UNIVERSAL EUBACTERIA NUCLEIC ACID PROBES AND METHODS

Abstract: Nucleic acid probes capable of hybridizing to rRNA of eubacteria and not to rRNA of non-eubacteria are described along with methods utilizing such probes for the detection of eubacteria in clinical and other samples. Preferred embodiments include probes capable of distinguishing between gram-positive and gram-negative bacteria.

Description: UNIVERSAL EUBACTERIA NUCLEIC ACID PROBES AND METHODS Field of the Invention This invention relates to detection of bacteria in clinical and other samples. Methods for the detection of bacteria in ordinarily aseptic bodily tissues or fluids such as blood, urine, and cerebrospinal fluid fluid - where the presence of any bacterium may be life threatening are of particular importance. The present invention provides nucleic acid probes and compositions along with methods for their use for the specific detection of any bacterium in such samples. Background of the Invention The term ■• eubacteria 1 ' as used herein, refers to the group of prokaryotic organisms (bacteria) as described in, for example, Bergey's Manual of Systematic Bacteriology (N.R. Krieg and J.G. Holt, ed., 1984, Williams & Vilkins, Baltimore). As a group, the eubacteria comprise all of the bacteria which are known to cause disease in humans or animals and are of most concern with respect to detection. The only other described group of bacteria, the archaebacteria, are biologically and genetically distinct from the eubacteria (C.R. Voese, Scientific American, 1981, Volume 244, .pages 98-102). Archaebacteria as a group occupy a variety of "extreme" environments such as hot springs, strongly oxygen-depleted muds, salt brines, etc., which generally do not support the growth of eubacteria. There are no known archaebacterial pathogens and, consequently, their detection is of little clinical significance. Eukaryotic organisms comprise the third fundamental genetic lineage which, together with the eubacteria and archaebacteria, include all known life forms (Figure 1). Eukaryotes include humans, animals, plants and a host of organizationally less complex, free-living and parasitic "protists," including: protozoans, fungi, ciliates, etc. In a clinical context, it is particularly important that eubacteria be distinguished from eukaryotic, e.g. fungal and protozoan, infections which nay present the same symptoms but require a significantly different regime of antimicrobial or chemo-therapy. These genetic distinctions thus are clinically significant from the point of view of diagnosis and antimicrobial chemotherapy. It is an aspect of the present invention to provide nucleic acid probes which discriminate between eubacterial, human (including human ■itochondrial) and fungal rRNA molecules. It is another aspect of the present invention to provide probes and probe sets which provide a basis for discriminating between Gram positive and Gram negative eubacteria. Methods for detecting, identifying and enumerating bacteria in normally sterile body fluids vary with the type of sample and the suspected pathogen. No currently available method is optimal for the detection of all pathogens. Often a combination of methods must be used to increase the likelihood that the pathogen will be detected. All commonly used methods for detection of, for example, bactere ia or bacterial septiceraia rely on the in vitro cultivation of microbes from clinical samples. Generally, a blood sample is drawn from a patient and incubated in a rich artificial culture medium and monitored for 1 to 14 days. During this time, the medium is examined or blindly sub- cultured (plated), or assayed chemically or isotopically for evidence of bacterial growth or fermentative processes. Clinicians generally draw two or three samples of 10 milliliters of blood which may yield as few as one to ten colony forming units of bacteria for a positive diagnosis. Following the isolation of individual colonies of bacteria on diagnostic solid media and/or by Gram-staining, presumptive identification of the bacteria (or fungus) is made. All cultivation methods suffer a number of serious shortcomings, including the following: - High material costs; - Labor intensive; - Technologists extensively handle dangerous bodily fluids; - False positives due to handling; - False negatives due to low viable cell numbers; - False negatives due to fastidious media requirements of many potential pathogens; and - Relatively long time to positive diagnosis and identification. Because of the relatively long time required by current methods to achieve a diagnosis and because of the potentially life threatening nature of such infections, antimicrobial therapy often is begun empirically before the results of such tests can be known. Therefore, it is another aspect of the present invention to provide nucleic acid probes which are broadly specific for all eubacteria and which preferably do not react with other eukaryotic pathogens, especially fungi, which may be present in sampled materials. It is yet another aspect of the present invention to provide probes which may be used in a variety of assay systems which avoid many of the disadvantages associated with traditional, multi-day culturing techniques. It is still another aspect of the present invention to provide probes that are capable of hybridizing to the ribosomal ribonucleic acid (rRNA) of the targeted eubacterial organisms under normal assay conditions. While Kohne et al. (Biophysical Journal 8:1104-1118, 1968) discuss one method for preparing probes to rRNA sequences, they do not provide the teaching necessary to make broad-specificity eubacterial probes. Pace and Campbell (Journal of Bacteriology 107:543-547, 1971) discuss the horaology of ribosomal ribonucleic acids from diverse bacterial species and a hybridization method for quantitating such homology levels. They do not identify particular nucleic acid sequences shared by bacteria, but absent in eukaryotes. Woese (Microbiological Reviews 51:221-271, 1987) describes the breadth of the eubacteria, in terms of rRNA sequence, but does not indicate sequences of interest for complete bacterial indusivity. These references, however, fail to relieve the deficiency of Kohne's teaching with respect to eubacterial probes and, in particular, do not provide eubacterial specific probes useful in assays for detecting eubacteria in clinical or other samples. Giovannoni et al. (Journal of Bacteriology 170:720-726, 1988) describe a number of probes which are claimed to be useful for the identification of broad groups of eubacteria, archaebacteria and eukaryotes. However, Giovannoni et al. do not disclose the probes of the present Invention. Nor do they provide the teaching necessary to design such probes. Hogan et al. (European patent publication WO 88/03957) describe a number of probes which are claimed to hybridize to a broad representation of eubacteria. However, Hogan et al. do not teach the probes of the present invention and also fail to relieve the deficiency of Kohne's teaching with respect to these probes. Riboso es are of profound importance to all organisms because they serve as the only means of translating genetic information into cellular proteins. A clear manifestation of this importance is the observation that all cells have ribosomes. Actively growing bacteria may have 20,000 or more ribosomes per-cell. This makes ribosomes one of the most abundant macromolecular entities in a cell, and an attractive diagnostic assay target. Ribosomes contain three distinct RNA molecules which in Escherichia coli are referred to as 5S, 16S and 23S rRNAs. These names historically are related to the size of the RNA molecules, as determined by their sedimentation rate. In actuality, however, ribosomal RNA molecules vary in size between organisms. Nonetheless, 5S, 16S, and 23S rRNA are commonly used as generic names for the homologous RNA molecules in any bacteria, and this convention will be continued herein. Discussion will be confined to 16S and 23S rRNAs. As used herein, probe(s) refer to synthetic or biologically produced nucleic acids (DNA or RNA) which, by design or selection, contain specific nucleotide sequences that allow them to hybridize under defined predetermined stringencies, specifically (i.e., preferentially, see below - Hybridization) to target nucleic acid sequences. In addition to their hybridization properties, probes also may contain certain constituents that pertain to their proper or optimal functioning under particular assay conditions. For example, probes may be modified to improve their resistance to nuclease degradation (e.g. by end capping), to carry detection ligands (e.g. fluorescein, 32- Phosphorous, biotin, etc.), or to facilitate their capture onto a solid support (e. g., poly-deoxyadenosine "tails"). Such modifications are elaborations on the basic probe function which is its ability to usefully discriminate between target and non-target organisms in a hybridization assay. Hybridization traditionally is understood as the process by which, under predetermined reaction conditions, two partially or completely complementary strands of nucleic acid are allowed to come together in an antiparallel fashion (one oriented 5' to 3', the other 3' to 5') to form a double-stranded nucleic acid with specific and stable hydrogen bonds. (Note that nucleic acids do have a polarity; that is, one end of a nucleic acid strand is chemically different from another. This is defined by the polarity of the chemical linkages through the asymmetric sugar moiety of the nucleotide components. The terms 5' and 3' specifically refer to the ribose sugar carbons which bear those names. Except in rare or unusual circumstances, nucleic acid strands do not associate through hydrogen bonding of the base moieties in a parallel fashion. This concept is well understood by those skilled in the art.) The stringency of a particular set of hybridization conditions is defined by the base composition of the probe/target duplex, as well as by the level and geometry of ispairing between the two nucleic acids. Stringency may also be governed by such reaction parameters as the concentration and type of ionic species present in the hybridization solution, the types and concentrations of denaturing agents present, and/or the temperature of hybridization. Generally, as hybridization conditions become more stringent, longer probes are preferred if stable hybrids are to be formed. As a corollary, the stringency of the conditions under which a hybridization is to take place (e. g., based on the type of assay to be performed) will dictate certain characteristics of the preferred probes to be employed. Such relationships are well understood and can be readily manipulated by those skilled in the art. As a general matter, dependent