric substrate. The samples can also be visualized using other techniques suitable for hybridization reactions, such as by radiolabeling. The ISH probes can also be subsequently indirectly visualized by bright-field microscopy following reaction of a colorimetric dye (INT/BCIP), for example, with antibody-conjugated alkaline phosphatase. Two approaches can be used together or sequentially (e.g., confocal microscopy and bright- field microscopy) to corroborate results and thereby diminish the possibility of false positive results.

In one preferred embodiment, the ISH method of the present invention is performed as follows: 16s and 23 s Oligo probe hybridization

• Species- or phylogenetic-specific oligonucleotide probes according to the present invention are used. In addition, a bacteria group-specific probe, such as EUB338, can be applied simultaneously with specific probes in order to assess the extent of permeabilization. • Hybridization buffer consisted of 90OmM NaCI, 2OmM Tris pH 8. 0.01% SDS, and

20% Formamide. (this buffer was adapted for the present method from Amman et al.)

• Both probes are added to hybridization buffer at a final concentration of 2 ng/uL (adapted from Amman et al.)

• Frame-Seal (MJ Research, Inc.) incubation chambers for slides are used to prevent evaporation.

• Hybridization slides are placed in an MJ Research. Inc. Slide Thermocycler and are heated at 94⁰C for 3 minutes and then brought down to 4O⁰C for 6-12 hours.

• Slides are washed with 225 mM NaCl, 5mM EDTA, 0.01% SDS, and 20 mM Tris pH 8 for 20 minutes at 41⁰C. (adapted from Amman et al) • Slides are dipped briefly in cold 20 mM Tris pH 8 in order to remove excess salt and then mounted with Citifluor antifading reagent. Muorescehce 'Microscopy^{" '}

• Slides are visualized under epifluorescence using either a Nikon Eclipse E600 microscope or a Leica laser scanning confocal microscope

The permeabilization method and the hybridization method of the present invention can be combined, as described above, to provide a complete method for the in situ hybridization of microbial cells, or a method to detect microbial cells, and particularly, mycobacterial cells, in a sample. In one embodiment, the method can be used to diagnose a mycobacterial infection in a human or other animal subject, by detecting the presence of mycobacterium in the subject, and by further detecting the particular species or phylogenetic group of mycobacteria that is infecting the patient. Based on this diagnosis, a patient can be diagnosed long before positive identification of the causative species can be accomplished by other methods known prior to the present invention, and treatment can be immediately administered, with significantly improved opportunity to improve the health of the patient and clear the infection. The methods of the present invention are particularly useful for diagnosing infections that cause or are associated with a variety of diseases or conditions including, but not limited to, tuberculosis, skin infections, lymphandenitis, meningitis, Acquired Immune Deficiency Syndrome (AIDS), infection following transplant surgery, emphysema, cystic fibrosis, "hot tub lung", "life guard lung", granulomatous diseases (e.g., sarcoidosis and Crohn's disease), and Johne's disease (JD).

In addition to the methods of the present invention, additional testing can be performed to corroborate the results obtained by the present method, including, but not limited to, morphological analysis (highly recommended), acid-fast staining protocols, in vitro cultivation, polymerase chain reaction protocols, and sequencing of mycobacterial- specific genes. However, even used by itself, the method and probes of the present invention are powerful new tools that will greatly enhance the detection of microbes, and particularly mycobacteria, in a sample.

Various aspects of the present invention are described in the following experiments. These experimental results are for illustrative purposes only and are not intended to limit the scope of the present invention.

Examples The following Materials and Methods were used in Examples 1-4 below. CuϊtuFe^' preparations, cultures were acquired from Dr. Leonid Heifets at The National Jewish Medical and Research Center (Denver, CO) (M avium ssp. paratuberculosis, M. intracellular, M. kansasii, M. tuberculosis) and Dr. Mark Hernandez at the University of Colorado (Boulder, CO) (M avium ssp. avium, M. phlei, M. parafortuitum, B. subtilis, E. colt, Rhodococcus sp., Corynebacterium sp.). All cultures were fixed in 10% formalin for 4-10 hours, washed with phosphate buffered saline (PBS) (Sambrook, 1989) pH 8.0, and stored in 70% Ethanol/30%PBS at -2O⁰C. Fixed cultures were applied to silane prepared slides (Sigma- Aldrich). Slides were dipped in 100% xylene for 5 min and rehydrated in an ethanol series (100, 70, 30, 0% ethanol in 10 mM Tris pH 7.5). Cultures were then treated with lysozyme (1 mg/mL, Sigma- Aldrich) and achromopeptidase (30 units/mL, Sigma-Aldrich) in 10 mM Tris pH 7.5 at 37°C for 25 min. Cultures were washed for 5 min in 10 mM Tris pH 7.5 and then dehydrated in an ethanol series (30, 70, 100% ethanol in 10 mM Tris pH 7.5). Slides were allowed to air dry before hybridization.

Tissue preparation. Bovine mesenteric lymph nodes and ileal tissues, from diseased and normal dairy cows, were obtained from Dr. Randall Basaraba at Colorado State University (Fort Collins, CO). The presence of MAP in tissues from cows with Johne's disease was confirmed by Ziehl-Neelsen staining (Murray et al., 1994), IS900 quantitative PCR (Bull et al., 2003), and MAP specific rRNA quantitative PCR (De Groote, unpublished). MAP was not detected in negative control samples, obtained from healthy cattle, by either acid-fast stain microscopy or PCR. The National Jewish Medical and Research Center (Denver, CO) provided archived small bowel sections from a pediatric CD patient and adult lung tissues from patients infected with M avium and/or M. intracellular. Swedish Medical Center (Denver, CO) provided an archived hand biopsy specimen from a patient with tenosynovitis, secondary to M. avium. Denver Health Medical Center (Denver, CO) provided acid-fast positive sputum specimens from an elderly smoker with a pulmonary mycobacterial infection. All tissue samples were de-identified.

Tissues were fixed in 10% formalin for 24 hours, washed with PBS, and stored in 70% ethanol/30% PBS at -2O⁰C. Tissue was embedded in paraffin wax and 4 micron sections were placed on silane prepared slides (Sigma-Aldrich). Tissue was dewaxed in 100% xylene for 20 min and then rehydrated in an ethanol series (100, 70, 30, 0% ethanol in 10 mM Tris pH 7.5). Sections were then treated with lysozyme (1 mg/mL, Sigma-Aldrich) and achromapeptidase (30 units/mL, Sigma-Aldrich) in 10 mM Tris pH 7.5 at 37⁰C for 25 min. Tissue was washed for 5 min in 10 mM Tris pH 7.5 and dehydrated in an ethanol series "(30" 70,^''i00^'%^JetMnόrin 10 ^"mM Tris pH 7.5). AU sections were allowed to air dry before hybridization.

