Method for detecting legionella genus

Abstract

The present invention provides a method of detecting Legionella genus for determining whether a patient exhibiting a pneumonia-like symptom will benefit from treatment with a therapeutic agent that inhibits Legionella sp. The present invention provides a method of detecting Legionella genus for determining whether a patient exhibiting a pneumonia-like symptom will benefit from treatment with a therapeutic agent that inhibits Legionella sp. These methods are based on the detection of Legionella species and / or Legionella pneumophila (Legionella pnemophila) in a biological sample. Kits for practicing the methods are also provided.

Description

This application is a divisional application of the invention application with a filing date of January 30, 2020, Chinese application number 202080023742.X, and invention name “Method for detecting Legionella”. Technical Field

[0001] The present disclosure provides methods for determining whether a patient exhibiting pneumonia-like symptoms will benefit from treatment with a therapeutic agent that inhibits Legionella sp. and / or Legionella pneumophila. These methods are based on detecting Legionella sp. and Legionella pneumophila in a biological sample by determining the presence of ssrA genes and 16S genes, respectively. A kit for practicing the method is also provided. Background Art

[0002] The following description of the background art of the present disclosure is provided only to help readers understand the present disclosure, and it is not admitted that the description describes or constitutes the prior art of the present disclosure.

[0003] Legionellae are facultative intracellular Gram-negative bacteria found in soil and water environments where they can parasitize and proliferate within protozoa. As a result, they are common contaminants of man-made water systems, including air conditioning systems, cooling towers, and hot tubs. Legionellae are also able to replicate in mammalian alveolar macrophages and epithelial cells. Once aerosolized, the bacteria enter the human respiratory tract and cause community-acquired, travel-acquired, and hospital-acquired pneumonia. In some cases, Legionellae can cause Legionnaires' disease (a severe form of pneumonia) or Pontiac fever (a milder flu-like illness).

[0004] There are currently 50 known species in the genus Legionella, which encompass approximately 70 different serogroups. Serogroup 1 of Legionella pneumophila accounts for the majority of human infections, but more than 20 species within this genus have been reported to be associated with human disease. Identification of infections caused by L. pneumophila is particularly important because this species of Legionella is associated with disease that causes severe morbidity and mortality.

[0005] Culture is considered the "gold standard" for detecting Legionella. However, Legionella is a slow-growing and fastidious organism. Serologic diagnosis is also commonly used; however, serologic diagnosis can only be performed retrospectively and rarely affects patient treatment. Urine antigen testing is a rapid test for diagnosing Legionnaires' disease, but is limited by its ability to detect only a limited number of L. pneumophila serogroups. Existing nucleic acid amplification tests are prone to detecting non-Legionella bacteria, including Pseudomonas species and Enterobacter species.

[0006] Therefore, there is a great need for more robust, sensitive, and specific methods that can rapidly detect and differentiate Legionella species and L. pneumophila in a single biological sample. Summary of the invention

[0007] The present disclosure provides compositions and methods for detecting and distinguishing Legionella species and Legionella pneumophila in a single biological sample. In another aspect, the methods and compositions of the present technology can be used to select the best treatment for a subject exhibiting pertussis-like symptoms. Nucleic acid amplification tests (including assays using real-time PCR) are attractive tools for detecting Legionella in clinical specimens because they are able to detect all Legionella and provide rapid results. These tests are also able to distinguish Legionella pneumophila from non-Legionella pneumophila species. This is important in view of the greater morbidity and mortality that have been observed in the case of Legionella pneumophila.

[0008] Thus, in some aspects, provided herein are methods for detecting the presence of at least one Legionella species in a biological sample, the methods comprising, consisting of, or consisting essentially of: (a) providing a first primer pair suitable for amplifying a ssrA target nucleic acid; providing a second primer pair suitable for amplifying a 16S rRNA target nucleic acid; amplifying the ssrA target nucleic acid and the 16S rRNA target nucleic acid, if present; and detecting one or more amplification products produced in step (c); wherein the presence of the ssrA target nucleic acid identifies the presence of at least one Legionella species, and the presence of the 16S rRNA target nucleic acid identifies the presence of Legionella pneumophila.