upon probe length, such persons understand stringent conditions to mean approximately 35°C-65 β C in a salt solution of approximately 0.9 molar. Summary of the Invention In accordance with the various principles and aspects of the present invention, there are provided nucleic acid probes and probe sets comprising deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) sequences which hybridize, under specific conditions, to the ribosomal RNA molecules (rRNA), rRNA genes (rDNA), and certain amplification and in vitro transcription products thereof of eubacteria but which do not hybridize, under the same conditions, to the rRNA or rDNA of eukaryotic cells which may be present in test samples. In addition, certain of the probes and probe sets described herein may be used as primers for the specific amplification of eubacterial rRNA or rDNA sequences which may be present in a sample by such methods as the polymerase chain reaction (US 4,683,202) or transcriptional amplification systems (e.g. TAS, Kwoh et al., 1989, Proceedings of the National Academy of Science 86:1173- 1177). The probes of the present invention advantageously provide the basis for development of valuable nucleic acid hybridization assays for the specific detection of eubacteria in clinical samples such as blood, urine, cerebrospinal fluid, biopsy, synovial fluid, or other tissue or fluid samples from humans or animals. The probes also provide the basis for testing, for example in quality control, substances that are presumed sterile, e.g., pharmaceuticals. The probes described herein are specifically complimentary to certain highly conserved bacterial 23S or 16S rRNA sequences. The detection of bacteria by nucleic acid hybridization constitutes enhanced performance capability compared to the available culture- dependent tests for several reasons including: a) increased sensitivity; i.e., the ability to detect said bacteria in a given sample more frequently; b) potentially significant reductions in assay cost due to the use of inexpensive reagents and reduced labor; c) accurate detection of even nutritionally fastidious strains of bacteria; d) faster results because such tests do not require the isolation of the target bacterium from the sample prior to testing; e) the ability to screen, in a batch mode, a large number of samples, and only culture those identified as "hybridization positive"; f) potential detection of phagocytized organisms eliminating the need for multiple, punctuated blood samples in order to sample the cyclical "window" of viable organisms (which probably depends on host immunological cycles); g) some reduction of technologist handling of potentially infectious body fluids; h) the ability to detect very low numbers of targets by amplifying either the bacterial signal or target using in vitro nucleic acid amplification. It has been discovered that other advantages incurred by directing the probes of the present invention against rRNA include the fact that the rRNAs detected constitute a significant component of cellular mass. Although estimates of cellular ribosome content vary, actively growing Escherichia coli, for example, may contain upwards of 50,000 ribosomes per cell, and therefore 50,000 copies of each of the rRNAs (present in a 1:1:1 stiochiometry in ribosomes). The abundance of ribosomes in other bacteria particularly under other, less favorable, metabolic conditions may be considerably lower. However, under any circumstances, rRNAs are among the most abundant cellular nucleic acids present in all cell types. In contrast, other potential cellular target molecules such as genes or RNA transcripts thereof, are less ideal since they are present in much lower abundance. A further unexpected advantage is that the rRNAs (and the genes specifying them) appear not to be subject to lateral transfer between contemporary organisms. Thus, the rRNA primary structure provides an organism-specific molecular target, rather than a gene-specific target as would likely be the case, for example of a plasmid-borne gene or product thereof which may be subject to lateral transmission between contemporary organisms. Additionally, the present invention provides probes to eubacterial rRNA target sequences which are sufficiently similar in most or all eubacteria tested that they can hybridize to the target region in such eubacteria. Advantageously, these same rRNA target sequences are sufficiently different in most non-eubacterial rRNAs that, under conditions where the probes hybridize to eubacterial rRNAs they do not hybridize to most non-eubacterial rRNAs. These probe characteristics are defined as inclusivity and exclusivity, respectively. The discovery that probes could be generated with the extraordinary inclusivity and exclusivity characteristics of those of the present invention with respect to eubacteria was unpredictable and unexpected. ι0 Brief Description of the Figures Further understanding of the principles and aspects of the present invention may be made by reference to the tables wherein: Figure 1 - Shows an evolutionary "tree" of the major genetic "kingdoms" of life (Woese, 1987, Microbiological Reviews 51:221- 271). The branching patterns represent the utational distances between the 16S rRNA sequences of the represented organism. Such comparisons readily distinguish the eubacteria from the archaebacteria and eukaryotes. Figure 2 - Shows a more detailed evolutionary tree of the eubacterial kingdom (ibid.). So far about 10 major divisions/phyla have been defined based on 16S rRNA sequence comparisons. Certain discriminations among eubacterial divisions can be important in a clinical context and certain of the probes of the present invention do exhibit preferential hybridization to one or more or the eubacterial divisions. Therefore, the test organisms listed in Tables 3, 4 and 5 are grouped according to the divisions shown in Figure 2 so that significant patterns of hybridization may be most easily discerned. Brief Description of the Tables Table 1 - Shows alignment of the nucleotide sequences of the preferred 16S rRNA-targeted probes of the present invention with their target nucleotide sequences in E. coli 16S rRNA. Very extensive sequence comparison to some 350 aligned 16S and 18S rRNA sequences were performed during the development of the probes of the present invention. It simply is not practical to show this analysis in detail. However, a consensus sequence (CONS-90%) of highly conserved 16S rRNA nucleotide positions is provided as a summary of the patterns of // nucleotide sequence variation discovered among representative eubacteria. A nucleotide on the C0NS-90Z line indicates that that nucleotide is found at the homologous position in 902 or greater of the eubacterial sequences inspected. Note that the probe target regions all correspond to clusters of high sequence conservation among the eubacterial 16S and 23S rRNA molecules. Since the E. coli 16S and 23S rRNA sequences were among the first full rRNA sequences obtained, the assigned position numbers have become a convenient and commonly accepted standard for explicitly identifying the homologous regions in other rRNA sequences under consideration. In Table 1, the E. coli RNA (target) sequence is written 5' to 3'. Probe sequences are DNA and written 3' to 5', except for probes 1638, 1642 and 1643 which are designed to hybridize to the rRNA-complementary sequence rather than the rRNA itself. These latter probes have the same "sense" (i. e. polarity) as the rRNA and are written 5' to 3'. Table 2 - Shows alignment of the nucleotide sequences of the preferred 23S rRNA-targeted probes of the present invention with their target nucleotide sequences in E. coli 23S rRNA. As in Table 1 the E. coli sequence numbering is used as a standard in order to identify the homologous probe target sequences in all 23S rRNAs. C0NS-902. has the same meaning as in TABLE 1. For the 23S rRNA analyses only about 30 sequences were available. However, these represent most of the major eubacterial divisions shown in Figure 2. In the probe 1730 sequence, "R" - a 1:1 mixture of A and G at that position. Table 3 - Exemplifies the inclusivity and exclusivity behavior of a number of the preferred 16S rRNA-targeted probes toward a representative sampling of eubacterial and non-eubacterial rRNAs in a dot blot hybridization assay. ιZ Table 4 - Exemplifies the inclusivity and exclusivity behavior of a number of the preferred 23S rRNA-targeted probes toward a representative sampling of eubacterial and non-eubacterial rRNAs in a dot blot hybridization assay. Table 5 - Exemplifies the inclusivity and exclusivity behavior of a number of additional preferred 16S and 23S rRNA-targeted probes toward a representative sampling of eubacterial and non-eubacterial rRNAs in a dot blot hybridization assay. These probes exhibit useful patterns of hybridization to specific subgroups of eubacteria - notably Gram positive and Gram negative bacteria. Detailed Description of the Invention and Best Mode Probe Development Strategy: The first step taken in the development of the probes of the present invention involved identification of regions of 16S and 23S rRNA which potentially could serve as target sites for eubacteria specific nucleic acid probes. This entailed finding sites which are: 1) highly conserved (few nucleotide changes, deletions, or insertions) among eubacterial rRNA sequences, and 2) substantially different in non-eubacterial rRNA sequences. For this analysis, precise alignments of available 16S and 23S rRNA sequences were developed. A number of 16S and 23S rRNA sequences were determined as part of this effort. Such nucleotide sequences were determined by standard laboratory protocols either by cloning (Maniatis et al., 1982, Molecular Cloning; A Laboratory Manual, Cold Spring Harbor Laboratory, New York, pp 545) and sequencing (Maxam and Gilbert, 15 1977, Proceedings of the National Academy of Science, USA 74:560-564: Sanger et al., 1977, Proceedings of the National Academy of Science, USA 74:5463-5467) the genes which specify the rRNAs, and/or by direct sequencing of the rRNAs themselves using reverse transcriptase (Lane et al., 1985, Proceedings of the National Academy of Science, USA 82:6955- 6959; Lane, manuscript in preparation). A computer algorithm, operating on the aligned set of 16S and 23S rRNA sequences, was used to identify regions of greatest similarity among eubacteria. Nucleic acid probes to such regions will hybridize most widely among diverse eubacteria. Such regions of homology among eubacteria next were assessed for differences with non-eubacterial rRNA sequences. In particular, sequence differences between eubacterial and human, fungal, and mitochondrial sequences were sought. Forty one probes were designed based on these analyses; 22 targeting 23S