16S and 23S oligonucleotide probe hybridization. Probes used were: EUB338 (5' CTG CTG CCT CCC GTA GGA GT 3'; SEQ ID NO:1) (Amann, 1990; DeLong, 1989; Giovannoni, 1988), MAVP187ssu (5' TGC GTC TTG AGG TCC TAT CC 3'; SEQ ID NO:2), MAVP5151su (5' TGT CCA TGC ATG CGG TTT 3'; SEQ ID NO:3), MAC25431su (5¹ ACG CCA CTA CAC CCC AAA 3'; SEQ ID NO:4), MIN351ssu (5¹ AGG TAG AGC TGA GAT GTA TCC T 3'; SEQ ID NO:5) and MIN15861su (5' CCC CGA AAC TCC ATG CCC 3'; SEQ ID NO:6). All oligonucleotides were obtained from Integrated DNA Technologies, Inc. and labeled at the 5' and 3' ends with either 6' FAM or Cy3. All probes described above, except for EUB338, were designed using the probe design function of ARB (Ludwig et al., 2004) and databases of small and large subunit rRNA genes obtained from GenBank. Hybridization buffer consisted of 900 mM NaCl, 20 mM Tris pH 8.0, 0.01% SDS, and 20% formamide. Probes were added to the hybridization buffer at a final concentration of 1 ng/μL and the probe/hybridization buffer were applied to the specimens. Frame-Seal incubation chambers (MJ Research, Inc.) for slides were used to prevent evaporation. Hybridization slides were placed in an MJ Research, Inc. slide thermocycler and were heated at 94⁰C for 5 min. Slides were incubated at either 36°C (MIN3511su, MIN15861su, and MAC25431su) or 4O⁰C (MAVP187ssu, MAVP5151su, and EUB338) for 6-18 hours. Following hybridization, slides were washed with 225 mM NaCl, 5 mM EDTA, 0.01% SDS, and 20 mM Tris pH 8.0 for 20 min at either 37⁰C or 41⁰C. Slides were then dipped briefly in cold 20 mM Tris pH 8.0 in order to remove excess salt and finally mounted with Citifluor (Electron Microscopy Sciences) antifading reagent. Slides were visualized under epifluorescence with a Nikon Eclipse E600 Microscope or with a Leica Laser Scanning Confocal Microscope.

Anti-fluorescein alkaline phosphatase (AP) visualization. The hybridization of 6' FAM labeled oligonucleotide probes was carried out as described above. Hybridization was followed by incubation of slides in blocking buffer (3% Bovine Serum Albumin, 100 mM Tris pH 7.5, 150 mM NaCl, 0.3% Triton X-100) at 4⁰C for 30 min. Fresh blocking buffer that contained 2 mU/uL of anti-fluorescein- AP Fab fragments (Roche) was then added to the sections. Slides were incubated at 4⁰C for 2-3 hours. Slides were then washed for 15 min with buffer 1 (100 mM Tris pH 7.5, 150 mM NaCl, 0.3% Triton X-100) followed by another wash with buffer 2 (100 mM Tris pH 9.5, 150 mM NaCl, 50 mM MgCl₂) for 15 min. Slides were placed in AP buffer (100 mM Tris pH 9.5, 150 mM NaCl, 50 mM MgCl₂, 0.2 mg/ml " tevamis61^'"{Sϊgma-AI(fcchj" anaO.1% Tween) for 10 min. BCIP/INT (Roche) was then added to fresh AP buffer (75 μl INT7BCIP per 10 ml AP buffer), applied to the slides and then incubated in the dark at 25°C for 1-2 hours. The reaction was stopped by washing briefly in water. Sections were counterstained with methylene blue, mounted with Faramount (DakoCytomation), and observed under bright field microscopy.

IS900-biotin and IS900-fluorescein hybridization. The IS900-biotin and IS900- fluorescein labeled probes were prepared according to Sechi et al. (Sechi et al., 2001), with the exception that fluorescein- 12-dUTP (Roche) was incorporated into the IS900-fluorescein probe. Hybridization was performed according to Hulten et al. (Hulten et al., J. Microbiol. Methods, 2000) with the exception that biotin rather than digoxigenin was used to label the 241 base pair double stranded DNA probe. Tissue was deparaffinized in 100% xylene, rehydrated through a graded ethanol series (100, 70, 30% ethanol in PBS) and washed with PBS pH 7.4. Tissues were then incubated with proteinase K (Sigma- Aldrich) in PBS at concentrations ranging from 0 to 1 mg/ml for 20 min at 37⁰C. Proteinase K was inactivated with 0.2% glycine in PBS. Sections were then dehydrated through a graded ethanol series and allowed to air dry. Sections were hybridized with 1 ng/μl of labeled probe in hybridization buffer (50% deionized formamide, 2X SSC (Sambrook, 1989), 10% dextran sulfate, 0.25 μg/μl yeast tRNA (Sigma- Aldrich), 0.5 μg/μl denatured salmon sperm DNA (Sigma- Aldrich) and IX Denhardt's solution (Sigma- Aldrich)). Probe was boiled for 10 min and cooled on ice for 10 min before application. Sections with probe were heated for 10 min at 95⁰C and chilled on ice for 10 min. Hybridization was performed overnight at 37°C. Washing steps included 2X SSC for 15 min. at room temperature (RT), IX SSC for 15 min. at RT, 0.3X SSC for 15 min. at 40⁰C and 0.3X SSC for another 15 min. at RT. Washes were followed by incubation of slides in blocking buffer at room temperature for 30 min. Fresh blocking buffer that contained 2 mU/μL of anti-biotin-AP Fab fragments (Roche) was added to the slides and slides were incubated at room temperature for 2-3 hours. Slides were then washed for 15 min. with buffer 1 followed by another wash with buffer 2 for 15 min. Slides were then placed in AP buffer for 10 min. BCIP/INT (75 μl per 10ml AP buffer) was added to fresh AP buffer and applied to the slides that were incubated in the dark at room temperature for 4 to 12 hours. The reaction was stopped by washing in distilled water. Slides were mounted with Faramount (DakoCytomation) and observed under bright field microscopy. IS900-fluorescein hybridizations were performed as described for IS900 biotin except that epifluorescence microscopy was used for direct visualization of specimens. Dot" blot hybridization ^"was used to confirm that the IS900 biotin and IS900 fluorescein probes were synthesized correctly. Briefly, MAP genomic DNA, E. coli genomic DNA, IS900 PCR product, and E. coli 16S PCR product were blotted to nitrocellulose (Hybond+®, Amersham Biosciences) according to manufacturer procedures. The dot blot was hybridized with either probe according to the procedures above. The blots were visualized with either anti-biotin AP Fab fragments (Roche) or anti-fluorescein AP Fab fragments (Roche). The AP and INT/BCIP colorimetric reaction was incubated for 20 min at RT. The probes hybridized only to MAP genomic DNA and IS900 PCR product (data not shown). Example 1

The following example describes the development of ISH protocols and characterization of MAC rRNA ISH probes.

A variety of potential MAC-specific ISH oligonucleotide probe sequences were chosen using the "Probe Design" function of the ARB software package (Ludwig et al., 2004) and an alignment of small- and large-subunit rRNA sequences obtained from GenBank. MAP and MAA rRNA sequences are identical, and so it was impossible to design probes that would differentiate between the two sub-species. As a first test of specificity, fluorescently labeled probes were applied to pure cultures of cognate and non-cognate species, including M. avium spp. paratuberculosis, M. avium spp. avium, M. intracellulare, M. tuberculosis, M. kansasii, M. parafortuitum, M. phlei, Escherichia coli, Bacillus subtilis, Rhodococcus sp., and Corynebacterium sp. A broad-range bacterial 16S probe, EUB338-6¹ FAM, was included in every hybridization experiment in order to test the effectiveness of permeabilization and hybridization protocols.

