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United States Patent m
Pelletier et al.
 NUCLEIC ACID PROBES FOR THE
DETECTION OF BACTERIA OF THE GENUS
LEGIONELLA AND METHODS FOR THE
DETECTION OF THE ETIOLOGICAL
AGENTS OF LEGIONNAHtES' DISEASE
 Inventors: Dale A. Pelletier, Southborough;
William G. Weisburg, Milford, both of
 Assignee: Amoco Corporation, Chicago, 111.
 Appl. No.: 309,560
 Filed: Sep. 20,1994
Related U.S. Application Data
 Continuation of Ser. No. 66,373, May 24,1993, abandoned.
 Int. CI.6 C12Q 1/68; C07H 21/04
 U.S. CI 435/6; 536/24.3; 536/24.32;
 Field of Search 435/6; 536/24.32,
536/23.1, 23.7, 24.3, 24.33; 935/78
 References Cited
U.S. PATENT DOCUMENTS
[ii] Patent Number: 5,569,586
 Date of Patent: Oct. 29, 1996
FOREIGN PATENT DOCUMENTS
WO88/03957 6/1988 WIPO .
Fox et al., Syst. Appl. Microbiol. 11, 135-139 (1989).
Fox et al., Syst. Appl. Microbiol 14, 52-56 (1991).
FEMS Microbiol. Lett., Bottger, vol. 65, 171-176 (1989).
Am. J. Clin. Path., Fain et al., vol. 95(5), 719-724 (1991).
Primary Examiner—Kenneth R. Horlick
Attorney, Agent, or Firm—Norval B. Galloway
Nucleic acid sequences which hybridize preferentially to the 16S or 23S rRNA or rDNA of Legionella sp., L. pneumophila, L. micdadei, a L. pneumophila, or a Legionella subset are taught. These organisms are the etiological agents of Legionnaires' disease, Pontiac fever, Pittsburgh pneumonia and other infections. The nucleic acids are useful in the detection of these pathogenic microorganisms. Probes based on these sequences and kits containing the probes are also disclosed.
19 Claims, 1 Drawing Sheet
Legionella were first discovered in 1976 following an outbreak of 182 cases of pneumonic illness (termed Legionnaires' disease) occurred at a state convention of the American Legion in Philadelphia, Pa., which resulted in 29 deaths. 20 What is now known as L. pneumophila serotype 1 was originally isolated by standard techniques for the isolation of rickettsiae and subsequently shown to be the etiological agent for Legion-naires' disease, a form of atypical pneumonia with other non-respiratory complications. Since that 25 first outbreak, Legionellae have been implicated in Pontiac fever and Pittsburgh pneumonia. Pontiac fever is a nonpneumonic febrile self-limiting illness caused by L. pneumophila. Pittsburgh pneumonia is a pulmonary legionellosis caused by L. micdadei. 30
Legionellae are gram negative, aerobic, facultative intracellular, parasitic bacteria found to be practically ubiquitous in fresh water supplies (including evaporative condensers, cooling towers, and potable water). It is believed that Legionellae cause disease when contaminated water is 35 inhaled, often leading to epidemic or clustered outbreaks. In addition to community-acquired cases, legionellae may be a major cause of nosocomial infections. Along with hospitalization, host risk factors include smoking, advanced age, chronic lung disease, and immunosuppression. 40
The family Legionellaceae contains the single genus Legionella which includes some 29 species, 21 serogroups and 5 tentatively named species. According to DNA homology studies, L micdadei is the most distant relative of L. pneumophila, and there is some who argue that L. micdadei 45 should be properly classified as a member of the genus Tatlockia.
L. pneumophila, the primary cause of Legionnaire's disease, is the most common human isolate. The presence of 5Q legionellae in human clinical samples always provides clinically relevant information, as the bacteria are not considered normal human microflora.
Legionellae are slow growing organisms which are difficult to culture. Thus, isolation of L. pneumophila by labo- 55 ratories can be difficult and time-consuming. Given the serious nature of the diseases, and the need to prescribe correct antibiotics, it is highly desirable for a physician to make a rapid and accurate diagnosis of the presence of these organisms. Current methods of detection of Legionellae go include (a) culture; (b) direct fluorescence antibody (DFA); (c) nucleic acid probes for culture confirmation; and (d) serology (TEA). Serology is currently the most sensitive and specific test method. It is limited however, in that antibodies may persist in an individual's serum for years after infection. 55
Certain probes which are based on Legionella rRNAs are disclosed in WO 88/03957 "Nucleic Acid Probes for Detec
tion and/or Quantification of Non-Viral Organisms", published Jun. 2,1988, Applicant: Gen-Probe, Inventors: Hogan et al. This application however, discloses a mixture of three different probes which can be used to differentiate Legionella bacteria from non-Legionella bacteria. No probes suitable for solitary use are reported. It would be desirable to have probes which, when used singly or in pairs can be used in various diagnostic assays involving Legionella.