rRNA and 19 targeting 16S rRNA. The hybridization behavior of these probes toward extensive panels of eubacteria was determined by hybridization analysis in a dot blot format. Physical Description of the Probes: The foregoing probe selection strategy yielded a number of probes useful for identifying eubacteria in samples and include the following preferred oligonucleotide probes: 16S rRNA-targeted probes: Probe 1638: 5'-AGAGTTTGATCCTGGCTCAG-3' Probe 1642: 5'-AGAGTTTGATCATGGCTCAG-3' Probe 1643: 5'-AGAGTTTGATCCTGGCTTAG-3' Probe 1738: 5'-CTGAGCCAGGATCAAACTCT-3' Probe 1744: 5' -CAGCGTTCGTCCTGAGCCAGGATCAAACT-3' Probe 1659: 5'-CTGCTGCCTCCCGTAGGAGT-3' Probe 1660: 5'-CTGCTGCCTCCCGTAGGAGTTTGGGCCGTGTCTCAGTTCCAGTGT-3' Probe 1661: 5'-TATTAC∞CGGCTGCTGGCACGGAGTTAGCCG-3' Probe 1739: 5'-^CGTGGACTACC∞GGTATCTAATCCTGTTTGCTCCCCACGCTTTCG-3' Probe 1740: 5'-GGGTTGCGCTCGTTGCGGGACTTAACCCGACATCTCACGGCACGAGCT GACGACAGCCATGCAT-3 ' Probe 1741: 5'-CTCACGGCACGAGCTGACGACAGCCATGCAT-3' Probe 1742: 5'-GGGTTGCGCTCGTTGCGGGACTTAACCCGACAT-3' Probe 1745: 5'-ΔGCTGACGACAACCATGCACCACCTGT-3' Probe 1746: 5'TCATAAGGGGCATGATGATTTGACGTCAT-3' Probe 1743: 5'-GTACAAGGCCCGGGAACGTATTCACCG-3' Probe 1637: 5'-AAGGAGGTGATCCAGCC-3' Probe 1639: 5'-ACGGTTACCTTGTTACGACTT-3' Probe 1640: 5'-ACGGCTACCTTGTTACGACTT-3' Probe 1641: 5'-ACGGATACCTTGTTACGACTT-3' 23S rRNA-targeted probes: Probe 1730: 5'-CTTTTCTCCTTTCCCTCRCGGTACTGGTTCRCTATCGGTC'3 Probe 1731: 5'-CTTTTCGCCTTTCCCTCGCGGTACTGGTTCGCTATCGGTC'3 Probe 1658: 5'-TCTTTAAAGGGTGGCTGCTTCTAAGCCAACATCCTGGTTG-3' Probe 1656: 5'-CTACCTGTGTCGGTTTGCGGTACGGGC-3' Probe 1657: 5'-GGTATTCTCTACCTGACCACCTGTGTCGGTTTGGGGTACG-3' Probe 1653: 5'-CCTTCTCCCGAAGTTACGGGGGCATTTTGCCTAGTTCCTT-3' Probe 1654: 5'-CCTTCTCCCGAAGTTACGGGGTCATTTTGCCGAGTTCCTT-3' Probe 1655: S'-CCTTCTCCCGAAGTTACGGCACCATTTTGCCGAGTTCCTT-S' Probe 1651: 5'-CTCCTCTTAACCTTCCAGCACCGGGCAGGC-3' Probe 1652: 5'-TTCGATCAGGGGCTTCGCTTGCGCTGACCCCATCAATTAA-3' Probe 1512: 5'-TTAGGACCGTTATAGTTACGGCCGCCGTTTACTGGGGCTT-3' Probe 1256: 5'-GGTCGGAACTTACCCGACAAGGAATTTCGCTACCTTAG-3' Probe 1398: 5'-GGTCGGTATTTAACCGACAAGGAATTTCGCTACCTTAG-3' Probe 1511: 5'-CGTGCGGGTCGGAACTTACCCGACAAGGAATTTCGCTACC3' Probe 1595: 5'-CGATATGAACTCTTGGGCGGTATCAGCCTGTTATCCCCGG-3' Probe 1600: 5'-CAGCCCCAGGATGAGATGAGCCGACATCGAGGTGCCAAAC-3' Probe 1601: 5'-CAGCCCCAGGATGTGATGAGCCGACATCGAGGTGCCAAAC-3' Probe 1602: 5'-CAGCCCCAGGATGCGATGAGCCGACATCGAGGTGCCAAAC-3' Probe 1598: 5'-CGTACCGCTTTAAATGGCGAACAGCCATACCCTTGGGACC-3' Probe 1599: 5'-CGTGCCGCTTTAATGGGCGAACAGCCCAACCCTTGGGACC-3' Probe 1596: 5'-GATAGGGACCGAACTGTCTCACGACGTTTTGAACCCAGCT-3' Probe 1597: 5'-GATAGGGACCGAACTGTCTCACGACGTTCTGAACCCAGCT-3' The specific behaviors of the aforementioned probes are dependent to a significant extent on the assay format in which they are employed. Conversely, the assay format will dictate certain of the optimal design features of particular probes. The "essence" of the probes of the invention is not to be construed as restricted to the specific string of nucleotides in the named probes. For example, the length of these particular oligonucleotides was optimized for use in the dot blot assay (and certain other anticipated assays) described below. It is well known to those skilled in the art that optimal probe length will be a function of the stringency of the hybridization conditions chosen and hence the length of the instant probes may be altered in accordance therewith. Also, in considering sets comprised of more than one probe, it is desirable that all probes behave in a compatible manner in any particular format in which they are employed. Thus, the exact length /7 of a particular probe will to a certain extent, reflect its specific intended use. Again, given the probes of the instant invention, these are familiar considerations to one of ordinary skill in the art. The "essence" of the probes described herein resides in the discovery and utilization of the specific sequences described above and given in Table 1 and Table 2. Hybridization Analysis of Probe Behavior: The sequence comparisons which led to the discovery of the disclosed target sequences suggested that many of the probes should hybridize to a significant number of eubacteria. For the 16S rRNA analyses, some 350 sequences were considered in designing the probes; for the 23S rRNA analyses only about 30 eubacterial sequences were available. Since it is impossible to test every eubacterial strain, greater sequence variation might exist in other eubacterial strains not inspected by sequence analysis which might reduce or eliminate hybridization by the prospective probes to such untested eubacteria. As can be seen in Tables 3, 4 and 5, some probes of extremely broad inclusivity nevertheless fail to hybridize to certain bacteria. Therefore, carefully documenting the hybridization behavior to a large and representative number of eubacteria is an important element in documenting that such probes are capable of detecting all eubacteria or, failing that, for documenting which eubacteria are not detected. .Such "failures" may not be clinically significant or alternatively, may be compensated for by appropriate inclusion of other probes of the instant inventions. Equally as important as the inclusivity behavior of the probes, is their exclusivity behavior, i.e., their reactivity toward non- eubacteria. As mentioned, demonstrating a lack of hybridization to human and fungal rRNAs is of paramount importance in the types of clinical applications envisioned for such probes. Therefore, the IS behavior of the probes toward representative eubacterial, human and fungal rRNAs was determined by hybridization analysis using a dot blot procedure. Example 1: Dot-blot analysis of probe hybridization behavior. Dot-blot analysis, in accordance with well known procedures, involves immobilizing a nucleic acid or a population of nucleic acids on a filter such as nitrocellulose, nylon, or other derivatized membranes which readily can be obtained commercially, specifically for this purpose. Either DNA or RNA can be easily immobilized on such a filter and subsequently can be probed or tested for hybridization under any of a variety of conditions (i.e., stringencies) with nucleotide sequences or probes of interest. Under stringent conditions, probes whose nucleotide sequences have greater complementarity to the target sequence will exhibit a higher level of hybridization than probes containing less complementarity. For most of the oligonucleotide probes described herein, hybridization to rRNA targets at 60 β C for 14- 16 hours (in a hybridization solution containing 0.9 M NaCl, 0.12 M Tris-HCl, pH 7.8, 6 M EDTA, 0.1 M KP04, 0.1% SDS, O.tt pyrophosphate, 0. 002% ficoll, 0.02% BSA, and 0.0023. polyvinylpyrrolidine), followed by standard post-hybridization washes to remove unbound and non- specifically hybridized probe (at 60°C in 0.03 M NaCl, 0. 004 M Tris- HCl, pH 7.8, 0.2 mM EDTA, and 0.1* SDS), would be sufficiently stringent to produce the levels of specificity demonstrated in Tables 3, 4 and 5. The exceptions to these conditions are probe 1738 (which was hybridized at 37°C), and probe 1746 (which was hybridized at 37°C and washed at 50°C). Techniques also are available in which DNA or RNA present in crude (unpurified) cell lysates can be immobilized without first having to purify the nucleic acid in question (e.g. Maniatis, T., Fritsch, E. F. and Sa brook, J., 1982, Molecular Cloning:A Laboratory Manual). The dot-blot hybridization data shown in Tables 3, 4 and 5 were generated by hybridization of the indicated probes to purified RNA preparations from the indicated eubacterial, fungal and human specimens. Bacterial and fungal RNAs were purified from pure cultures of the indicated organisms. Mouse RNA was purified from L cells (a tissue culture cell line). Wheat germ RNA was purified from a commercial preparation of that cereal product. Human blood and stool RNAs were purified from appropriate specimens obtained from normal, healthy individuals. Purified RNA was used, rather than cell lysates for a number of simple technical reasons. The most important of these relate to proper interpretation of the relative signal arising from the hybridization of any particular probe to individual organisms. RNA content per cell is known to vary widely among different bacteria and varies even more between bacteria and eukaryotic cells. In addition, the specific metabolic status of cells at the time of harvest can have a profound influence on the amount and integrity of the RNA recovered. Some bacteria, for example, begin to degrade their RNA very rapidly upon reaching the stationary growth phase. The organisms represented in Tables 3, 4 and 5 comprise an extremely diverse collection in every respect. Represented are Gram positive and Gram negative bacteria, photosynthetic and chemosynthetic, heterotrophic and lithotrophic, and anaerobic and aerobic metabolisms. By using known, equivalent amounts of purified RNA in the individual "dots," relative levels of hybridization of each probe to each organism can be meaningfully compared without regard to the idiosyncracies of nucleic acid preparation from individual types of bacteria represented. RNA was prepared by a variation on standard published methods which has been developed in our laboratory (W. Weisburg, unpublished). The method rapidly yields bulk high molecular weight RNA in a highly purified but relatively unfractionated form. Little or no DNA, or low molecular weight RNA species are found in RNA prepared in this fashion. Zό A large proportion of the RNA is 16S and 23S rRNA (18S and 28S rRNA in eukaryotes) as is true of the RNA in the intact cells. The method is rapid and convenient, but otherwise is not relevant to interpretation of the dot-blot results presented in Tables 3, 4 and 5. Most other currently accepted methods available in the literature which yield RNA of reasonable intactness will yield equivalent hybridization results. For the hybridization experiments reported in Tables 3, 4 and 5, probes were end-labeled with radioactive 32-phosphorous , using standard procedures. Following hybridization and washing as described above, the hybridization filters were exposed to X-ray film and the intensity of the signal evaluated with respect to that of control RNA spots containing known amount of target RNA of known sequence. A scale of hybridization intensity ranging from ++++ (hybridization signal equivalent to that of control spots) to + (barely detectable even after long exposure of the x-ray film) has been used to compare hybridization signals between organisms and probes. +++ signal indicates a very strong signal only slightly less intense than control spots. ++ indicates a clearly discernible hybridization signal, but one that is noticeably weaker than the control spots. Note that while more "quantitative" ways to record hybridization signal are available, they are much more cumbersome to employ and, in our experience, not really any more useful for probe evaluation than the method employed in Tables 3, 4 and 5. In fact, because of certain uncontrollable variables in spotting exactly equivalent amounts of target RNA (of equivalent intactness) from such disparate organisms, numerically more precise counting methods are only deceptively more quantitative. In our experience, an organism generating a ++ or greater signal to a particular probe is easily distinguished from one generating a "-" signal. This is true of a variety of assay formats that have been tested. 