In initial experiments, none of the ISH probes, including EUB338, successfully hybridized to any of the mycobacterial cells. The complex, lipid-rich cell walls of mycobacteria are notoriously refractory to the lysis steps of standard ISH protocols (DeLong et al., 1989). Consequently the inventors tested a number of techniques to develop a protocol that would enhance the permeabilization of mycobacterial cells. Enzymatic digestion of fixed cells with proteinase K (Sechi et al., 2004; Sechi et al., 2001) failed to improve the fluorescent signal upon hybridization with EUB338 or the MAC-specific probes. Enzymatic digestion with lysozyme and achromopeptidase (Sekar et al., 2003) resulted in a fluorescent signal in only a small fraction of fixed cells from culture. The inventors found, however, that a brief incubation of fixed mycobacterial cells in 100% xylene, combined with subsequent lysozyme and achromopeptidase digestions, resulted in strong fluorescent hybridization 'signals. '"^'Tfeaϊnϊeht with, all three reagents (xylene, lysozyme, and achromopeptidase) was determined to be required for successful ISH of mycobacterial cells. Although each oligo probe was individually tested (Table 1), the inventors found that the combination of the species-specific probes for ISH resulted in a stronger fluorescent signal, even though the final probe concentration remained the same (data not shown). Table 1. Specificity of MAC ISH probes

MAP: Mycobacterium avium spp. paratuberculosis MAA: Mycobacterium avium spp. avium MIN: Mycobacterium intercellulare MTB: Mycobacterium tuberculosis MKA: Mycobacterium kansasii

Using this enhanced ISH protocol, the Cy3 conjugated probes MAVP187ssu and

MAVP5151su (Table 1) hybridized well with both MAA and MAP cultures (Figs. IA and IB., respectively) but not to M. intrαcellulαre (Fig. ID), M. tuberculosis (Fig. IF) or other related and unrelated bacterial species (listed above; data not shown). In Figs. 1A-1R, the scale bars equal 1 micron, and the original magnification is 100OX.

Hybridization with probe EUB338-6' FAM clearly labeled the M. intrαcellulαre and M. tuberculosis cells (Figs. 1C and IE, respectively), indicating that cell-wall permeabilization was effective. As expected, probe MAC25431su hybridized to all three members of the MAC complex: M. intrαcellulαre, M. avium spp. avium and M. avium spp. _f paratuberculosis, but failed to hybridize to the other species tested (Figs. IG-L and Table 1). Finally, probes MIN3511su and MIN15861su specifically hybridized to M. intracellular cells, but not to other species tested, including other members of MAC, M. avium spp. avium and M. avium spp. paratuberculosis (Figs. IM-R and Table 1). Example 2

The following example describes the application of MAC ISH probes to tissue samples.

Well-characterized bovine Johne's disease and healthy tissues were used in order to establish the ability of the MAC-specific probes to stain MAC cells embedded within tissues. The^" presence or absence of MAP in infected and uninfected mesenteric lymph nodes (MLN) was first determined by Ziehl-Neelsen staining and quantitative PCR (data not shown) (Bull et al., 2003; De Groote et al, unpublished data). Both negative and positive control MLN samples were then probed with a combination of Cy3 labeled MAVP187ssu and MAVP5151su fluorescent oligonucleotides. The negative control MLN showed no sign of fluorescent bacilli when probed with MAVP187ssu and MAVP5151su oligos (Fig. 2A). In contrast, hybridization of the positive control MLN resulted in an intense and abundant fluorescent signal (Fig. 2B) in an aggregate pattern similar to the acid fast staining pattern (Fig. 4B). The MIN351ssu and MIN15861su probes did not hybridize to either MLN specimens (data not shown). The MAC25431su probe labeled the MAP positive MLN specimen but not the MAP negative MLN specimen (data not shown). Because the ISH protocol requires extensive digestion of the mycobacterial cell wall for probe accessibility, the inventors did not find conditions that would allow dual staining with Ziehl-Neelsen and rRNA probes. Fig. 2C demonstrates the ability of confocal laser scanning microscopy to detect clearly defined bacilli hybridized with the MAVP187ssu- and MAVP5151su-Cy3 probes in MAP infected bovine MLN tissue. The nuclei of lymphatic cells are stained with DAPI and appear blue (dark stain in black and white). The confocal microscope was able to resolve bacilli in different focal planes within the tissue, indicating that the probe had penetrated the entire tissue section. As expected for M. avium spp. paratuberculosis, the labeled cells are rods approximately one micron in length. In general, tissue autofluorescence did not greatly impact the labeling and detection of MAP cells in most bovine or human tissues. However, high levels of autofluorescence were observed when erythrocytes, collagen, and/or elastic fibers were abundant, such as in lung samples. The inventors consequently developed a non- fluorescent approach to probe detection that uses alkaline-phosphatase (AP), conjugated to anti-fluorescein antibodies, to enzymatically amplify a colorimetric dye (INT/BCIP; Materials and Methods). In addition to avoiding background autofluorescence, this approach also allows the use of tissue staining and light microscopy to visualize hybridized microbes and co-localize them with mammalian tissue pathology. A representative micrograph of a Johne's ileum specimen, probed with the MAVP oligos, is shown in Fig. 2D. Arrows indicate the MAP bacilli. Example 3

The following example illustrates the use of MAC ISH probes detect MAC species in human clinical specimens. fϊ-e^{^}MAC-specϊfic ISH ^"probes were applied to a variety of archival tissues from human patients with MAC infections in order to determine the applicability of these probes for identification and diagnosis of mycobacterial infections. As shown in FIGS. 3A, 3B, 3C, and 3D, tissues were probed with the MAA/MAP-specific oligos MAVP187ssu and MAVP5151su and visualized by either fluorescence microscopy (Fig. 3B and 3C) or light microscopy following treatment with anti-fluorescein coupled AP (Fig. 3A and 3D). Fig. 3A is a tissue section from a lung resection of a patient infected with M. avium spp. avium, as determined by culture and Accuprobe (GenProbe). The MAVP probes were able to detect many bacilli (Fig. 3A, arrows) throughout the tissue. The MIN probes did not hybridize to the same resected lung tissue (data not shown). Tissue from a patient with tenosynovitis of the hand was negative by acid-fast staining, but M. avium spp. avium positive by culture after several weeks of growth; Accuprobe (GenProbe) assay confirmed the presence of M. avium spp. avium. The MAVP probes were clearly able to detect isolated bacilli (Figure 3B) of the appropriate size and morphology. Figs. 3C and 3D are tissue sections from the duodenum and small bowel, respectively, of a pediatric CD patient. In these specimens, the MAVP probes hybridized to bacilli located predominantly within cells of the lamina propia.