DESCRIPTION OF THE INVENTION
One aspect of this invention is to provide nucleic acids complementary to unique nucleic acid sequences within the ribosomal RNA (rRNA) of legionellae, and which can be used either singly or in pairs. It is a further aspect of the invention to provide for probes which either (1) specifically discriminate between L. pneumophila and other Legionellas; (2) specifically discriminate between a group of bacteria comprised of L. pneumophila and other species of Legionella (a Legionella cluster) and other bacteria; (3) specifically discriminate between L. micdadei and other Legionelias; or (4) specifically discriminate between Legionella and other genera.
Bacterial ribosomes contain three distinct RNA molecules which, at least in Escherichia coli are referred to as 5S, 16S, and 23S rRNAs. In eukaryotic organisms, there are four distinct rRNA species, generally referred to as 5S, 18S, 28S and 5.8S. These names are historically related to the size of the RNA molecules, as determined by their sedimentation rate. In actuality, however, rRNA molecules vary substantially in size between organisms. This notwithstanding, 5S, 16S and 23 S rRNA are art-recognized names referring to rRNA molecules in any bacteria, including the legionellae and this convention will be used herein.
The probes of the present invention target either the 16S or the 23S rRNA molecules of various organisms of the genus Legionella.
DESCRIPTION OF THE FIGURES
FIG. 1 is a diagram of a sandwich assay.
As used throughout the application and claims, the term "probe" will refer to synthetic or biologically produced nucleic acids, between 10 and 250 base pairs in length, which by design or selection, contain specific nucleotide sequences that allow specific and preferential hybridization under predetermined conditions to target nucleic acid sequences, and optionally contain a moiety for detection or for enhancing assay performance. A minimum of ten nucleotides is generally necessary in order to statistically obtain specificity and form stable hybridization products, and a maximum of 250 nucleotides generally represents an upper limit for sequences in which reaction parameters can be adjusted to determine mismatched sequences and preferential hybridization. Therefore, in general, a preferred length of a probe will be between 10 and 250 nucleotides. Probes may optionally contain certain constituents that pertain to their proper or optimal functioning under certain assay conditions. For example, probes may be modified to improve their resistance to nuclease degradation (such as by end-capping), to carry detection ligands (such as fluorescein, 32p, biotin, etc.) or to facilitate their capture onto a solid support (e.g. poly-deoxyadenosine "tails").
"Preferential hybridization" or "hybridizing preferentially" means that hybridization with the intended target nucleic acid results in a hybridization reaction product which is more stable than any hybridization reaction prod15
ucts resulting from hybridization with a non-target nucleic acid under identical conditions. It is well within the skill of the ordinary artisan to compare stability of hybridization reaction products and evaluate which one is more stable, i.e. determine which one has bound "preferentially". 5
As used herein, the terms "homology" and "homologous to" are meant to refer to the degree of similarity between two or more nucleic acid sequences, and is not meant to imply any taxonomic relatedness between organisms. The degree of similarity is expressed as a percentage, i.e. 90% homol- 10 ogy between two sequences will mean that 90% of the bases of the first sequence are identically matched to the bases of the second sequence.
"Legionella cluster" means at least two members of the genus Legionella. Probes which identify a Legionella cluster will typically hybridize to rRNA of a plurality of Legionella species tested, (although not all Legionella).
"L. pneumophila cluster" means at least two strains of the species L. pneumophila. Probes which identify a L. pneu- 2Q mophila cluster will typically hybridize to rRNA of a plurality of L. pneumophila strains tested (although not all L. pneumophila).
"Specific" means that a nucleotide sequence will hybridize to a predetermined target sequence and will not substan- 25 tially hybridize to a non-target sequence.
"Specifically discriminate" means that a probe will substantially hybridize to a predetermined target sequence and will not substantially hybridize to a non-target sequence.
"Hybridization" is a process by which, under predeter- 30 mined reaction conditions, two partially or completely complementary strands of nucleic acid are allowed to come together in an antiparallel fashion to form a double stranded nucleic acid with specific and stable hydrogen bonds, following explicit rules pertaining to which nucleic acids bases 35 may pair with one another.