2/ As is evident in Tables 3 and 4, 23S rRNA-targeted probes 1600, 1602, 1596, 1256 and 1512 and 16S rRNA-targeted probes, 1738, 1660, 1639, 1739, 1740, 1741 and 1743 hybridize most extensively among the eubacteria and are thus the most preferred. Other probes hybridize in a variety of patterns to subgroups of eubacteria and would be preferred for the detection of those subgroups or as components of more broadly inclusive probe sets. For example, probes 1599, 1656, 1744, 1745 and 1746 hybridize preferentially to Gram positive bacteria. Probes 1657, 1598 and 1595 hybridize preferentially to gram-negative bacteria, particularly to members of the so-called "purple bacterial" division (Figure 2 and Table 5). Other probes exhibit other useful patterns of hybridization as is evident upon inspection of the data in Tables 3, 4 and 5. These probes can be combined in a variety of ways to create probe sets which exhibit the combined hybridization properties of the component probes. An example of one such hybridization format is given below (Example 2). Alternatively, the probes could be used in a variety of subtractive hybridization schemes in which specific rRNA molecules are removed from the pool present in a mixed population of organisms prior to or simultaneous with the target organism-specific probes (e.g. Collins, European Patent Application 87309308.2). Example 2: Dual Probe, Sandwich Hybridization Assay The probes of the present invention or derivatives thereof can be advantageously employed in a variety of other hybridization formats. One such format is a dual probe, sandwich-type hybridization assay such as that described, for example, in USSN 277,579; USSN 169,646, or USSN 233,683. In such a dual probe application, one probe (for example, probe 1602 or a derivative) would be ideally modified at its 3' terminus to contain a tract of about 20 - 200 deoxyadenosine (dA) residues. This would be used to "capture" the target rRNA (following liquid hybridization) from the test sample onto a solid support (e.g., beads, plastic surface, filter, etc.) which had been suitably derivatized with poly-deoxythymidine (dT) for this purpose. A second probe (for example, probe 1596 or derivative) would then be advantageously used as the detection probe and would be suitably derivatized with some detectable ligand (e.g. 32-P, fluorescein, biotin, etc.). Detection of the presence of the target nucleic acid in a test sample then would be indicated by capture of the detection ligand onto the solid surface through the series of hybridization interactions: SOLID I % (Target Nucleic Acid) , 1-rττττττττττn \ / SUPPORT I limmi 1111111111111 nAAAAAAAAAAA(Capture Probe) (Detection Probe) I I Ligand Ligand This could occur only if the target nucleic acid is present in the test sample. In principle, the above scheme could be employed with multiple capture and detection probes (probe sets) for the purpose of, for example, improving inclusivity or enhancing sensitivity of the assay. Example 3: PCR Amplification of 16S rRNAs. The polymerase chain reaction (PCR) is a well known method for amplifying target nucleic acid by "copying" the nucleic acid sequences located between two target sequences (US 4,683,202). The PCR process could be useful in an assay for the diagnosis of, for example, a non- viral pathogen by amplifying the genes encoding the pathogen's rRNA or rRNA genes and subsequently detecting that product. Implementation of this diagnostic strategy requires the invention of primers capable of V> amplifying the rRNA of the targeted organism(s). A second important application of such primers is in cloning amplified rRNA genes, and a third application is the direct sequencing of amplified rRNA genes. Probes 1638, 1642, 1643, 1637, 1639, 1640 and 1641 may be ideally used as primers for enzymatically copying and/or amplifying eubacterial 16S rRNAs or the genes encoding them. Details of the PCR procedure vary slightly depending on whether the target nucleic acid is single or double stranded, and whether it is DNA or RNA. However, the principle is the same in either case. Briefly, the steps are as follows: 1) Double-stranded DNA is denatured, 2) Oligonucleotide primers complimentary to each of the sister DNA strands are annealed, and 3) deoxynucleotide triphosphate precursors are incorporated into newly synthesized sister DNA strands by extension of the primers from their 3' termini using DNA polymerase and/or reverse transcriptase. Thus, a pair of oligonucleotide primers are required for the PCR reaction, one complementary to each strand within the target gene. They are positioned such that the newly synthesized product of one primer is also a target/template for the other primer. Thus the target nucleotide sequence located between the two primer annealing sites may be amplified many fold by repeating the steps listed above 20 to 30 times. Probes 1638, 1642, 1643, 1637, 1639, 1640 and 1641 are suitable for use as primers for enzymatically copying and/or amplifying eubacterial 16S rRNAs or the genes encoding them. That is, as a set, they will anneal very broadly among eubacterial rRNAs and rRNA genes and so will amplify any eubacterial rRNA sequences present in a sample. #} Probes 1637, 1639, 1640 and 1641 hybridize to the 16S rRNA (or rRNA- like strand of the ribosomal RNA gene) near its 3' end (Table 1). The template strand is read in the 3' to 5' direction producing an rRNA- complementary strand with the primer itself incorporated at its 5' terminus. Probes 1638, 1642, and 1643 hybridize near the 5' end of the rRNA- co plementary strand of the rRNA gene or to such a complement produced as described immediately above. Individually, the above-described 16S rRNA amplification primers have approximately the following specificities: 5' primers: Probe 1638: most eubacteria Probe 1642: enterics and relatives Probe 1643: Borrelia spirochetes 3' primers: Probe 1637: most eubacteria Probe 1639: enterics, Deinococcus, Campylobacter Probe 1640: most eubacteria Probe 1641: fusobacteria, some Bacillus species In test samples where the target bacterium is known, specific primers can be used. Where the target organism is not specifically known (for example, any eubacterium) all of the above mentioned primers can be used as a set. The above described primers have been designed to amplify nearly the entire 16S rRNA sequence. Any of the other probes of the present invention or derivatives thereof can be used to amplify sub-segments of the 16S and 23S rRNAs or genes in a fashion similar to that just described. Any such primers can be modified in a great number of ways to, for example, incorporate RNA polymerase promoters, cloning sites, etc. into the amplified transcripts. While the description of the invention has been made with reference to detecting rRNA, it will be readily understood that the probes described herein and probes complementary to those described herein also will be useful for the detection of the genes (DNA) which specify the rRNA and, accordingly, such probes are to be deemed equivalents to the described probes and encompassed within the spirit and scope to the present invention and the appended claims. TABLE It 16S rRNA-TARGETED PROBES AND TARGET SEQUENCES E. colifs 8 38 I I CONS-90% AGAGUUUGAUC UGGCUCAG GAACGCUGGCG E. coll 5 ' -AAAUUGAAGAGUUUGAUCAUGGCUCAGAUUGAACGCUGGCG Probe 1738 3 ' -TCTCAAACTAGGACCGAGTC-5 ' Probe 1744 3 ' --TCAAACTAGGACCGAGTCCTGCTTGCGAC-5 ' Probe 1638 5 ' -AGAGTTTGATCCTGGCTCAG-3 ' Probe 1642 . 5 * -AGAGTTTGATCATGGCTCAG-3 » Probe 1643 5 ' -AGAGOTTGATCCTGGCTTAG-3 ' E. coll#s 313 357 I I C0NS-90* G CACA UGG ACUGAGACACGG CCA ACUCCUACGGGAGGCAGCAGU E. coli GCCACACUGGAACUGAGACACGGUCCAGACUCCUACGGGAGGCAGCAGUGG Probe 1660 3'-TGTGACCTTGACπX^mxrCGGGTTTGAGGATCCCσrcCGOXrGTC ' Probe 1659 3'-TGAGGATGCCCTCCGTCGTC-5' E. coli#s 504 535 I I CONS-90% C CGGCUAACU GUGCCAGCAGCCGCGGUAAUAC E. coli CACCGGCUAACUCCGUGCCAGCAGCCGCGGUAAUACGG Probe 1661 3*-GCCGATTGAGGCACGGTCGTCGGCGCCATTAT-5' E. coli#s 764 810 I I CONS-90* G CGAAAGCGUGGGGAGC AACAGGAUUAGAUACCCUGGUAGUCCACGC U E. coli GUGCGAAAGCGUGGGGAGCAAACAGGAUUAGAUACCCUGGUAGUCCACGCCGU Probe 1739 3'-GCTTTCGCACCCCTCGTTTGTCCTAATCTATGGGGCCATCAGGTGCG-5' TABLE 1 (cont'd): 16S rRNA-TARGETED PROBES AND TARGET SEQUENCES E. coli#s 1044 1114 I I CONS-90% ACAGGUG UGCAUGG UGUCGUCAGCUCGUG CGUGAG UGUUGGGUUAAGUCCCGCAACGAGCGCAACCC E. coli GAGACAGGUGCUGCAUGGCUGUCGUCAGCUCGUGUUGUGAAAUGUUGGGUUAAGUCCCGCAACGAGCGCAACCCUUA Probe 1745 3 * -TGTCCACCACGTACCAACAGCAGTCGA-5 ' Probe 1740 3 ' -TACGTACCGACAG(^GTCGAGCACGGCACtt^ACAGCCCAATTCAGGGCGTTGCTCGCGTTGGG-5 ' Probe 1741 3 ' -TACGTACCGACAGCAGTCGAGCACGGCACTC-5 ' Probe 1742 3 ' -TACAGCCC TTCAGGGCGTTGCTCGCGITGGG-5 ' E. coli*s 1188 1216 I I CONS-90% GGA GACGUCAA UC UCAUG CCCUUA G E. coli GGGAUGACGUCAAGUCAUCAUGGCCCUUACGACCA Probe 1746 3'-TACTGCAGTTTAGTAGTACGGGGAATACT-5' E. coli#a 1369 1395 I I CONS-90% CGGUGAAUACGUUC CGGG CUUGUACACA E. coli CCACGGUGAAUACGUUCCCGGGCCUUGUACACλ Probe 1743 3'-GCCACTTATGCAAGGGCCCGGAACATG-5' E. colifs 1492 1541 I I CONS-90% AAGUCGUAACAAGGUA CC UA GAA UG GG UGGAU ACCUCCUUU E. coli GUGAAGUCGUAACAAGGUAACCGUAGGGGAACCUGCGGUUGGAUCACCUCCUUA-3' Probe 1637 3'-CCGACCTAGTGGAGGAA-5' Probe 1639 3'-TTCAGCATTGTTCCATTGGCA-5* Probe 1640 3'-TTCAGCATTGTTCCATCGGCA-5' Probe 1641 3'-TTCAGCATTGTTCCATAGGCA-5' 9 TABLE 2 : 23S rRNA-TARGETED PROBES AND TARGET SEQUENCES E. coli #s 442 481 I I I CONS- 90* GACCGAUAG G AC AGUACCGUGAGGGAAAGG GAAAAG AC E. coli 23S ACUGACCGAUAGUGAACCAGUACCGUGAGGGAAAGGCGAAAAGAAC Probe 1730 3 ' -CTCGC__ATra(_TTGGTCATGG(_ECT Probe 1731 3 ' -CTGGCTATCGCTTGGTCATGGCG(_TCCCTT^ E. coli #s 1049 1088 I I I CONS-90% A ACA C AGGA GUUGGCUUAGAAGCAGCCA C UU AAAGA G E.coli 23S AGACAGCCAGGAUGUUGGCUUAGAAGCAGCCAUCAUUUAAAGAAAG Probe 1658 3 ' -GTTGGTCCTAO^CCGAATCπTCGTCGGTGGGAAATTTCT-5 ' E. coli #B 1597 1639 I I I CONS-90% CGUACC AAACCGACACAGGU G G A A C AG E.coli 23S UCAAAUCGUACCCCAAACCGACACAGGUGGUCAGGUAGAGAAUACCAAG Probe 1656 3' -∞GGCATGGC_GTTTGGCTGTGTCCATC-5' Probe 1657 3 ' -GCATGGGG-rrTGGCTGTGTCCACCACT^ E. coli #s 1664 1703 I I I CONS-90% AAGGAACU GCAAA U CCGUAACUUCGG A AAGG E. coli 23S GUGAAGGAACUAGGCAAAAUGGUGCCGUAACUUCGGGAGAAGGCAC Probe 1653 3 ' -TTCCITGATCCGTTTTACGGGGGC^ , _?TGAAGCCC7 CTTCC-5 ' Probe 1654 3 ' *-TTCCI_TGAGCCGTTTTACTGGGGCATTGAAGCCCTCTTCC-5 ' Probe 1655 3 ' -TTCCTTGAGC(X. * I?TTTACCACGGCATTGAAGCCCTC_ rCC-_^ ' E. coli #s 1831 1860 I I I CONS-90% GAC CCUGCCC GUGC GGAAGGUUAA G E.coli 23S GACGCCUGCCCGGUGCCGGAAGGUUAAUUGAUGGGG Probe 1651 3'-α_«ACGGGCC_U:GAC-_^raCCAATT_TCCTC-5' E. coli *s 1851 1890 I I I CONS-90-fe AGGUUAA G U G AAG A GAAGCC E.coli 23S AGGUUAAUUGAUGGGGUUAGCGCAAGCGAAGCUCUUGAUCGAAGCC Probe 1652 3'-AATTAACTACCCCAGTCGCGTTCGCTTCGGGGACTAGCTT-5' * <\ TABLE 2 (cont'd): 23S rRNA-TARGETED PROBES AND TARGET SEQUENCES E. coli #ε 1889 1928 I I I CONS-90* GAAGCCCC GU AACGGCGGCCGUAACUAUAACGGUCCUAAGGU E.coli 23S UCGAAGCCCCGGUAAACGGCGGCCGUAACUAUAACGGUCCUAAGGU Probe 1512 3'-TTCGGGGTCATTTGCCGCCGGCATTGATATTGCCAGGATr-5' E. coli #E 1925 1968 I I I CONS-90* GUCCUAAGGUAGCGAAAUUCCUUGUCGGGUAAGUUCCGACC GCACGAA E.coli 23S GUCCUAAGGUAGCGAAAUUCCUUGUCGGGUAAGUUCCGACCUGCACGAAU Probe 1256 3'-GATTCCATCGCTTTAAGGAACAGCCCATTCAAGGCTGG-5' Probe 1511 3'-CCATCGCTTTAAGGAACAGCCCATTCAAGGCTGGGCGTGC-5' Probe 1398 3'-GAI CCATCGCTTTAAGGAACAGCCAATTTATGGCTGG-5' E. coli #s 2442 2481 CONS I-90% AC CI GGGGAUAACAGGCU AU C CC AG GU CA AUCGI CG E.coli 23S ACUCCGGGGAUAACAGGCUGAUACCGCCCAAGAGUUCAUAUCGACG Probe 1595 3'-GGCCCCTATTGTCCGACTATGGCGGGTTCTCAAGTATAGC-5' E. COli #8 2490 2529 I I I CONS-90% GUUUGGCACCUCGAUGUCGGCUC UC CAUCCUGGGGCUG AG E.coli 23S GGUGUUUGGCACCUCGAUGUCGGCUCAUCACAUCCUGGGGCUGAAG Probe 1600 3'-CAAACCGTGGAGCTACAGCCGAGTAGAGTAGGACCCCGAC-5' Probe 1601 3'-CAAACCGTGGAGCTACAGCCGAGTAGTGTAGGACCCCGAC-5' Probe 1602 3'-CAAACCGTGGAGCTACAGCCGAGTAGCGTAGGACCCCGAC-5' Ξ. coli #s 2535 2574 I I I CONS-90% G GGUCCCAAGGGU GGCUGUUCGCC UUAAAG GG ACG GA E.coli 23S GUAGGUCCCAAGGGUAUGGCUGUUCGCCAUUUAAAGUGGUACGCGA Probe 1598 3*-CCAGGGTTCCCATACCGACAAGCGGTAAATTTCGCCATGC-5' Probe 1599 3'-CCAGGGTTCCCAACCCGACAAGCGGGTAATTTCGCCGTGC-5' E. coli #s 2577 2616 CONS I-90* G GAIGCUGGGUU A AACGUCGUGAGACAGUU GGUC CUAUCI E.coli 23S GCGAGCUGGGUUUAGAACGUCGUGAGACAGUUCGGUCCCUAUCUGC Probe 1596 3'-TCGACCCAAGTTTTGCAGCACTCTGTCAAGCCAGGGATAG-5' Probe 1597 3'-TCGACCCAAGTCTTGCAGCACTCTGTCAAGCCAGGGATAG-5' TABLE 3: DOT BLOT HYBRIDIZATION of 16S rRNA-TARGETED PROBES PROBE HYBRIDIZATION Genus species strain div 1738 1739 1659 1660 1661 1740 1741 1742 1743 Acinetobacter calcoaceticus GT0002 Purple ++++ ++++ ++++ ++++ *♦*+++ +-M-+ ++++ ++++ ++++ Aeromonas sobria GT0007 gamma ++++ Alteromonas putrefaciens G GTT11994455 " ++++ ++++ ++++ ++++ ++++ ++++ ++++ ++++ ++++ Citrobacter atnalonaticus G GTT00669900 " ++++ ++++ ++++ ++++ ++++ ++++ +++•*■ T 1T1T1T1 Citrobacter diversus G GTT00003300 " ++++ -r-M- ++++ +++•!• ++++ ++++ ++++ ++++ Citrobacter freundii GTl0U6D8O7/ " TTTT TTTT TTTT TTTT TT T TTTT TTTT ++++ Edwardsie11a tarda G GTT00556699 " ++++ ++++ ++++ ++++ ++++ +TT+ ++++ ++++ ++++ Enterobacter agglomerans G GTT00668833 ** ++++ TTTT TTTT TTTT TTTT TTTT TTTT ++++ Enterobacter cloacae G GTT00668866 "" +« • +- ++++ ++-H- ++++ ++++ ++++ + • * ■ ++ ++++ Enterobacter sakaza ii G GTT00006622 " ++++ A TXTT.JT. TiTTT, TiTTlT TlTuTTx 11 T 1 T 1 T 1111 T ++++ ++++ Escherichia coli G GTT11666655 " ++++ ++++ ++++ ++++ ++++ +••++ ++++ ++++ Escherichia coli G GTT11559922 " ++++ ++++ ++++ ++++ ++++ ++++ +++ ++++ Escherichia coli G GTT11665599 " +++ ++++ ++++ ++++ ++++ ++++ ++ • ++ ++++ Haemophilus influenza G GTT00224444 " -H- ++++ ++++ ++++ ++++ ++++ ++++ ++++ ++++ Haeπtophilus duc eyi G GTT00224433 " ++++ ++++ ++++ ++++ ++++ • ++++ ++++ ++++• ++++ Hafnia alvei G GTT00224411 " ++++ ++++ ++++ ++++ ++++ ++++ ++++ ++++ Morgenella morganii G GTT00330033 " ++++ ++++ ++++ ++++ ++++ ++++ ++++ ++++ ++++ Klebsiella pneumoπiae G GTT11550000 " ++++ ++++ ++++ ++++ ++++ ++++ ++•++ ++++ ++++ Proteus mirabilis G GTT11449966 " ++++ ++++ ++++ ++++ ++++ ++++ -H-++ ++++ ++++ Frovidencia alcalifaciens G GTT00337711 "" ++++ ++++ ++++ ++++ ++-H- ++++ ++++ ++++ ++++ Pseudomonas aeruginosa G GTT11990099 " ++++ ++++ ++++ ++++ ++++ ++++ ++++ ++++ ++++ Salmonella arizona G GTT00779999 "" ++++ ++++ ++++ ++++ ++++ ++++ -M-H- ++++ ++++ Salmonella typhimurium G GTT00338899 " ++++ ++++ ++++ ++++ ++++ ++++ ++++ ++++ ++++ Serratia marcescens G GTT00339922 " ++++ ++++ ++++ ++++ ++++ ++++ ++++ + +++++++ ++++ Shigella flexneri G GTT00779988 " ++++ ++++ ++++ ++++ ++++ ++++ +-H-+ ++++++++ ++++ Vibrio parahaemolyticus G GTT00556688 " ++++ ++++ -H-++ ++++ ++++ ++++ ++++ ++++++++ •++++ Xanthorαonas maltophilia G GTT00441177 " ++++ ++++ ++++ ++++ ++++ ++++ ++++ ++++++++ ++++ Yersinia enterocolitica G GTT00441199 " ++++ ++++ ++++ ++++ ++++ ++++ ++++ ++++ ++++ Alcaligenea faecalis GT0610 Purple ++++ 4+++ ++++ ++++ ++++ ++++ ++++ ++++ ++++ Branhamella cabarrhalis GT0014 beta +++ ++++ ++++ -H-++ ++++ ++++ ++++ ++++ ++++ Chromobacteriura violaceum G GTT22002222 " ++++ ++++ ++++ ++++ ++++ ++++ ++++ +++•*• ++++ Kingella indologenes 0 0224466 " ++++ ++++ ++++ ++++ ++++ ++++ ++++ ++++ ++++ Moraxella oβloenεis G GTT00330011 " ++++ ++++ ++++ ++++ ++++ -M- - ++*f+ ++++ ++++ Moraxella phenylpyruvica G GTT00330022 " +++ ++++ ++++ ++++ ++++ ++-»"♦• ++++ ++++ ++++ TABLE 3 (cont'd): DOT BLOT HYBRIDIZATION of 16S rRNA-TARGETED PROBES PROBE HYBRIDIZATION Genus species strain div 1738 1739 1659 1660 1661 1740 1741 1742 1743 Borrelia burgdorferi Spiro ++++ ++++ 4444 4444 4444 4444 4444 4444 4444 Borrelia turicatae M TTTT TTTT 4444 4444 4444 4444 4444 4444 4444 Leptospira interrogans-pomona II ++++ +++ 4444 ++++ ++++ 4444 4444 4444 44 Leptosplra biflexa (Patoc-Patoc) a ++++ ++ 4444 ++++ ++++ 4444 4444 4444 4 Leptospira biflexa (CDC) M ++++ +++ 4444 4444 ++4+ 4444 4444 4444 44 Spirochaeta aurantia H 4444 ++++ 4444 ++++ 4444 4444 4444 4444 4444 Bacteroides fragilis 25285 Bact 4444 +++ ++++ ++++ 4444 4444 4444 - 4444 Bacteroides fragilis 29771 M ++++ 444 ++++ ++++ 4444 4444 4444 - 4444 Bacteroides thetaiotaomicron 0572 II 4444 44 4444 +++4 4444 4444 4444 - 4444 Bacteroides melaninogenicus 0011 H ++++ ++ 4444 4444 4444 4444 4444 - 4444 Flavobacteriu meningosepticum 0237 M ++++ ++++ 4444 4444 4444 4444 4444 4444 4444 Chlaπtydia psittaci Chlam ++++ 444 4444 4444 4444 4444 4444 Chlamydia trachomatis » 4444 444 4444 4444 4444 4444 4444- Chlorobiu liroicola Misc ++++ ++++ 4444 4444 4444 4444 4444 4444 4 Chlσroflexus aurantiacus Y400 H 4444 ++++ 4444 4444 4444 4444 4444 4444 4444 CM Deinococcus radiodurans 2608 II ++++ ++++ 4444 4444 4444 4444 4444 4444 4444 Planctomyces marls 2577 II ++++ 4444 4444 4444 4444 4444 - - Normal Stool RNA ++++ ++++ 4444 4444 4444 4444 4444 4444 4444 Mouse L-Cell + 44 - - Wheat Germ - - - - 44 - - - - Normal Human Blood - 44 - - — • Candida lusitaniae 403-87 - 4444 - - - Candida parapsilosis 882-88 _. 4444 - - - Candida tropicalis 224-87 - 4444 - - - Candida albicans 1008-88 - 4444 - - — Candida albicans 223-87 - 4444 - - - Candida albicans 819-88 - 4444 _. - - A Inclusivity and Exclusivity data was determined after overnight exposures. A* Each organism is represented by lOOng of CsTFA purified RNA. AAA Probe 1738 - hybridizations and washes were carried out at 37 C. 4444 = positive control level of hybridization, 4 = barely detectable and - = zero, ND = not done. TABLE 3 (cont'd): DOT BLOT HYBRIDIZATION of 16S rRNA-TARGETED PROBES PROBE HYBRIDIZATION Genus species strain div .1738 1739 1659 1660 1661 1740 1741 1742 1743 Mycoplasma pneumoniae ATCC15531 II 4444 4444 4444 4444 - 4444 4444 4444 - Mycoplasma putrefaciens ATCC15718 M 4444 4444 4444 4444 4444 4444 4444 4444 - Peptostreptococcus productus ATCC27340 M 44444444 4444 4444 4444 4444 4444 4444 4444 Planococcus citreus ATCC14404 M 4444 4444 4444 4444 4444 4444 4444 4444 4444 Staphlyococcus aureus GT0399 H 44444444 4444 4444 444 4444 4444 4444 4444 Staphylococcus aureus . GT1711 II TTTT TTTT TTTT TTTT TTTT TTTT TTTT 'f" I'T TTTT Staphylococcus epidermidis GT0401 N 44444444 4444 4444 4444 4444 4444 4444 4444 Staphylococcus haemolyticus ATCC29970 H , 4444 4444 4444 4444 4444 4444 4444 4444 4444 Streptococcus agalactiae GT0405 N 4444 4444 4444 4444 4444 4444 4444 4444 4444 Streptococcus bovis GT0668 M 44444444 4444 4444 4444 4444 4444 4444 4444 Streptococcus faecalis GT0406 n 44444444 4444 4444 4444 4444 4444 4444 4444 Streptococcus morbillorum GT2194 H 44 4444 4444 44444444 4444 4444 4444 4444 Streptococcus utans GT0412 n 444 4444 4444 4444 4444 4444 4444 4444 4_444 Streptococcus pneumoniae GT0408 « 444 4444 4444 4444 4444 4444 4444 4444 4444 Streptococcus salivarius GT0410 II 44444444 4444 4444 4444 4444 4444 4444 4444 6* Streptococcus sanguis GT0411 II 4444 4444 4444 4444 4444 4444 4444 4444 ND Bifidobacteriu dentiu GT0012 !hiG4C 4444 4444 4444 4444 4444 4444 4444 4444 Corynebacterium genitalium GT0045 Gm 4 4444 4444 4444 4444 4444 4444 4444 4444 4444 Corynebacterium glutamicum GT2120 II 4444 4444 4444 4444 4444 4444 4444 4444 4444 Corynebacter. pseudodiphtheriticuumm GGTT22111199 n 4444 4444 4444 4444 4444 4444 4444 4444 4444 Corynebacterium pεeudotuberculosiiss GGTT22112222 II 4444 44444444 4444 4444 4444 4444 4444 4444 Corynebacterium pyogenes GT2121 II 4444 4444 4444 4444 4444 4444 4444 4444 4444 Corynebacterium xerosis GT0046 H 44 444 4444 4444 4444 4444 4444 4444 4444 Mycobacterium bovis BCG 44 4444 4444 4444 4444 4444 4444 4444 4444 Mycobacterium kansasii 4444 4444 4444 4444 4444 4444 4444 4444 4444 Nocardia asteroides GT2191 n 4444 4444 4444 4444 4444 4444 4444 4444 4444 Rhodococcus rhodochrous Misc 4444 4444 4444 4444 4444 4444 4444 4444 4444 Aerococcus viridans GT2116 Gm 4 4444 4444 4444 4444 4444 4444 4444 4444 - FuEobacterium necrophorum GT0238 4444 4444 4444 4444 4444 4444 4444 4444 4444 Fusobacteriu prausnitzii 1 111 * T-_."-_."