Fig. 3E is a representative micrograph of lung tissue containing large granulomas that was probed with the M. infracellulare-specific oligos MIN3511su and MIN15861su. Hybridization signal was visualized with the anti-fluorescein AP colorimetric assay. The lung tissue was acid-fast positive, with one to two bacilli located in granulomas per tissue section; M. intracellular was cultured from a section of this tissue and confirmed with Accuprobe (GenProbe). The MIN-6' FAM probes were able to detect more M. intracellular bacilli (Fig. 3E, arrows) in the granulomas than were detected by acid-fast staining. In contrast, the MAVP probes did not hybridize to this lung section (data not shown). The in situ hybridization methodology also works well with sputum samples. Fig. 3 F shows a micrograph of a sample of acid-fast positive sputum from a patient with a history of smoking, chronic cough and an upper zone cavity with a diagnosis of pulmonary MAC two years prior. Accuprobe identification was inconclusive and broad-range 16S rRNA sequence data detected Corynebacterium sp. and small numbers of M. intracellular (data not shown). In situ hybridization with the MAC-specific probe MAC2543-6' FAM revealed sparse, labeled bacilli among a background of DAPI labeled bacteria of corynebacterial morphology. Example 4

The following example shows a comparison of rRNA ISH and IS900 ISH methodologies. Some recently published studies have used double stranded DNA probes that target genomic DNA for localization of mycobacteria in situ (Hulten et al., 2001; Sechi et al., 2001). Because ribosomes, the targets of the ISH probes described in this study, are present in much greater cellular abundance than are genomic DNA loci, a direct comparison of the DNA- and rRNA-based approaches was in order. To this end, MLN tissues from healthy and Johne's diseased cows were used as negative and positive controls for ISH. Ziehl-Neelsen staining determined the absence or presence of acid-fast bacilli in each tissue sample (Figs. 4A and 4B, respectively). MAP infection of the positive control tissue was also demonstrated by IS900 and 16S PCR (data not shown). In Figs. 4A-4H, arrows indicate signal positive bacilli in (A), (B), (C), and (D) and amorphous nonspecific signal in (E), (F), (G), and (H). For technical reasons, it was necessary to probe different tissue sections of the same sample block separately in each panel.

As expected, the MAC rRNA probe MAVP187ssu-6' FAM stained only the positive control tissue, as indicated by precipitate (the INT/BCIP AP product) with the morphology of clearly defined bacilli and a size and morphology consistent with MAP (Fig 4D). No signal was evident in the negative control tissue (Fig. 4C). In contrast, application of IS900 probes to bovine tissue sections resulted in the appearance of a variety of rod-like and amorphous forms in both the negative (Fig. 4E) and the positive (Fig. 4F) control MLNs. Under high- power oil immersion magnification, however, these objects had a granular, amorphous appearance not necessarily suggestive of mycobacteria, compared to the clear morphological boundaries evident in cells stained with either Ziehl-Neelsen or rRNA probes (Figs. 4B and 4D), structures that resemble microorganisms.

To determine whether the false positive results that were obtained with the IS900 probe were due to non-specific precipitation or aggregation of the colorimetric dye, the inventors synthesized a fluorescein labeled IS900 probe (IS900-fluorescein) that allowed direct epifluorescent detection of cells. In situ hybridization of positive and negative control bovine MLNs with this IS900-fluorescein probe also resulted in significant non-specific staining in both control tissues (Figs. 4G and 4H). Indeed, application of the IS900 ISH published protocol to organisms other than MAP that were grown in pure culture resulted in strong non-specific signals. Fig. 5 A, for example, shows B. subtilis that was dual probed, via the IS900 ISH procedure, with IS900-FITC (lighter, rounder structures) and EUB338-Cy3 (rod-like, darker structures). Although the rRNA probe is uniformly distributed within the cells, consistent with the expected distribution of ribosomes, the IS900-FITC probe results in a punctate staining pattern that is localized to the outside of the cells. Similarly, Figs. 5B and 5C show ϊaϊse-positive hybridization of the IS900 biotin probe to Rhodococcus sp., and M. kansasii. Of the bacteria tested, only Gram-negative microbes, such as E. coli and P. aeruginosa, were resistant to the non-specific hybridization of IS900 biotin probe (data not shown). Fig. 5D is an example of M. avium ssp. avium probed with EUB338-6' FAM and visualized enzymatically with anti-fluorescein AP Fab fragments and INT7BCIP. The signal is distributed evenly throughout the cell with expected morphology. Thus, both the present inventors' permeabilization protocol and the use of small oligonucleotide rRNA probes of the present invention, rather than large DNA probes such as IS900, are necessary for successful ISH of mycobacteria. Example 5

The following example demonstrates the development of oligonucleotide probes specific for rRNA from Mycobacteria tuberculosis (MTB ISH probes) and the use of MTB ISH probes detect MTB species in tissue specimens. Materials and Methods Cultures were acquired from Dr. Leonid Heifets (National Jewish Medical and

Research Center, Denver, CO) and Dr. Mark Hernandez (University of Colorado, Boulder, CO) and were fixed in 10% formalin for 4-10 hours. Denver Health Medical Center (Denver, CO) provided acid fast positive sputum from a de-identified patient with a history of tuberculosis. M. avium ssp. paratuberculosis (MAP) infected bovine mesenteric lymph node tissue and lung tissue sections from guinea pigs and mice infected with Mtb H37Rv were obtained from Dr. Randall Basaraba (Colorado State University, Fort Collins, CO). Tissues were fixed and processed as previously described (Taylor, 2003; Turner, 2003). Tissue and cultures were pretreated with xylene, lysozyme and achromapeptidase according to St. Amand et al. (Examples 1-4 and St. Amand, 2005). Probes used were: EUB338 (DeLong, 1989), MTB770 (5¹-

CACTATTCACACGCGCGT-3'), MTB226 (5'-CCACACCGCTAAAG-3^l)_J MTB 187 (5'- TGCATCCCGTGGTCCTATCC-S') and MAVP187 (Examples 1-4). Oligonucleotides were obtained from Integrated DNA Technologies, Inc. and labeled at the 5' and 3' ends with either 6'-carboxyfluorescein (FAM), Cy3, or Cy5. All ISH protocols were performed according to Examples 1-4 (see also St. Amand., 2005) with the following exceptions: Specimens were incubated in a 20% formamide hybridization buffer at either 38°C (MTB770, MTB226, and MTB187) or 4O⁰C (MAVP187 and EUB338) for 6-12 hours. Following hybridization, slides were washed with the appropriate wash buffer for 20 minutes at 39⁰C or 41⁰C. Colorimetric detection ^"of fluorescein probes using anti-fluorescein-AP antibodies and INT/BCIP was carried out as previously described (Examples 1-4 and St. Amand, 2005).

Results

MTB-specific ISH oligonucleotide probe sequences were chosen using the "Probe Design" function of the ARB software package (Ludwig, 2004) and an alignment of rRNA sequences obtained from GenBank. As a first test of specificity, fluorescent probes were applied to pure cultures, including: Mtb H37Rv, M. avium, M. intracellulare, M. kansasii, M. absecessus, M. parafortuitum, M. phlei, Escherichia coli, Bacillus subtilis, Rhodococcus sp., and Corynebacterium sp. The bacterial 16S probe EUB338-FAM was included in each ISH experiment to monitor the effectiveness of permeabilization and hybridization protocols.