[0009] In some embodiments of the methods provided herein, the first primer pair comprises at least one degenerate primer. In some embodiments, the first primer pair comprises a first forward primer, and the first forward primer comprises 5'TCGACGTGGGTTGCRAAACG 3' (SEQ ID NO: 1) or its complement. According to the method according to any one of the preceding claims, the first primer pair comprises a first reverse primer, and the first reverse primer comprises 5'TATGACCGTTGATTCGATACC 3' (SEQ ID NO: 2) or its complement. In some embodiments, the second primer pair comprises at least one degenerate primer. In some embodiments, the second primer pair comprises a second forward primer, and the second forward primer comprises 5'TACCTACCCTTGACATACAGTG 3' (SEQ ID NO: 4) or its complement. In some embodiments, the second primer pair comprises a second reverse primer, and the second reverse primer comprises 5'CTTCCTCCGGTTTGTCAC 3' (SEQ ID NO: 5) or its complement.

[0010] In some embodiments, the method further comprises contacting the biological sample with one or more oligonucleotide probes capable of specific hybridization with an amplification product or its complement. In some embodiments, the oligonucleotide probe is detectably labeled. In some embodiments, the detectable label is a fluorescent label. In some embodiments, the fluorescent label is selected from fluorescein, Cy3, Cy5, Cy5.5, tetrachloro-6-carboxyfluorescein, 2,7-dimethoxy-4,5-dichloro-6-carboxyl-fluorescein, Yajiwa yellow (Yakima Yellow), Texas Red, TYE 563, ROX, TEX 615, TYE 665, TYE 705 and hexachloro-6-carboxyfluorescein. In some embodiments, the oligonucleotide probe also includes at least one quencher. In some embodiments, the quencher is selected from TAMRA, Black Hole quencher, Deep Dark quencher, ZEN, Iowa Black FQ, Iowa Black RQ and DABCYL. In some embodiments, the oligonucleotide probe specifically hybridizes to the ssrA amplification product, and wherein the oligonucleotide probe comprises 5'TAAATATAAATGCAAACGATGAAAACTTTGC3' (SEQ ID NO: 3) or its complement. In some embodiments, the oligonucleotide probe specifically hybridizes to the 16S rRNA amplification product, and wherein the oligonucleotide probe comprises 5'CCAGCATGTGATGGTGGGGACTCTA 3' (SEQ ID NO: 6) or its complement.

[0011] In some embodiments, the method further comprises mixing the exogenous control DNA with the biological sample. In some embodiments, the method further comprises contacting the biological sample with a third primer suitable for amplifying an exogenous control target nucleic acid, and amplifying the exogenous control target nucleic acid. In some embodiments, the exogenous control target nucleic acid comprises SEQ ID NO: 20. In some embodiments, the third primer is made up of a third forward primer comprising 5'GCTTCAGTACCTTCGGCTTG 3' (SEQ ID NO: 17) and a third reverse primer comprising 5'TTGCAGGCATCTCTGACAAC 3' (SEQ ID NO: 18). In some embodiments, the method further comprises contacting the biological sample with a third oligonucleotide probe, wherein the third oligonucleotide probe is detectably labeled and comprises 5'TGGCTCTTGGCGGTCCAGATG 3' (SEQ ID NO: 19).

[0012] In some embodiments of the methods provided herein, real-time PCR amplification is performed in a direct amplification plate in conjunction with an integrated thermal cycler.

[0013] In some embodiments of the methods provided herein, the biological sample is a bronchoalveolar lavage sample, a bronchial wash sample, a sputum sample, a nasopharyngeal (NP) aspirate or wash sample, a nasal swab, or a bacterial isolate.

[0014] In another aspect, provided herein is a kit for detecting the presence of at least one Legionella species in a biological sample, the kit comprising, consisting of, or consisting essentially of: (a) a first primer pair that amplifies a ssrA target nucleic acid; (b) a second primer pair that amplifies a 16S rRNA target nucleic acid; (c) a first oligonucleotide probe that is capable of specifically hybridizing to a segment of the ssrA target nucleic acid; and (d) a second oligonucleotide probe that is capable of specifically hybridizing to a segment of the 16S rRNA target nucleic acid; wherein the first oligonucleotide probe and the second oligonucleotide probe are detectably labeled.

[0015] In some embodiments of the kits provided herein, the kits further comprise a third primer pair for amplifying a control target nucleic acid. In some embodiments, the first primer pair is capable of specifically hybridizing to a ssrA target nucleic acid comprising nucleotides at least 85%-95% identical to SEQ ID NO:7 or its complement. In some embodiments, the second primer pair is capable of specifically hybridizing to a 16S rRNA target nucleic acid comprising nucleotides at least 85%-95% identical to SEQ ID NO:8 or its complement.