"Substantial hybridization" means that the amount of hybridization observed will be such that one observing the results would consider the result positive in a clinical setting Data which is considered "background noise" is not substantial hybridization.
"Stringent hybridization conditions" means approximately 35° C. to 65° C. in a salt solution of approximately 0.9 molar NaCl. Stringency may also be governed by such 45 reaction parameters as the concentration and type of ionic species present in the hybridization solution, the types and concentrations of denaturing agents present, and the temperature of hybridization. Generally as hybridization conditions become more stringent, longer probes are preferred if 5Q stable hybrids are to be formed. As a rule, the stringency of the conditions under which a hybridization is to take place 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. 55
"Legionella sp." refers to any member of the genus Legionella, regardless of the species.
In accordance with this invention, there are provided nucleic acids having approximately 10 to 250 nucleotides which hybridize preferentially to rRNA or rDNA of a target 60 organism selected from the group consisting of 1) L. pneumophila, 2) L. micdadei, 3) a Legionella cluster, or 4) a L pneumophila cluster, or 5) all species of the genus Legionella. Under those same hybridization conditions, the nucleic acids of this invention do not substantially hybridize 65 to the rRNA or rDNA of non-target organisms, or the host or environmental matrix which may be present in test samples.
Probes which specifically discriminate between L. pneumophila and other Legionella species are useful in the diagnosis of Legionnaires' disease or Pontiac fever. Probes which specifically discriminate between L. micdadei and other Legionella species are useful in the diagnosis of Pittsburgh pneumonia. Probes which specifically hybridize to a Legionella cluster are useful in detecting the presence of one or more organisms which makes up the particular cluster of bacteria. Probes which specifically discriminate between members of the genus Legionella and non-Legionella bacteria are useful in detecting the presence or absence of one or organisms belonging to the genus Legionella. Probes which specifically hybridize to a L. pneumophila cluster are useful in determining which strains (serotypes) of L. pheumophila are present. Probes which are either complementary to or at least 90% homologous to at least ten consecutive nucleic acids of the aforementioned nucleotides also form another aspect of this invention.
One embodiment of the nucleic acids and probes of this invention are those which are complementary to, at least 90% homologous with, or hybridize preferentially with regions of 16S rRNA or rDNA of either 1) L. pneumophila, 2) L. micdadei, 3) a Legionella cluster, 4) a L. pneumophila cluster, or 5) all Legionella species. The regions of 16S rRNA of particular interest include those indicated below. The numbering of these regions is by reference to the numbering used for E. coli rRNA designations.
L. pneumophila 16S rRNA positions 60 to 110, 1105 to 1165, and 1250 to 1315;
L. micdadei 16S rRNA positions 60 to 110, and 815 to 875;
Legionella sp. 16S rRNA positions 205 to 255, 425 to 475, 715 to 765, and 845 to 895 (for Legionella cluster probes);
Legionella sp. 120 to 175, and 800 to 870 (for Legionella genus probes).
Another embodiment of this invention includes nucleic acids and probes which are complementary to, at least 90% homologous with, or hybridize preferentially with regions of 23 S rRNA or rDNA of either 1) a L. pneumophila cluster, 2) L. micdadei, 3) a Legionella cluster, or 4) all Legionella species. The regions of 23S rRNA of particular interest include:
L. micdadei 23S rRNA positions 285 to 335, 1195 to 1245, 1485 to 1565, and 1705 to 1755.
Legionella sp. 23S rRNA positions 285 to 335, 1485 to 1560 and 1705 to 1755 (for Legionella cluster probes)
Legionella sp. 23S rRNA positions 1565 to 1615 and 2270 to 2310 (for Legionella genus probes).
Preferably the nucleic acid composition is complementary to or homologous with at least 90% of a sequence comprising any ten consecutive nucleotides within sequences selected from the group of sequences defined by the group of probes consisting of: 2701,2703,2704,2705,2697,2690, 2698, 2695, 2696, 2693, 2708, 2699, 2924, 2926, 2930, 2932, 2956, 2958, 2963, 2968, 2928, 2957, 2927, 2929, 2954, 2955, and 2959. The sequences of these probes are presented below.
A further embodiment of this invention includes a kit for the detection of either 1) L. pneumophila, 2) L. micdadei, 3) a Legionella cluster, 4) a L. pheumophila cluster, or 5) any Legionella species. The kit comprises a set of nucleic acids comprising at least two nucleic acids. Each nucleic acid is 10 to 250 nucleotides in length and is of a different base sequence composition. Each nucleic acid is complementary