Cy3 conjugated probes MTB770, MTB226, and MTB 187 (MTB probes) hybridized with Mtb H37Rv culture (Fig. 6E) but not to M. intracellulare (Fig. 6F), M. avium (Fig. 6G), M. kansasii (Fig. 6H) or other bacterial species (Table 1). Hybridization with probe EUB338-FAM clearly labeled all the cultures tested (Figs. 6A, 6B, 6C, and 6D; Table 1) indicating that MTB probe negative cells were sufficiently permeabilized.

The MTB probes were also tested against a mixed culture containing Mtb, M. avium, M. intracellulare, and Corynebacterium sp. (Fig. 61). Probes EUB338-Cy5 and MAVP187- Cy3 (M avium subspecies specific) were also included in the hybridization experiments. EUB338-Cy5 hybridized to all bacteria in the culture showing cell permeabilization. MTB- FAM hybridizes to long Mtb rods while the MAVP187-Cy3 hybridizes to short M. avium rods. The inventors tested the MTB-probes on a clinical sputum sample that was AFB positive but culture negative. Figs. 6J and 6N shows the ability of the MTB probes to detect bacilli (arrow) in a sputum sample. The sputum's MTB-ISH positive result was confirmed by IS6110 PCR with extracted genomic DNA from the sputum (data not shown). To assess MTB ISH in tissue samples, the inventors applied the probes to well- characterized Mtb H37Rv infected guinea pig and mouse lungs. MAP infected bovine lymph node was used to establish the specificities of the MTB probes when hybridized to tissue sections. Guinea pig and mouse tissue sections were probed with a combination of MTB- FAM probes and EUB338-Cy3. The bovine lymph node was probed with both the MTB- FAM and MAVPl 87-Cy3.

The presence of mycobacteria in tissue was initially demonstrated by AFB staining (Murray, 1994). Figs. 6K, 6L and 6M are the results of AFB staining on mycobacteria infected guinea pig lung, mouse lung, and bovine lymph node, respectively. Fig. 60 is a representative micrograph of positive hybridization with the MTB-FAM probes to the Mtb feacillϊ lό&tiMήih^'e granulomas of guinea pig lung tissue. Although tissue autofluorescence did not generally impact the detection of mycobacteria, occasionally it was necessary to visualize the MTB-FAM probes with a non-fluorescent approach. This method utilizes anti- flourescein-AP conjugated antibodies that bind to the FAM moieties on the probe. The colorimetric dye INT/BCIP was used as a substrate for the alkaline phosphatase. A representative micrograph of a mouse lung probed with the MTB-FAM probes and visualized with INT/BCIP is shown in Fig. 6P (note staining bacilli). Additionally, the MTB probes did not hybridize with the MAP present in the bovine lymph node tissue while the MAVP 187- Cy3 did hybridize to the MAP (Fig. 6Q), as expected. Using the present inventors' methodology, the ISH probes specifically labeled the Mtb organisms in culture, sputum and tissue. ISH probes offer researchers and clinicians a fast and accurate method for identifying members of the MTB-complex in tissue and culture. Example 6

The following example provides additional examples of mycobacterial probes applied to human clinical samples.

Tissue from a patient with bronchiectasis and lung destruction requiring a lobectomy was probed with the M. avium probe, MAC2543 (SEQ ID NO:4). After standard FISH probe assay, the tissue was processed with anti-FITC antibody conjugated to alkaline phosphatase and developed enzymatically as previously described (see Examples 1-4 and also St. Amand et al., 2005). An image was obtained using light microscopy, which is shown in Figs. 7 A and 7B (Arrow denotes a -1.5 μm long rod embedded in the cilia of a large airway (Fig. 7A) and numerous organisms from the center of a granuloma (Fig. 7B)).

These results demonstrate the presence organisms in the epithelial layer of an airway and from the center of a granuloma. In contrast, the clinical pathology lab detected only a rare acid-fast bacillus (2-3 per entire tissue) using standard AFB stain, while M. avium was grown from the tissue. The finding of numerous probe-positive organisms is in keeping with data that mycobacteria may have a common non-acid fast form (Wayne, 1994). This example highlights the high sensitivity of the probes of the present invention. Example 7 The following example provides additional examples of mycobacterial probes applied to human clinical samples.

The inventors have examined several M. abscessus and M. avium infected cystic fibrosis patient samples with progressive, unexplained declining lung function (in collaboration with Dr. Frank Accurso at our Children's Hospital) and found probe positive resuM^'s when "smears have been negative. Fig. 8A shows a clump of M. abscessus probe (Mabsc-1 (SEQ ID NO:10)and Mabsc-2 (SEQ ID NO: H)) positive rods in expectorated sputum from a chronically infected patient with M. abscessus. Fig. 8B demonstrates no rods in a non-infected control CF specimen (patient has no known mycobacterial disease). Fig. 8C demonstrates acid fast (Carbol Fuchsin method) done with attention to differentiation, where the inventors were able to detect only one acid-fast rod in >300 high-powered fields examined (100OX oil). The clinical pathology lab failed to detect any acid-fast bacilli using standard AFB tissue stain.

Fig. 8D shows the result of using an CY3 M. abscessus probe (Mabsc-1 (SEQ ID NO: 10) and Mabsc-2 (SEQ ID NO: 11)) in a patient with known M. avium pulmonary disease, and Fig. 8E shows the CY5 M. avium probe (MAVP 187 SEQ ID NO:9) from a known M. avium cystic fibrosis patient. This patient also had a negative AFB stain.

Optimum probe performance of M. abscessus dictates that these should be mixed together in equal amounts, as they work better in a mixture than separately. There is no discrimination between M. abscessus and M. chelonae.

Table 2 lists many of the probes described in the present invention.

Table 2 ISH Probes *

Example^'s

The following example describes the development of Locked Nucleic Acid (LNA) oligonucleotide probes.

A Locked Nucleic Acid (LNA) is a novel type of nucleic acid modification that contains a 2'-O, 4'-C methylene bridge. This bridge restricts the flexibility of the ribofuranose ring and locks the sugar into the N-type conformation of the A-form DNA. LNA monomers also induce the adjacent DNA or RNA nucleotides to adopt the N type conformation. Addition of LNA monomers to specific oligonucleotides leads to increases in specificity and thermal stability (+4⁰C to +8⁰C increase in melting temperature) when hybridized to the DNA or RNA target. Specificity of oligonucleotide promotes can be increased by placing LNA nucleotides at positions that discriminate target sequences from non-target sequences. This enhanced specificity and duplex stability of LNA containing oligonucleotides makes them ideal for a myriad of molecular applications particularly, in situ hybridization (ISH). The present inventors experimented with various lengths and amounts of LNA monomer in the BAC338 oligonucleotide probe (SEQ ID NO:1), a commonly used ISH probe that targets all bacterial small subunit rRNAs. Figs. 9A-9C is a comparison of the ability of the different probes to hybridize to their targets. The hybridizations were performed in parallel using the methodology described in Examples 1-4 and in St. Amand et al., J. Clin. Microbiol, 2005, 43(4):1505-1514, with the following changes. Briefly, fixed E. coli culture was placed on a slide and subsequently dehydrated in ethanol. Three variations of the BAC338 probe were applied to different E. coli cultures. Hybridization was done at 43°C and in a buffer that contained 50% formamide and 900 mM NaCl for 2 hours. Slides were washed at 43⁰C with wash buffer containing 28 mM NaCl and 5 mM EDTA. Samples were then mounted with Citiflour anti-fading reagent. Pictures were taken using a Nikon E600 microscope using a Cy3 cube and at 400X. Each picture was taken at a 60 milliseconds exposure for direct comparison.