._ϊ_*/77/CDPO 4444 4444 4444 4444 4444 4444 4444 4444 4444 Ge ella haemolyεans GT2118 II 44444444 4444 4444 4444 4444 4444 4444 4444 Heliobacillus mobilis 4444 4444 4444 4444 4444 4444 4444 4444 4444 Phormidium ectocarpi Cyano 4444 44 4444 4444 4444 4444 4444 4444 4444 Plectonema boryanum 4444 44 4444 4444 4444 4444 4444 4444 4444 TABLE 3 (cont'd) t DOT BLOT HYBRIDIZATION of 16S rRNA-TARGETED PROBES PROBE HYBRIDIZATION Genus species strain div 1738 1739 1659 1660 1661 1740 1741 1742 1743 Neisseria gonorrhoeae GT0315 H 444444444444444444444444 44444444 4444 Neisseria meningitidis GT0349 N 444444444.4444444 444444444444 4444 4444 Pseudomonas acidovorans GT0376 M 444444444444444444444444 444444444444 Fseudomonas cepacia GT2015 ■ 444444444444 444444444444444444444444 Rhodocyclus gelatinosa ATCC17013 N 444444444444444444444444444444444444 Vitreoscilla stercorarla M 4444444444444444 44444444 Achromobacter xerosis GT0810 Purple 444444444444 4444 444444444444 44444444 Acidiphilium cryptum ATCC33463 alpha 4444444444444444444444444444 +4444444 Agrobacterium tumefaciens GT2021 H 4444444444444444 444444444444 44444444 Brucella abortus ATCC23448 444444444444 4444 44444444 44444444 Flavobacterium capεulatum GT2025 4444444444444444 44444444 4444 4444 4 M coplana bullata GT2023 444444444444 4444444444444444 4444 Pseudomonas di inuta GT2020 4444 44444444 444444444444444444444444 Rhodobacter sphaeroides ATCC17023 H 444444444444 4444444444444444 44444444 Rhodospirillu rubrum ATCC25903 II 4444 44444444 4444 44444444 4444 44444444 Thiobacillus versutus ATCC25364 4444 44444444 4444 444444444444 44444444 Desulfovibrio desulfuricans ATCC 7757 delta 444444444444 4444 4444 4444 4444 4444 4444 Cardiobacterium hominis GT2095 Gm - 4444 44444444 4444 44444444 4444 4444 4444 Francisella tularensis GT2172 Gm - 4444 44444444 44444444 4444 44444444 CM Campylobacter jejuni GT0022 Campy 4444 4444 4444 444444444444 4444 44444444 Ca pylobacter pylori GT0026 44444444 4444 44+4 444444444444 44444444 Campylobacter sputorum GT0027 44444444 4444 4444 4444 4444 Bacillus brevis GT0803 lowG4C 44444444 4444 4444 4444 4444 4444 ++++ 4444 Bacillus subtilis GT0804 Gm 4 444444444444 44444444 4444 +4444444 Clostridiu clostridioforme ATCC25537 44444444 4444 44444444 4444 4444 44444444 Cloεtridium βordellii GT0567 4444 4444 4444 4444 4444 44444444 44444444 Clostridium sporogenes ATCC 3587 4444 4444 4444 4444 4444 444444444444 Clostridium histolyticum ATCC19401 4444 4444 4444 4444 4444 ++++ +4444444444+ Clostridium perfringens ATCC13124 44444444 4444 44+4 4444 4444 4444 4444 ++++ Cloεtridium ramoεum ATCC25582 +444 4444 4444 4444 4444 4+++ ++++ 4+++ ++++ Kurthia zopfii ATCC33403 4444 4444 4444 44++ 4444 44++ 4444 4444 ++++ Lactobacilluε acidophilus GT0256 +444 4+++ 44++ ++++ 4444 ++++ 4444 +++4 ++++ Lactobacillυs plantarum GT0258 +444 444+ +444 ++++ 4444 ++++ 4444 4444 ++++ Listeria monαcytogeneε IG3299 4+++ 444+ +++4 44++ ++++ 44++ 4444 ++++ TABLE 4: DOT BLOT HYBRIDIZATION OF 23S rRNA-TARGETED PROBES PROBE HYBRIDIZATION Genus species strain div 1730 1731 1658 1653 1654 1655 1651 Acinetobacter calcoaceticus GT0002 Purpl 1 4444 444 4444 4444 4444 4444 444 Aeromonas sobria GT0007 gamma n TTiTlT nTTiTTl 4444 444 4444 4444 - Altero onas putrefaciens GT1945 H 4444 444 4444 444 4444 4444 44 Citrobacter amalonaticus GT0690 II 4444 444 4444 444 4444 4444 444 Citrobacter diversus GT0030 H 4444 444 44 4444 4444 4444 44 Citrobacter freundii GT0687 It 4444 4444 4444 444 4444 4444 44 Edwardsiella tarda GT0569 M 4444 4444 4444 4444 4444 4444 4444 Enterobacter agglomeranα GT0683 II 444 444 4444 4444 4444 4444 44 Enterobacter cloacae GT0686 N 4444 4444 4444 4444 4444 4444 444 Enterobacter sakazakii GT0062 N . 444 44 4444 4444 4444 4444 44 Escherichia coli GT1665 M 4444 444 TTTT TTTT TTTT TTTT TTT Escherichia coli GT1592 M 4444 4444 4444 4444 4444 4444 4444 Escherichia coli GT1659 U 44 44 44 4444 444 - 4444 Haemophilus influenza GT0244 N 444 4 44 44 44 - 4444 * Haemophilus influenza ATCC33391 II ND ND ND ND ND ND ND Haemophilus ducreyi GT0243 H 4444 4444 4444 4444 4444 4444 4444 Hafnia alvei GT0241 •1 4444 4444 4444 4444 4444 4444 44 Morganella m rganii GT0303 M 4444 4444 4444 4444 4444 4444 44 -fc. Klebsiella pneumoniae GT1500 H 4444 4444 4444 4444 4444 4444 444 Proteus mirabilis GT1496 H 4444 4444 4444 4444 4444 4444 4444 Providencia alcalifaciens GT0371 N 4444 4444 4444 4444 4444 4444 4444 Pseudomonas aeruginosa GT1909 II 4444 4444 44 4444 4444 4444 444 Salmonella arizona GT0799 II 4444 444 4444 4444 444 4444 444 Salmonella typhimuriu GT0389 II 444 444 4444 4444 444 4444 444 Serratia marcescens GT0392 H 4444 4444 4444 4444 444 4444 4444 Shigella flexneri GT0798 II 4444 4444 4444 444 444 4444 4444 Vibrio parahaemolyticus GT0568 II 4444 4444 4444 444 444 4444 44 Xanthomonas maltophilia GT0417 II 4444 4444 4444 444 444 - 444 Yersinia enterocolitica GT0419 II 4444 4444 4444 4444 444 4444 444 Alcaligines faecalis GT0610 Purple +++ +++ 4 - 444 4444 44 Branha ella catarrhalis GT0014 beta +++ 444 4444 4444 444 4444 4444 Chromobacterium violaceum GT2022 II 4444 44++ 4+++ - 444 4444 444 Klngella indologenes 0246 II +444 444 444 44 44 4444 4 Moraxella osloenβis GT0301 •1 4444 4444 4444 444+ 444 4444 44 TABLE 4 (cont'd): DOT BLOT HYBRIDIZATION OF 23S rRNA-TARGETED PROBES PROBE HYBRIDIZATION Genus species _______ strain div 1730 1731 1658 1653 1654 1655 1651 Morexella phenylpyruvica GT0302 444 444 4 444+ 444 4444 4444 Neisseria gonorrhoeae GT0315 II 444 444 4444 4444 4 Neisseria meningitidis GT0349 H 444444444444 44444444 Pseudomonas acidovorans GT0376 M 44444444 4444 44444444 Pseudomonas cepacia GT2015 M 4444444 444 44 Rhodocycluε gelatinosa ATCC17013 444 444 444 44 Vitreoscilla stercorarla 44444444444 444 Achromobacter xerosis GT0810 Purple 444 444 444 44 Acidiphllium cryptum ATCC33463 alpha 4 - 44 444 Agrobacteriu tumefaciens GT2021 44 44 4444 - 444 Brucella abortus ATCC23448 444444444444 - 4444 Flavobacterium capsulatm GT2025 444 444 4444 - Mycoplana bullata GT2023 4444444 4 Pseudomonas diminuta GT2020 444 444 4 Rhodobacter sphaeroides ATCC17023 444444444444 - - 4444 Rhodospirillum rubrum ATCC25903 44444444444444444444 4444 Thiobacillus versutus ATCC25364 4444 4444444 444 Desulfovibrio desulfuricans ATCC 7757 delta 444 4 44 4 444 444 Cardiobacterium hominis GT2095 Gm - 444 444 444 4 Franciεella tularenεiε GT2172 Gm - 444444444444 44 Campylobacter jejuni GT0022 Campy 444 4 4444 - Campylobacter pylori GT0026 II 44 44 4444 - 444 Campylobacter sputorum GT0027 H 444 44 4444 - Bacillus brevls GT0803 lowG4C 444 444 444444444444 4444 4444 Bacillus subtilis GT0804 Gm 4 444 444 444444444444 4444 4444 Clostridium clostridioforme ATCC25537 4 444444444444 4444 Clostridium sordellii GT0567 4444 4444 44444444 4444 444 Clostridium εporαgenes ATCC 3587 444 444 444444444444 4444 4444 Cloεtridium histolyticum ATCC19401 4444 44 444 444 4444 44 444 Clostridium perfringens ATCC13124 4444 444 444 4444 4444 Clostridium ramoεum ATCC25582 4444 444 4444 Kurthia zopfii ATCC33403 4444 4444444 44444444 444 Lactobacillus acidophilus GT0256 444 444 4 4444 4444 Lactobacillus plantarum GT0258 4444 444 444 44444444 444 444 TABLE 4 (cont'd): DOT BLOT HYBRIDIZATION OF 23S rRNA-TARGETED PROBES PROBE HYBRIDIZATION Genus species strain div 1730 1731 1658 1653 1654 1655 1651 Listeria monocytogenes IG3299 4444 444 4444 4444 4444 444 Mycoplasma pneumoniae ATCC15531 Mycoplasma putrefaciens ATCC15718 Peptostreptococcus productus ATCC27340 44 4444 4444 4444 4444 Planococcus citreus ATCC14404 " Staphylococcus aureus GT0399 " 4444 4444 4444 4444 4444 Staphylococcus aureus GT1711 4444 4444 4444 4444 4444 444 444 Staphylococcus epidermidis GT0401 TTTT 4444 4444 444 4444 444 4444 Staphylococcus haemolyticus ATCC29970 4444 TTTT TTTT 4444 Streptococcus agalactiae GT0405 T*f"T 4444 4444 444 Streptococcus bovis GT0668 444 444 4444 444 4444 Streptococcus faecalis GT0406 TTT 4444 4444 Streptococcus morbillorum GT2194 " 44 444 4444 4444 Streptococcus rautans GT0412 44 4444 4444 444 Streptococcus pneumoniae GT0408 " 4444 TTT -" 444 4444 4444 Streptococcus salivarius GT0410 44 4444 4444 444 GJ Streptococcus sanguis GT0411 444 4444 4444 444 4444 6^ Bifidobacterium dentium GT0012 hiG4C 444 4444 Corynebacterium genitalium GT0045 Gm + 444 4444 4444 44 4444 Crynebacterium glutamlcum GT2120 4444 444 4444 4444 44 4444 Corynebacterium pseudodiphtheriticum GT2119 4444 444 4444 4444 4444 4444 Corynebacterium pseudotuberculosis GT2122 44 4444 44 4444 Corynebacterium pyogenes GT2121 4444 4444 444 444 Corynebacterium xerosis GT0046 4444 4444 44 444 Mycobacterium bovis BCG 4444 4444 44 444 Mycobacterium kansasii 4 444 4444 4444 444 4444 Nocardia asteroides GT2191 4444 4444 444 4444 Rhodocσccus rhodochrouε 444 444 444 4444 444 44 .++++ Aerococcus viridans GT2116 Misc 4444 4444 444 444 444 44 4444 Fusobacterium necrophorum GT0238 Gm 4 4444 4444 - 444 444 4 Fusobacterium prausnitzii ATCC27768 444 44 44 444 4444 Ge ella haemolysans GT2118 4444 4444 4444 44 4444 4444 4444 Heliobaclllus mobilis 444 44 444 4444 4444 Phormidium ectocarpi Cyano 4 444 4444 4444 TABLE 4 (cont'd) : DOT BLOT HYBRIDIZATION OF 2 S rRNA-TARGETED PROBES PROBE HYBRIDIZATION Genus species strain div 1730 1731 1658 1653 1654 1655 1651 Plectonema boryanum n 44 44 - 44 4444 444 - Borrelia burgdorferi Spiro 444 44 44 - - - 444 Borrelia turlcatae ■1 4444 444 4444 - — - 444 Leptospira interrogans-pomona H 444 44 4444 - - - 44 Leptospira biflexa (Patoc-Patoc) It 444 44 4444 - - - 4444 Leptospira biflexa (CDC) n 444 44 4444 - - - 4444 Spirochaeta aurantia II 4444 44 4444 - - ~ 4444 Bacteroides fragilis 25285 Bact 44 44 4 - - - 4444 Bacteroides fragilis 29771 N 44 44 4 - - - 4444 Bacteroides thetaiotaomicron 0572 H 4 4 4 - - - 4444 Bacteroides melaninogenicus 0011 II - - 4 44 44 - 4444 Flavobacterium meningosepticura 0237 4 4 * -** "• 444 Chlamydia psittaci Chlaiα Chla ydia trachomatis LGV II 444 444 - - - - - Chlorobium limicola Misc. 4444 444 4 4444 4444 444 444 Chloroflexus aurantiacus Y400 n 4 - 44 - 44 - 4 G* Deinococcus radiodurans 2608 II 4444 444 444 - - — 4 -_ Planctomyces maris 2577 H - - 444 - - - Normal Stool RNA 444 4+ 444 44 444 44 444 Mouse L-Cell Wheat Germ 44 Normal Human Blood Candida lusitaniae 403-87 Candida parapsilosis 882-88 Candida tropicalis 224-87 Candida albicans 1008-88 Candida albicans 223-87 Candida albicans 819-88 A Inclusivity and Exclusivity data was determined after overnight exposures. AA Each organism is represented by lOOng of CsTFA purified RNA. 4444 = positive level of hybridization, 4 = barely detectable and - =» zero, ND - not done. TABLE 4 (cont'd): DOT BLOT HYBRIDIZATION OF 23S rRNA-TARGETED PROBES PROBE HYBRIDIZATION Genus species strain div 1512 1256 1398 1600 1601 1602 1596 1597 Acihetobacter calcoaceticus GT0002 Purple 44444444 4444 4444 4444 4444 Aero onas sobria GT0007 gamma 4444 4444 4444 4444 4444 4444 Altero onas putrefacienε GT1945 H 4444 4444 44 4444 4444 4444 4444 444 Citrobacter amalonaticus GT0690 M TTTT TTT 4 4444 444 TTTT TTT 444 Citrobacter diversus GT0030 M TTTT TTTT 4 4444 TTT 4444 444 444 Citrobacter freundii GT0687 N 4444 4444 4 4444 444 4444 444 4444 Edwardsiella tarda GT0569 II 4444 4444 4 4444 4444 4444 444 444 Enterobacter agglomerans GT0683 n TmTTTi nTTiTlT 4 444 444 4444 444 44 Enterobacter cloacae GT0686 H 4444 4444 4 4444 444 4444 444 44 Enterobacter sakazakii GT0062 II TTTT TTT 44 444 444 4444 444 44 Escherichia coli GT1665 n 4444 4444 4 444 444 4444 444 4444 Escherichia coli GT1592 ■ 4444 4444 4 4444 4444 4444 4444 4444 Escherichia coli GT1659 w 4444 444 4 444 44 4444 444 -44 Haemophilus influenza GT0244 It 44 444 4 444 44 4444 444 44 Haemophilus influenza ATCC33391 N ND ND ND 444 444 444 444 444 Haemophilus ducreyi GT0243 M 0» 4444 4444 44 ND 44 4444 4444 44 oca Hafnia alvei GT0241 n 4444 444 4 4444 4++ 4444 4444 444 Morganella morganii GT0303 H 4444 4444 4 4444 4444 4444 444 444 Klebsiella pneumoniae GT1500 n 4444 4444 4 4444 444 444 444 444 Proteus mirabilis GT1496 H 4444 4444 4 4444 4444 4444 444 444 Providencia alcalifaciens GT0371 « 4444 4444 4 4444 4444 4444 444 444 Pseudomonas aeruginoεa GT1909 M 4444 4444 44 4444 4444 4444 444 44 Salmonella arizona GT0799 M 4444 4444 4 4444 444 4444 444 44 Salmonella typhimurlum GT0389 H 4444 4444 4 4444 444 4444 444 444 Serratia marcescens GT0392 H 4444 4444 4 4+++ 444 4444 444 44 Shigella flexneri GT0798 « 4444 4444 44 4444 4444 4444 4444 44 Vibrio parahaemolyticus GT0568 II 4444 444 44 444 444 4444 4444 44 Xanthomonas maltophilia GT0417 II 4444 444 44 4444 44 4444 4444 44 Yerεinia enberocolitica GT0419 •1 4444 4444 44 4444 4444 4444 4444 4444 Alcaligenes faecalis GT0610 Purple 4444 444 4 444 4 444 444 44 Branhamella catarrhalis GT0014 beta 4444 444 4 4444 44 444 444 444 Chromobacterium violaceum GT2022 n 4444 4444 .4 4444 4444 444 444 444 Kingella indologenes 0246 II 4444 44 4 444 444 444 444 44 Moraxella osloensis GT0301 H 4444 444 4 4+++ 444 4444 444 44+ TABLE 4 (cont'd): DOT BLOT HYBRIDIZATION OF 23S rRNA-TARGETED PROBES PROBE HYBRIDIZATION Genus species strain div 1512 1256 139 8 1600 1601 1602 1596i 1597 Morexella phenylpyruvica GT0302 4444 4444 4 4444 44 444 444 444 Neisseria gonorrhoeae GT0315 •I 44444444 4 4444 44 4444 4444 • 44 Neisseria meningitidis GT0349 II 4444 4444 4 4444 444 4444 4444 44 Pseudomonas acidovorans GT0376 II 4444 4444 4 4444 444 4444 4444 44 Pseudomonas cepacia GT2015 II 4444 444 4 4444 444 4444 444 44 Rhodocyclus gelatinosa ATCC17013 H 4444 444 4 444 444 4444 444 44 Vitreoscilla stercoraria II 4444 44 4 4444 444 4444 4444 4444 Achromobacter xerosis GT0810 Purple 4444 44 4 444 444 4444 444 444 Acidiphilium cryptum ATCC33463 alpha 4444 44 4 4444 444 4444 4444 4444 Agrobacterium tumefaciens GT2021 II 4444 44 4 44 44 4444 44 444 Brucella abortus ATCC23448 II 4444 4444 4 444 444 4444 444 444 Flavobacterium capsulatm GT2025 II 4444 444 4 4444 444 4444 44 444 Mycoplana bullata GT2023 II 4444444 4 4444444 4444 444 4444 Pseudomonas diminuta GT2020 •1 4444444 4 4444 444 4444 444 444 Rhodobacter sphaeroides ATCC17023 n 4444 444 44 4444 444 4444 444 4+4 Rhodospirilium rubrum ATCC25903 tt 4444 4444 4 4444 444 4444 444 444 Thiobacillus versutus ATCC25364 II 4444 444 4 4444 4444 4444 4444 4444 Desulfσvibrio desulfuricans ATCC 7757 delta 4444 444 4 44444444 4444 4 4 Cardiobacteriu hominis GT2095 Gm - 4444 444 4 4444 444 4444 44 444 Francisella tularensis GT2172 Gm - 4444 444 4 4444 444 4444 4444 4444 Campylobacter jejuni GT0022 ι Campy 4444 - 4444 444 44 4444 444 444 Campylobacter pylori GT0026 II 4444 - 444 4444 444 4444 444 444 Campylobacter sputorum GT0027 II 4444 - 444 4444 444 4444 444 444 Bacillus brevis GT0803 :lowG4C 4444 444 4 4444 444 4444 444 444 Bacillus subtilis GT0804 Gm 4 4444 444 4 4444 444 4444 4444 444 Clostridium clostridioforme ATCC25537 H 4444 444 4 444 44 4444 4444 444 Clostridium sordellii GT0567 U 4444 444 4 444 444 4444 4444 444 Clostridium εporogenes ATCC 3587 II 4444 444 4 444 44 +444 4444 444 Clostridium histolyticum ATCC19401 •1 44++ +++ 4 4 4444 4444 44 Clostridium perf ingens ATCC13124 II ++++ - 4 444 444 4444 4444 44 Clostridium ramosu ATCC25582 H 4444 - 4 44 44 4444 4444 44 Kurthia zopfii ATCC33403 II 4444 4444 4 4444 444 4444 4444 44 Lactobacillus acidophilus GT0256 II 4444 444 4 4444 444 4444 4444 444 Lactobacillus plantarum GT0258 w 4444 444 4 4444 444 4444 444 44 TABLE 4 (cont'd) x DOT BLOT HYBRIDIZATION OF 23S rRNA-TARGETED PROBES PROBE HYBRIDIZATION Genus species _ strain <jv 1512 1256 13981600 1601 1602 1596 1597 Listeria monocytogenes IG3299 4444 444 4 4444 444 4444 44 4444 Mycoplasma pneumoniae ATCC15531 4444 - 4 44 Mycoplasma putrefaciens ATCC15718 4444 - 444 Peptostreptococcus produc us ATCC27340 4444 Planococcus citreus ATCC14404 4 4444 444 4444 4444 444 Staphylococcus aureus GT0399 N 4444 4444 4 4444 444 4444 444 Staphylococcus aureus GT1711 M 4 4444 444 444 444 Staphylococcus epider idis GT0401 II 4444 444 4 4444 444 4444 444 Staphylococcus haemolyticus ATCC29970 II 4444 444 4 4444 444 4444 444 444 Streptococcus agalactiae GT0405 II TTTT TTT 4 4444 444 444 4444 Streptococcus bovis GT0668 4444 44 4 4444 444 4444 444 4444 Streptococcus faecalis GT0406 4444 44 4 4444 4444 444 444 Streptococcus morbilloru GT2194 44+4 444 4 TT TTT 4444 44 +444 Streptococcus mutans GT0412 4444 444 4 4444 444 444 Streptococcus pneumoniae GT0408 4444 444 4 4444 4444 444 444 Streptococcus salivarius GT0410 " 4444 444 4 4444 4444 444 44 Streptococcus sanguis GT0411 ND ND 4 444 444 Bifidobacterium dentiu GT0012 hiG4C 4 4444 444 444 Corynebacterium genitaliura GT0045 Gm 4 4 4444 444 4444 4444 444 Crynebacterium glutamicum GT2120 4 4444 444 4444 4444 Corynebacterium pseudodiphtheriticum GT2119 4444 444 4 4444 444 4444 4444 444 Corynebacterium pεeudotuberculσsis GT2122 4444 4 4444 444 4444 4444 Corynebacterium pyogenes GT2121 44 4 4444 44 4444 ++++ 44++ Corynebacterium xerosis GT0046 4 44 4 44 4444 ++ +++ Mycobacterium bovis BCG 4 44 4 4 4444 ++ +++ Mycobacterium kansasii II 444 444 4 4444 444 4444 +++ +++ Nocardia asteroldes GT2191 4444 4 4444 4444 ++++ +++ Rhodococcus rhodochrous II 44 444 4 4444 44 4444 +++ Aerococcus viridans GT2116 Misc 4444 444 4 4444 44 4444 4444 ++++ Fusobacterium necrophorum GT0238 Gm 4 44 444 4 444 44 4444 4444 +444 Fusobacterium prauεnitzii ATCC27768 4444 4++ 4 4444 444 44++ 4444 4444 Gemella hae olysans GT2118 4444 ' 4 4 4444 444 ++++ 4444 4444 Heliobacillus mobilis •I 4444 444 4 4 ++++ 44 4444 Phormidium ectocarpi Cyano 4444 444 4 4 44 ++++ 44 44 TABLE 4 (cont'd): DOT BLOT HYBRIDIZATION OF 23S rRNA-TARGETED PROBES PROBE HYBRIDIZATION Genus species strain div 151212561398160016011602 15961597 Plectonema boryanum " 4444444 4 4 4 4444 44 44 Borrelia burgdorferi Spiro 4444444 4 444 444 4444444 44 Borrelia turicatae " 44444444 4 4444444444444444 44 Leptospira interrogans-pomona " 4444444 4 444 444 4444444 444 Leptospira biflexa (Patoc-Patoc) " 4444444 4 444 44 4444 44 44 Leptospira biflexa (CDC) " 4444444 4 444 44 4444 44 44 Spirochaeta aurantia " 4444-4444 4 444 44444444 44 44 Bacteroides fragilis 25285 Bact 4444444 4 444 44 4444 44 44 Bacteroides fragilis 29771 " 4444444 4 444 44 4444444 44 Bacteroides thetaiotaomicron 0572 M 4444444 4 4 4 4444 44 44 Bacteroides melaninogenicus 0011 " 4444444 4 . 4 44 4444444 4 Flavobacterium meningoεepticum 0237 " 44444444 4 4444444444444444.4 Chlamydia psittaci - Chlam - 444 - 44444444444 44 -4 Chlamydia trachomatis LGV " - 444 - 4444444 4444 44 4 Chlorobium limicola Misc. 44444444 4 444444444444 4 44 Chloroflexus aurantiacus Y400 " 44 444 4 44 44 4444 44 44 DelnoCOCCUS radiodurans 2608 " 4444 4444 4 4444 4444 4444 4 44 Planctomyces marls 2577 " 4444444 4 444444444444444 Normal Stool RNA 4444444 - 444 444 444444 44 Mouse L-Cell _ - _ -. _ -_. _ _ Wheat Germ - - - - - - - - Normal Human Blood - - - - - - - - Candida lusitaniae 403-87 + - - - - - - - Candida parapsilosis 882-88 + - - - - - - - Candida tropicalis 224-87 + - - - - - - - Candida albicans 1008-88 + - - - - - - - Candida albicans 223-87 + - - - - - - - Candida albicans 819-88 + - - - - - - - A Inclusivity and Exclusivity data was determined after overnight exposures. AA Each organism is represented by lOOng of CεTFA purified RNA. 4444 « poεitive level of hybridization, 4 • = barely detectable and - -» zero, ND - not done. TABLE 5: DOT BLOT HYBRIDIZATION - GRAM POSITIVE & GRAM NEGATIVE PROBES PROBE HYBRIDIZATION 16S RNA-TARGET 23S RNA-TARGET Genus species _ strain div 1744 1745 1746 1657 1656 1598 1599 1595 Acinetobacter calcoaceticus GT0002 Purple 4444 - Aero onas sobria GT0007 gamma 4444 Alteromonas putrefaciens GT1945 4444 - 4444 Citrobacter amalonaticus GT0690 44+4 - 4444 Citrobacter diversus GT0030 4444 Citrobacter freundii GT0687 ' 4444 - 4444 4444 Edwardsie11a tarda GT0569 " 4444 - 4444 4444 Enterobacter agglo erans GT0683 4444 - 44 Enterobacter cloacae GT0686 4444 - 44 4444 Enterobacter sakazakii GT0062 4444 - 4444 4444 Escherichia coli GT1665 4444 - 4444 4444 Escherichia coli GT1592 4444 - 4444 Echerichia coli GT1659 4444 4 44 4444 Haemophilus influenza ATCC33391 4444 4444 4444 4444 Haemophilus ducreyi GT0243 4444 - 4444 4444 Hafnia alvei GT0241 4444 - 4444 444 Morganella morganii GT0303 4444 - 4444 444 Klebsiella pneumoniae GT1500 4444 - 4444 44 Proteus mirabilis GT1496 4444 - 4444 4 444 Providencia alcalifaciens GT0371 4444 - 44 4 444 Pseudomonas aeruginosa GT1909 4444 - 44 4 4444 Salmonella arizona GT0799 4444 - 44 4 44++ Salmonella typhimurium GT0389 4444 - 4444 Serratia marcescens GT0392 4444 - 4444 +++ Shigella flexneri GT0798 4444 - 4444 +++ Vibrio parahaemolyticus GT0568 " 44++ - 4444 4 +++ Xantho onas maltophilia GT0417 4 4 4444 4 +++ Yersinla enterocolitica GT0419 " 4444 - 44+4 4 4444 Alcaligines faecalis GT0610 Purple 4444 4444 +++ Branhamella catarrhaliε GT0014 beta 44++ - 4+++ +++ Chromobacterium violaceura GT2022 ++++ +++ 4444 4 +++ Kingella indologenes 0246 ++++ - 444 +++ Moraxella osloensis GT0301 +4++ - 4444 4 + Morexella phenylpyruvica GT0302 ++++ - 44+4 ++++ TABLE 5: (cont'd) DOT BLOT HYBRIDIZATION - GRAM POSITIVE & GRAM NEGATIVE PROBES PROBE HYBRIDIZATION 16S RNA-TARGET 23S RNA-TARGET Genus species strain div 1744 1745174616571656 15981599 1595 Borrelia burgdorferi Spiro - 4 444 - 4444 44 Borrelia turicatae 44 4444 - 44444444 - 4 Leptospira interrogans-pomona " - 4 - - 44444444 44 4 Leptospira biflexa (Patoc-Patoc) " - 44444444 44 4 Leptospira biflexa (CDC) " - 4444444 44 4 Spirochaeta aurantia " 4444 - 4444 44 4444 4 Bacteroides fragilis 25285 Bact 4444 - - - - - ++++ + Bacteroides fragilis 29771 " ++++ - - - - - ++++ + Bacteroides thetaiotaomicron 0572 " + - - - - - 4444 4 Bacteroides melaninogenicus 0011 " 4444 - - - - - ++++ + Flavobacterium meningosepticum. 0237 " 4444 4 - 444 Chlamydia psittaci Chlamy - - - - - + + ++ Chlamydia trachomatis LGV " - - - - - 44 44. 