The shortest version of the BAC338 probe with the most LNA monomers, BAC338LNA1 Cy3 (SEQ ID NO:13), produced the brightest signal. The next best signal was the BAC338LNA2 Cy3 probe (SEQ ID NO: 14), while the original BAC338, the longest probe with no LNA monomers (SEQ ID NO:1), gave the weakest signal. These results thus indicate that incorporation of LNA bases into the oligonucleotide probes enhanced hybridization efficiency. Table 3 lists the LNA probes that have been developed by the present inventors and are undergoing testing, which include LNA versions of the olϊgbnucϊeotMes described in Examples 1-7 above. The probes are tested for efficacy in detecting tuberculous and non-tuberculous mycobacteria in human clinical specimens, including infected tissue samples.

Table 3. LNA Probes

Probe Sequence* SEQ ID NO Tm

BAC338LNAΪ CtGcCtCcCgTa SEQ ID NO:13 79^"

BAC338LNA2 CTgcctCcCgTagga SEQ ID NO:14 81

BAC338LNA3 tcCcGtAggAgt SEQ ID NO:15 72

MAVP187LNA GtcTTgAggtcctatcc SEQ ID NO:16 78

MTB187LNA AtcCCgTggtcctatcc SEQ ID NO:17 78

MTB226LNA CcAcaccgctaaaGCgc SEQ ID NO:18 81

MTB770LNA tattcAcAcgcCcGT SEQ ID NO:19 77

* Upper case letter represent LNA monomers

References

Amann et al. 1990. J. Bacterid. 172:762-70.

Ashford et al. 2001. Rev Sci Tech 20:325-37 Behr et al. 2004. Lancet Infect. Dis. 4:136-7.

Benson. 1994. Clin. Infect. Dis. 18 Suppl 3:S218-22. Bull et al. 2000. Microbiology 146 ( Pt 9):2185-97. Bull et al. 2003. J. Clin. Microbiol. 41:2915-23. Chacon et al. 2004. Annual Review Microbiology 58:329-363. Chandrasekhar et al. 1992. Tuber Lung Dis 73 :273-9

Chao et al. 2002. Otolaryngol Head Neck Surg 126: 176-9 Chiodini. 1989. Clin. Microbiol. Rev 2:90-117. Chiodini et al. 1993. J. Vet. Diagn. Invest. 5:629-31. Chiodini et al. 1986. J. Clin. Microbiol. 24:357-63. Collins et al. 2000. J. Clin. Microbiol. 38:4373-81. Cousins et al. 1999. MoI. Cell Probes 13:431-42.

De Groote, M. A. 2004. Pulmonary infection in non-HIV infected individuals. J. Bartram and A. Dufour (ed.), M. avium in water. U.S. EPAAVHO publication, London and New York. De Groote, M. A. 2004. Skin, bone, and soft tissue infections. In J. Bartram and A. Dufour

(ed.), M. avium in water. U.S. EPA/WHO publication, London and New York. De Groote et al. In preparation. Identification, characterization, and quantitation of

Mycobacterium avium subspecies paratuberculosis in bovine tissues of Johne's disease and healthy animals. D^"e Groo't^'e ^"et aϊ! ^'2003. Environmental Mycobacterial Infections. In J. G. Bartlett and N. R. Blacklow (ed.), Gorbach Textbook of Infectious Diseases. Sherwood Gorbach.

DeLong et al. 1989. Science 243:1360-1363.

Drake et al. 2002. Emerg. Infect. Dis. 8:1334-41. Drobniewski et al. 2000. J. Clin. Microbiol. 38:444-7.

Eccles et al. 1995. J. Assoc. Nurses AIDS Care 6:37-47; quiz 48-9. el-Zaatari et al. 1996. J. Clin. Microbiol. 34:2240-5.

El-Zaatari et al. 2001. Trends MoI. Med. 7:247-52.

Englund, S. 2003. Vet. Microbiol. 96:277-87. Falkinham, J. O., 3rd. 1996. Clin. Microbiol. Rev. 9: 177-215.

Falkinham, J. O., 3rd. 2003. Emerg. Infect. Dis. 9:763-7.

Fitzpatrick et al. 1996. J. La. State Med. Soc. 148:451-4.

Giovannoni et al. 1988. J. Bacterid. 170:720-6.

Greenstein, R. J. 2003. Lancet Infect. Dis. 3:507-14. Greenstein et al. 2004. Lancet 364:396-397.

Hance, A. J. 1998. Semin. Respir. Infect .13:197-205.

Hermon-Taylor et al. 1998. Bmj 316:449-53.

Hermon-Taylor et al. 2002. J. Med. Microbiol. 51:3-6.

Horsburgh, C. R., Jr. 1999. J. Infect. Dis. 179 Suppl 3:S461-5. Hulten et al. 2001. Am. J. Gastroenterol. 96:1529-35.

Hulten et al. 2000. Vet Microbiol. 77:513-8.

Hulten et al. 2000. J. Microbiol. Methods 42:185-95.

Iivanainen et al. 1999. Apmis. 107:193-200.

Kanazawa et al. 1999. J. Gastroenterol. 34:200-6. Kehinde et al. 2005. J Natl Med Assoc 97:394-6

Kilby et al. 1992. Chest 102:70-5.

Kon et al. 1997. Thorax 52 Suppl 3:S47-51.

Lambert, P. A. 2002. Symp. Ser. Soc. Appl. Microbiol. :46S-54S.

Lane et al. 1985. Proc. Natl. Acad. Sci. U S A 82:6955-9. Ludwig et al. 2004. Nucleic Acids Res. 32: 1363-71.

Mangiapan et al. 1995. Sarcoidosis 12:20-37.

McFadden et al. 1996. Soc. Appl. Bacteriol. Symp. Ser. 25:47S-52S.

Millar et al. 1996. Appl. Environ. Microbiol. 62:3446-52.

Mirmirani et al. 1999. J. Am. Acad. Dermatol. 41:285-6. Moss et al.1991. (iutl^'ϊ:395-8^".^"

Murray et al. 1994. Determinative and Cytological Light Microscopy, p. 32-33. In P. Gerhardt, R. G. E. Murray, W. A. Wood, and N. R. Krieg (ed.), General and Molecular Bacteriology. American Society for Microbiology, Washington, DC. Naser et al. 2002. Mo.l Cell Probes 16:41-8.

Oliver et al. 2001. Clin. Infect .Dis. 32:1298-303.

Primm et al. 3rd. 2004. Clin. Microbiol. Rev. 17:98-106.

Richter et al. 2002. Germany. Emerg. Infect. Dis. 8:729-31.

Roholl et al. 2002. J. Clin. Microbiol. 40:3112; author reply 3112-3. Rutherford et al. 2004. Chest 125:354.

Ryan et al. 2002. Gut 51:665-70.

Sambrook et al. 1989. Molecular Cloning, 2nd ed, vol. 3. Cold Springs Harbor Laboratory Press, Cold Springs Harbor, NY.

Sechi et al. 2001. J. Clin. Microbiol. 39:4514-7. Sechi et al. 2004. New Microbiol. 27:75-7.

Sekar et al. 2003. Microbiol 69:2928-35.

St. Amand et al. 2005. Journal of Clinical Microbiology 43:1505-1514

Taylor et al. 2003. Infect Immun 71:2192-8

Tortoli et al. 1996. J. Clin. Microbiol. 34:2838-40. Turner et al. 2003. Infect Immun 71 :864-71

U.S. Provisional Patent Application No. 60/606,766, filed September 2, 2004.

Van Kruiningen, H. J. 1999. Inflamm. Bowel. Dis. 5:183-91.

Vansnick et al. 2004. Vet Microbiol. 100:197-204.

Wall et al. 1993. J. Clin. Microbiol. 31:1241-5. Wang et al. 2001. [Observations of properties of the L-form of M. tuberculosis induced by the antituberculosis drugs]. Zhonghua Jie He He Hu Xi Za Zhi 24:52-5

Watterson et al. 2000. J. Clin. Pathol. 53:727-32.

Woods et al. 2001. J. Clin. Microbiol. 39:747-9.

Each of the references cited herein is incorporated herein by reference in its entirety.

While various embodiments of the present invention have been described in detail, it is apparent that modifications and adaptations of those embodiments will occur to those slcilϊeci in tne"^"arϊT It Ts to be expressly understood, however, that such modifications and adaptations are within the scope of the present invention, as set forth in the following claims.

Claims

What is claimed is:

1. An oligonucleotide probe consisting of a nucleic acid sequence that hybridizes under stringent conditions to the complement of an oligonucleotide sequence selected from the group consisting of any one of SEQ ID NOs:2-19; wherein the oligonucleotide probe also hybridizes under stringent conditions to rRNA hi a mycobacteria.

2. The oligonucleotide probe of Claim 1, wherein at least one nucleotide in the oligonucleotide probe has been modified by a Locked Nucleic Acid (LNA) modification. 3. The oligonucleotide probe of Claim 2, wherein the oligonucleotide probe comprises a nucleic acid sequence selected from the group consisting of any one of SEQ ID NOs:13-19.

4. The oligonucleotide probe of Claim 1 or Claim 2, wherein the oligonucleotide probe is between about 8 nucleotides and about 50 nucleotides in length.

5. The oligonucleotide probe of Claim 1 or Claim 2, wherein the oligonucleotide probe is between about 8 nucleotides and about 25 nucleotides hi length.

6. The oligonucleotide probe of Claim 1 or Claim 2, wherein the oligonucleotide consists of a nucleic acid sequence is at least 90% identical to any one of SEQ ID NOs:2-19.

7. The oligonucleotide probe of Claim 1 or Claim 2, wherein the oligonucleotide consists of a nucleic acid sequence is at least 95% identical to any one of SEQ ID NOs:2-19. 8. The oligonucleotide probe of Claim 1, wherein the oligonucleotide probe hybridizes under stringent conditions to rRNA from the Mycobacterium avium complex.

9. The oligonucleotide probe of Claim 8, wherein the oligonucleotide probe hybridizes under stringent conditions to rRNA from a mycobacterium selected from the group consisting of: Mycobacterium avium ssp. avium, Mycobacterium avium ssp. paratuberculosis, Mycobacterium avium ssp. silvaticum, Mycobacterium avium ssp. hominis, and Mycobacterium intracellular.

10. The oligonucleotide probe of Claim 8, wherein the oligonucleotide probe comprises a nucleic acid sequence of SEQ ID NO:4. IT The oligonucleotide probe of Claim I, wherein the oligonucleotide probe hybridizes under stringent conditions to rRNA from Mycobacterium intracellulare.

12. The oligonucleotide probe of Claim 11, wherein the oligonucleotide probe comprises a nucleic acid sequence selected from the group consisting of SEQ

ID NO:5 and SEQ ID NO:6.

13. The oligonucleotide probe of Claim 1, wherein the oligonucleotide probe hybridizes under stringent conditions to rRNA from Mycobacterium avium ssp. avium and Mycobacterium avium ssp. paratuberculosis. 14. The oligonucleotide probe of Claim 13, wherein the oligonucleotide probe comprises a nucleic acid sequence selected from the group consisting of SEQ ID NO:2, SEQ ID NO:3, and SEQ ID NO: 16.

15. The oligonucleotide probe of Claim 1, wherein the oligonucleotide probe hybridizes under stringent conditions to rRNA from Mycobacterium tuberculosis.

16. The oligonucleotide probe of Claim 15, wherein the oligonucleotide probe comprises a nucleic acid sequence selected from the group consisting of SEQ ID NO: 7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO: 17, SEQ ID NO: 18 and SEQ ID NO:19. 17. The oligonucleotide probe of Claim 1, wherein the oligonucleotide probe hybridizes under stringent conditions to rRNA from Mycobacterium abscessus. 18. The oligonucleotide probe of Claim 17, wherein the oligonucleotide probe comprises a nucleic acid sequence selected from the group consisting of SEQ

ID NO: 10 and SEQ ID NO: 11. 19. The oligonucleotide probe of Claim 1, wherein the oligonucleotide probe hybridizes under stringent conditions to rRNA from Mycobacterium kansasii.

20. The oligonucleotide probe of Claim 19, wherein the oligonucleotide probe comprises the nucleic acid sequence of SEQ ID NO: 12.

21. The oligonucleotide probe of any one of Claims 1-20, wherein the oligonucleotide probe is operatively linked to a detectable label.

22. The oligonucleotide probe of Claim 21, wherein the detectable label is selected from the group consisting of: a fluorescent label, a colorimetric label, biotin, an enzyme and a radiolabel. 2ST^"" ^{' ""'}A panel of oligonucleotide probes, comprising at least two oligonucleotide probes according to any one of Claims 1-22.

24. The panel of Claim 23, wherein the probes are immobilized on a substrate in an array. 25. An assay kit for the detection of mycobacteria in a sample, comprising at least one oligonucleotide probe according to any one of Claims 1-22.

26. A primer for polymerase chain reaction comprising the oligonucleotide probe of any one of Claims 1-20.

27. A method to detect mycobacteria in a sample, comprising: a) contacting a fixed sample suspected of containing mycobacterial cells with Xylene; b) rehydrating the sample; c) enzymatically digesting the sample with lysozyme and achromapeptidase; d) washing and drying the sample; e) contacting the permeabilized sample with at least one oligonucleotide probe according to Claim 21 in a hybridization buffer; f) heating the sample in the buffer at a temperature sufficient to dissociate polynucleotide strands; g) cooling the sample in the buffer at a temperature sufficient to allow polynucleotide strands to hybridize; h) washing the sample; and i) visualizing the sample to detect the presence or absence of mycobacteria in the sample. 28. The method of Claim 27, wherein the sample is selected from the group consisting of: a microbial cell sample, a tissue sample, a sputum sample, and an environmental sample.