44 Chlorobium limicola Misc 4444 - 444 - Chloroflexus aurantiacus Y400 " 4444 - 444 - 4444444 Deinococcu3 radiodurans 2608 " 444 - 444 - 4444 4 Planctomyces marls 2577 " - - - - - - - - Normal Stool RNA 444 4444 44 44 444 4 444 44 Mouse L-Cell - - - - - - - ' - Wheat Germ - - - - - - - - Normal Human Blood -, - Candida lusitanlae 403-87 - - - - - - - - Candida parapsilosis 882-88 - - - - - - - - Candida tropicaliε 224-87 - - - - - - - - Candida albicans 1008-88 - - - - - - - - Candida albicans 223-87 - - - - - - - - Candida albicans 819-88 - - - - - "" . ~ A Inclusivity and Exclusivity data was determined after overnight exposures. AA Each organism is represented by lOOng of CsTFA purified RNA. AAA ++++ - positive level of hybridization, + = barely detectable and - = zero. Probe 1746 was hybridized at 37 C and washes at 50 C. TABLE 5: (cont'd) DOT BLOT HYBRIDIZATION - GRAM POSITIVE & GRAM NEGATIVE PROBES PROBE HYBRIDIZATION 16S RNA-TARGET 233 RNA-TARGET Genus species strain div 1744 1745 1746 1657 1656 1598 1599 1595 Mycoplasma pneumoniae ATCC15531 4444 4444 Mycoplasma putrefaciens ATCC15718 4444 444 TTT Peptostreptococcus productus ATCC27340 4444 4444 4444 4444 Plaηococcu3 citreus ATCC14404 4444 4444 4444 4 4444 Staphylococcus aureus GT0399 4444 4444 4 4444 Staphylococcus aureus GT1711 4444 4444 4444 4 4444 Staphylococcus epidermidis GT0401 4444 4444 4444 Staphylococcus baemolyticus ATCC29970 4444 4+44 4444 4444 Streptococcus agalactiae GT0405 4444 4444 4444 4444 - Streptococcus bovis GT0668 4444 4444 4444 4444 Streptococcus faecalis GT0406 4444 4444 - Streptococcus morbillorum GT2194 " 444 4444 Streptococcus mutans GT0412 444 4444 Streptococcus pneumoniae GT0408 4444 4444 4444 4444 4444 Streptococcus salivarius GT0410 TTTT TTTT 4444 4444 4444 Streptococcus sanguis GT0411 4444 4444 4444 4444 4444 Bifidobacterium dentium GT0012 hiG4C 444 4444 4 Corynebacterium genitalium GT0045 Gm 4 4444 4444 4444 4444 4 Crynebacterium glut micum GT2120 4444 4444 4 Corynebacterium pseudodiphtheriticum GT2119 4444 4444 4 TTTT Corynebacterium pseudotuberculosis GT2122 4444 4444 - 444 Corynebacterium pyogenes GT2121 4444 4444 - 4 4444 444 Corynebacterium xerosis GT0046 4444 4444 44 4 Mycobacterium bovis BCG 4444 4444 4444 4444 4 Mycobacterium kansasii 4444 44 4444 4444 4 Nocardia asteroides GT2191 4444 4444 444 44 Rhodococcus rhodochrous 4444 4444 4444 - 4444 4 Aerococcus viridans GT2116 Misc 4444 4444 44 44 4444 Fusobacterium necrophorum GT0238 Gm 4 4444 4444 44 444 44 4444 Fusobacterium prausnitzii ATCC27768 4444 4444 44 44 4 4444 Gemella haemolysans GT2118 4444 4444 4444 - 4444 4444 4444 Heliobacillus mobili3 4444 4444 444 4444 - 4444 Phormidium ectocarpi Cyano 4444 4444 44 4444 Plectonema boryanum 4444 4444 4 - 4444 TABLE 5: (cont'd) DOT BLOT HYBRIDIZATION - GRAM POSITIVE & GRAM NEGATIVE PROBES PROBE HYBRIDIZATION 16S RNA-TARGET 23S RNA-TARGET Genus species strain div 1744 1745 1746 1657 1656 1598 1599 1595 Neisseria gonorrhoeae GT0315 - - 4444 4444 4444 - 4444 Neisseria meningitidis GT0349 H - - — 4444 4444 4444 - 444 Pseudomonas acidovorans GT0376 n - - - 4444 4444 +444 — 444 Pseudomonas cepacia GT2015 w - - 44 4444 4444 - 444 Rhodocyclus gelatinosa ATCC17013 M - - - 4444 - 444 Vitreoscilla stercoraria n - - - 4444 444 - 44 Achromobacter xerosis GT0810 Purple - - 4444 4444 4444 - 44 Acidiphilium cryptum ATCC33463 alpha - - - 444 - - - Agrobacterium tumefaciens GT2021 II 44 - 4444 - 4444 44 44 Brucella abortus ATCC23448 II 44 - 4444 - 4444 ■ 444 444 Flavobacterium capεulatum GT2025 H 4 - 4444 - 44 44 - Mycoplana bullata GT2023 II - - - 4444 - 44 - - Pseudomonas dimlnuta GT2020 M - - .4444 - 444 4 Rhodobacter sphaeroides ATCC17023 H - - 4444 .- 444 - 44 Rhodospirillum rubrum ATCC25903 •1 4444 - - 4444 - 4 4 4+4 Thiobacillus verεutus ATCC25364 n 44 - 4444 - 444 - 44 Desulfovibrio desulfuricans ATCC 7757 delta - - - 444 - 44 "Cardiαbacterium hominiε GT2095 Misc - - - 4444 - 444 - 4 Francisella tularensig GT2172 Gm - - - - 4444 - 444 - 4444 Campylobacter jejuni GT0022 Campy _ - 4444 444 44 - Campylobacter pylori GT0026 •1 - - - 4444 4444 44 4 Campylobacter sputoru GT0027 •I - — - 44 4444 44 - Bacillus brevis GT0803 :lowG4C 4444 4444 4444 4444 4 4444 - Bacillus subtilis GT0804 Gm 4 4444 4444 4444 4444 4 4444 - Clostridium clostrldioforme ATCC25537 II 4444 4444 4444 4444 - 4444 - Clostridium sordellii GT0567 H 4444 4444 4444 4444 - 4444 - Clostridium εporogenes ATCC 3587 II 4444 4444 4444 4444 - 4444 - Clostridium histolyticum ATCC19401 H 4444 4444 4444 4444 - 4444 - Clostridium perfringens ATCC13124 II 4444 4444 4444 4444 - 4444 - Clostridium ramosum ATCC25582 H 4444 44++ 4444 - - 4444 4 Kurthia zopfii ATCC33403 N +444 4444 4444 4444 - 4444 - Lactobacillus acldophilus GT0256 H 4444 4444 4444 444 - 4444 - Lactobacillus plantarum GT0258 M 4444 4444 4+++ 444 4 4444 - Listeria monocytogenes IG3299 II 4444 4444 4444 444 - 4444 -

Claims: What is claimed is: 1. A nucleic acid fragment capable of hybridizing to rRNA or rDNA of eubacteria. 2. The nucleic acid fragment of claim 1 wherein said fragment is not capable of hybridizing to rRNA or rDNA of Mouse L cells, wheat germ, human blood or Candida albicans. 3. The nucleic acid fragment of claim 2, wherein said fragment is complementary to at least 90)5. of a sequence comprising any ten consecutive nucleotides within probes selected from the group consisting of 1638, 1642, 1643, 1738, 1744, 1659, 1660, 1661, 1739, 1740, 1741, 1742, 1745, 1746, 1743, 1637, 1639, 1640, 1641, 1730, 1731, 1658, 1656, 1657, 1653, 1654, 1655, 1651, 1652, 1512, 1256, 1398, 1511, 1595, 1600, 1601, 1602, 1598, 1599, 1596, 1597. 4. The nucleic acid fragment of claim 2, wherein said fragment is homologous to at least 90% of a sequence comprising any ten consecutive nucleotides within probes selected from the group consisting of 1638, 1642, 1643, 1738, 1744, 1659, 1660, 1661, 1739, 1740, 1741, 1742, 1745, 1746, 1743, 1637, 1639, 1640, 1641, 1730, 1731, 1658, 1656, 1657, 1653, 1654, 1655, 1651, 1652, 1512, 1256, 1398, 1511, 1595, 1600, 1601, 1602, 1598, 1599, 1596, 1597. 5. A set of probes comprising at least two nucleic acids fragments, at least one of which is selected from the group consisting of 1638, 1642, 1643, 1738, 1744, 1659, 1660, 1661, 1739, 1740, 1741, 1742, 1745, 1746, 1743, 1637, 1639, 1640, 1641, 1730, 1731, 1658, 1656, 1657, 1653, 1654, 1655, 1651, 1652, 1512, 1256, 1398, 1511, 1595, 1600, 1601, 1602, 1598, 1599, 1596, 1597 and their complementary sequences. -77 6. The nucleic acid fragment of claim 1 which is probe 1638 or its complementary sequence. 7. The nucleic acid fragment of claim 1 which is probe 1642 or its complementary sequence. 8. The nucleic acid fragment of claim 1 which is probe 1643 or its complementary sequence. 9. The nucleic acid fragment of claim 1 which is probe 1738 or its complementary sequence. 10. The nucleic acid fragment of claim 1 which is probe 1744 or its complementary sequence. 11. The nucleic acid fragment of claim 1 which is probe 1659 or its complementary sequence. 12. The nucleic acid fragment of claim 1 which is probe 1660 or its complementary sequence. 13. The nucleic acid fragment of claim 1 which is probe 1661 or its complementary sequence. 14. The nucleic acid fragment of claim 1 which is probe 1739 or its complementary sequence. 15. The nucleic acid fragment of claim 1 which is probe 1740 or its complementary sequence. 16. The nucleic acid fragment of claim 1 which is probe 1741 or its complementary sequence. 17. The nucleic acid fragment of claim 1 which is probe 1742 or its complementary sequence. <5 18. The nucleic acid fragment of claim 1 which is probe 1745 or its complementary sequence. 19. The nucleic acid fragment of claim 1 which is probe 1746 or its complementary sequence. 20. The nucleic acid fragment of claim 1 which is probe 1743 or its complementary sequence. 21. The nucleic acid fragment of claim 1 which is probe 1637 or its complementary sequence. 22. The nucleic acid fragment of claim 1 which is probe 1639 or its complementary sequence. 23. The nucleic acid fragment of claim 1 which is probe 1640 or its complementary sequence. 24. The nucleic acid fragment of claim 1 which is probe 1641 or its complementary sequence. 25. The nucleic acid fragment of claim 1 which is probe 1730 or its complementary sequence. 26. The nucleic acid fragment of claim 1 which is probe 1731 or its complementary sequence. 27. The nucleic acid fragment of claim 1 which is pjrobe 1658 or its complementary sequence. 28. The nucleic acid fragment of claim 1 which is probe 1656 or its complementary sequence. < ? 29. The nucleic acid fragment of claim 1 which is probe 1657 or its complementary sequence. 30. The nucleic acid fragment of claim 1 which is probe 1653 or its complementary sequence. 31. The nucleic acid fragment of claim 1 which is probe 1654 or its complementary sequence. 32. The nucleic acid fragment of claim 1 which is probe 1655 or its complementary sequence. 33. The nucleic acid fragment of claim 1 which is probe 1651 or its complementary sequence. 34. The nucleic acid fragment of claim 1 which is probe 1652 or its complementary sequence. 35. The nucleic acid fragment of claim 1 which is probe 1512 or its complementary sequence. 36. The nucleic acid fragment of claim 1 which is probe 1256 or its complementary sequence. 37. The nucleic acid fragment of claim 1 which is probe 1398 or its complementary sequence. 38. The nucleic acid fragment of claim 1 which is probe 1511 or its complementary sequence. 39. The nucleic acid fragment of claim 1 which is probe 1595 or its complementary sequence. 40. The nucleic acid fragment of claim 1 which is probe 1600 or its complementary sequence. 56 41. The nucleic acid fragment of claim 1 which is probe 1601 or its complementary sequence. 42. The nucleic acid fragment of claim 1 which is probe 1602 or its complementary sequence. 43. The nucleic acid fragment of claim 1 which is probe 1598 or its complementary sequence. 44. The nucleic acid fragment of claim 1 which is probe 1599 or its complementary sequence. 45. The nucleic acid fragment of claim 1 which is probe 1596 or its complementary sequence. 46. The nucleic acid fragment of claim 1 which is probe 1597 or its complementary sequence. 47. A method of detecting the presence of eubacteria in a sample comprising: a) contacting said sample with at least one nucleic acid fragment under conditions that allow said fragment to hybridize to rRNA or rDNA of said eubacteria, if present in said sample, to form hybrid nucleic acid complexes not to rRNA or rDNA of non- eubacteria; and b) detecting said hybrid nucleic acid complexes as an indication of the presence of said eubacteria in said sample. 48. The method of claim 47 wherein said nucleic acid fragment of said contacting step is selected from the group of probes consisting of 1638, 1642, 1643, 1738, 1744, 1659, 1660, 1661, 1739, 1740, 1741, 1742, 1745, 1746, 1743, 1637, 1639, 1640, 1641, 1730, 1731, 1658, 1656, 1657, 1653, 1654, 1655, 1651, 1652, 1512, 1256, 1398, 1511, 1595, 1600, 1601, 1602, 1598, 1599, 1596, 1597. 5 / 49. The method of claim 47 wherein said eubacteria are gram-positive and said nucleic acid fragment is selected from the group of probes consisting of 1599, 1656, 1744, 1745 and 1746. 50. The method of claim 47 wherein said eubacteria are gram-negative and said nucleic acid fragment is selected from the group of probes consisting of 1599 . 1656, 1744, 1745 and 1746. 51. The method of claim 47 wherein said contacting step involves a nucleic acid fragment selected from the group consisting of probe 1638, probe 1642 and probe 1643 and said detecting step involves further contacting said sample with a second nucleic acid fragment selected from the group of probes consisting of 1637, 1639, 1640 and 1641 and amplifying eubacterial 16S rRNA or 16S rRNA gene sequences by the polymerase chain reaction.

Inventors: BUHARIN AMELIA, LANE DAVID J, SHAH JYOTSNA, WEISBURG WILLIAM G, BUHARIN, AMELIA, LANE, DAVID, J., SHAH, JYOTSNA, WEISBURG, WILLIAM, G.
Applicants: GENE TRAK SYSTEMS, GENE-TRAK SYSTEMS
Application year: 1990
Main classification: C12Q1
IPC codes: C12N15, C12Q1