29. The method of Claim 27, wherein the method is used to diagnose a mycobacterial disease in a subject. 30. The method of Claim 27, wherein step (e) comprises contacting the sample with two or more different oligonucleotide probes.

31. The method of Claim 30, wherein the two or more different oligonucleotide probes hybridize to rRNA from the same species or phylogenetic group of mycobacteria. ¹SE-¹¹ "^"41TRe method ^"of"^' Claim 30, wherein the two or more different oligonucleotide probes hybridize to rRNA from different species or phylogenetic groups of mycobacteria and are labeled such that the different species or phylogenetic groups can be differentiated. 33. A method to permeabilize microbial cells for use in in situ hybridization protocols, comprising: a) contacting fixed microbial cells with Xylene; b) rehydrating the microbial cells; c) enzymatically digesting the microbial cells with lysozyme and achromapeptidase; and d) washing and drying the microbial cells.

34. A method for in situ hybridization of microbial cells, comprising: a) contacting fixed microbial cells with Xylene; b) rehydrating the microbial cells; c) enzymatically digesting the microbial cells with lysozyme and achromapeptidase to produce permeabilized microbial cells; d) washing and drying the permeabilized microbial cells; e) contacting the permeabilized microbial cells with at least one oligonucleotide probe in a hybridization buffer; - f) heating the cells in the buffer at a temperature sufficient to dissociate polynucleotide strands; g) cooling the cells in the buffer at a temperature sufficient to allow polynucleotide strands to hybridize; h) washing the cells to remove excess hybridization buffer; and i) visualizing the cells.

35. The method of Claim 33 or Claim 34, wherein the microbial cells are mycobacterial cells.

36. The method of Claim 33 or Claim 34, wherein the microbial cells are in culture or in a tissue. 37. The method of Claim 33 or Claim 34, wherein the cells are not permeabilized using Proteinase K.

38. The method of Claim 33 or Claim 34, wherein steps (a)-(d) comprise: a) contacting fixed microbial cells with 100% Xylene for from about 1 to about 30 minutes; ⁽bj' ' ^'"""r^'enydfafing the microbial cells; c) enzymatically digesting the microbial cells with from about 0.1 mg/ml to about 10 mg/ml lysozyme, and from about 3 units/ml to about 300 units/ml achromapeptidase, for between about 5 and about 60 minutes; and d) washing and drying the microbial cells.

39. The method of Claim 38, wherein step (a) comprises contacting fixed microbial cells with 100% Xylene for from about 5 to about 20 minutes.

40. The method of Claim 38, wherein step (c) comprises enzymatically digesting the microbial cells with about 1 mg/ml lysozyme and from about 3 units/ml to about 300 units/ml achromapeptidase.

41. The method of Claim 38, wherein step (c) comprises enzymatically digesting the microbial cells with from about 0.1 mg/ml to about 10 mg/ml lysozyme, and about 30 units/ml achromapeptidase.

42. The method of Claim 38, wherein step (c) comprises enzymatically digesting the microbial cells with lysozyme and achromapeptidase for from about 15 to about 35 minutes.

43. The method of Claim 34, wherein the oligonucleotide probe is a species-specific or phylogenetic group-specific oligonucleotide that hybridizes under stringent conditions to rRNA in microbial cells. 44. The method of Claim 43, wherein the microbial cells are mycobacterial cells.

45. The method of Claim 34, wherein the oligonucleotide probe hybridizes under stringent conditions to rRNA from a mycobacterium species selected from the group consisting of: Mycobacterium avium ssp. avium, Mycobacterium avium ssp. paratuberculosis, Mycobacterium avium ssp. silvaticum, Mycobacterium avium ssp. hominis, Mycobacterium intracellular, Mycobacterium tuberculosis, Mycobacterium kansasii, and Mycobacterium abscessus.

46. The method of Claim 45, wherein the oligonucleotide probe does not cross-hybridize to rRNA from another mycobacterium species. 47. The method of Claim 34, wherein the oligonucleotide probe hybridizes under stringent conditions to rRNA from Mycobacterium avium complex.

48. The method of Claim 47, wherein the oligonucleotide probe does not cross-hybridize to rRNA from a non- Mycobacterium avium complex species. ^'''49T¹ The meffiod oϊ^'Claim 34, wherein the oligonucleotide probe is the oligonucleotide probe according to Claim 21.

50. The method of Claim 34, wherein steps (e)-(h) comprise: e) contacting the permeabilized microbial cells with at least one oligonucleotide probe in a hybridization buffer comprising NaCl, Tris, SDS and formamide; f) heating the cells in the buffer at a temperature of about 92°C to 98°C for from about 1 minute to about 5 minutes; g) cooling the cells in the buffer at a temperature of between about 37⁰C and about 6O⁰C for from about 30 minutes to about 12 hours; and h) washing the cells in a buffer comprising NaCl, EDTA, SDS and Tris.

49. The method of Claim 50, wherein step (f) comprises heating the cells in the buffer at a temperature of about 94°C for from about 1 minute to about 5 minutes.

50. The method of Claim 50, wherein step (g) comprises cooling the cells in the buffer at a temperature of between about 38⁰C and about 42⁰C, from about 30 minutes to about 12 hours.

51. The method of Claim 34, wherein steps (e)-(h) comprise: e) contacting the permeabilized microbial cells with at least one oligonucleotide probe at a concentration of about 2 ng/μl in a hybridization buffer comprising about 90OmM NaCl, about 2OmM Tris pH 8, about 0.01% SDS and about 20% formamide; f) heating the cells in the buffer at a temperature of about 94°C for about 3 minutes; g) cooling the cells in the buffer at a temperature of about 40°C for from about 6 hours to about 12 hours; and h) washing the cells in a buffer comprising about 225mM NaCl, about 5mM EDTA, about 0.01% SDS and about 2OmM Tris pH 8. 52. The method of Claim 34, wherein the cells are visualized by microscopy.

53. The method of Claim 34, wherein the cells are visualized using direct fluorescent microscopy or enzymatic amplification of a colorimetric substrate. ^tφj™. Y^ method "'^''OF' Claim 34, wherein the cells are visualized by radiolabeling.

55. A method to identify oligonucleotide probes for the species-specific or phylogenetic group-specific detection of mycobacteria in a sample, comprising: a) identifying or determining an rRNA sequence from a mycobacterial species or phylogenetic group; b) designing an oligonucleotide probe that is complementary to the rRNA sequence; c) contacting rRNA from mycobacterial cells of the mycobacterial species or phylogenetic group of (a) with the oligonucleotide probe of (b) using the method according to Claim 34; d) contacting rRNA from mycobacterial cells of a different mycobacterial species or phylogenetic group than the mycobacterial species or phylogenetic group of (a) with the oligonucleotide probe of (b) using the method according to Claim 34; e) selecting an oligonucleotide probe for the species-specific or phylogenetic group-specific detection of mycobacteria that hybridizes to the rRNA from (c) but does not cross-hybridize to the rRNA from (d).