[0016] In some embodiments of the kit provided herein, the first primer pair comprises at least one degenerate primer. In some embodiments, the first primer pair comprises a first forward primer, and the first forward primer comprises 5'TCGACGTGGGTTGCRAAACG 3' (SEQ ID NO: 1) or its complement. In some embodiments, the first primer pair comprises a first reverse primer, and the first reverse primer comprises 5'TATGACCGTTGATTCGATACC 3' (SEQ ID NO: 2) or its complement. In some embodiments, the second primer pair comprises at least one degenerate primer. In some embodiments, the second primer pair comprises a second forward primer, and the second forward primer comprises 5'TACCTACCCTTGACATACAGTG 3' (SEQ ID NO: 4) or its complement. In some embodiments, the second primer pair comprises a second reverse primer, and the second reverse primer comprises 5'CTTCCTCCGGTTTGTCAC 3' (SEQ ID NO: 5) or its complement. In some embodiments, the first nucleic acid probe comprises 5'TAAATATAAATGCAAACGATGAAAACTTTGC 3' (SEQ ID NO: 3) or its complement. In some embodiments, the second nucleic acid probe comprises 5'CAACCAGCCGCTGCTGACGGTC 3' (SEQ ID NO: 9) or its complement.

[0017] In some embodiments of the kit provided herein, the detectable label is a fluorescent label. In some embodiments, the fluorescent label is selected from fluorescein, Cy3, Cy5, Cy5.5, tetrachloro-6-carboxyfluorescein, 2,7-dimethoxy-4,5-dichloro-6-carboxyl-fluorescein, Yakiwa Yellow, Texas Red, TYE 563, ROX, TEX 615, TYE 665, TYE705 and hexachloro-6-carboxyfluorescein. In some embodiments, at least one oligonucleotide probe also includes at least one quencher. In some embodiments, the oligonucleotide probe includes two quenchers. In some embodiments, the quencher is selected from TAMRA, Black Hole quencher, Deep Dark quencher, ZEN, Iowa Black FQ, Iowa BlackRQ and DABCYL.

[0018] In one aspect, there is provided herein a composition comprising a detectably labeled oligonucleotide probe comprising 5'TAAATATAAATGCAAACGATGAAAACTTTGC 3' (SEQ ID NO: 3). In some embodiments, the detectable label is a fluorescent label. In some embodiments, the fluorescent label is selected from fluorescein, Cy3, Cy5, Cy5.5, tetrachloro-6-carboxyfluorescein, 2,7-dimethoxy-4,5-dichloro-6-carboxyfluorescein, Yakima Yellow, Texas Red, TYE 563, ROX, TEX 615, TYE 665, TYE 705 and hexachloro-6-carboxyfluorescein. In some embodiments, the oligonucleotide probe also includes at least one quencher. In some embodiments, the quencher is selected from TAMRA, Black Hole quencher, Deep Dark quencher, ZEN, Iowa Black FQ, Iowa Black RQ and DABCYL.

[0019] Also provided herein are methods for selecting a subject exhibiting pneumonia-like symptoms for treatment with a therapeutic agent that inhibits Legionella pneumophila, the method comprising, consisting of, or consisting essentially of: (a) contacting a sample isolated from the subject with a first primer pair suitable for amplifying a ssrA target nucleic acid; (b) contacting the sample with a second primer pair suitable for amplifying a 16S rRNA target nucleic acid; (c) amplifying the ssrA target nucleic acid and the 16S rRNA target nucleic acid, if present; and (d) detecting one or more amplification products produced in step (c); and (e) if an amplification product of the 16S rRNA target nucleic acid is detected, selecting the subject for treatment with a therapeutic agent that inhibits Legionella pneumophila.

[0020] In some aspects, provided herein are methods of treating a subject having a Legionella pneumophila infection, the methods comprising administering to a subject selected by a method comprising, consisting of, or consisting essentially of: (a) contacting a sample isolated from the subject with a first primer pair suitable for amplifying a ssrA target nucleic acid; (b) contacting the sample with a second primer pair suitable for amplifying a 16S rRNA target nucleic acid; (c) amplifying the ssrA target nucleic acid and the 16S rRNA target nucleic acid, if present; and (d) detecting one or more amplification products produced in step (c); and (e) if an amplification product of the 16S rRNA target nucleic acid is detected, selecting the subject for treatment with the Legionella pneumophila inhibitory therapeutic.

[0021] In some embodiments of the methods provided herein, the first primer pair comprises at least one degenerate primer. In some embodiments, the first primer pair comprises a first forward primer, and the first forward primer comprises 5'TCGACGTGGGTTGCRAAACG 3' (SEQ ID NO: 1) or its complement. According to the method according to any one of the preceding claims, the first primer pair comprises a first reverse primer, and the first reverse primer comprises 5'TATGACCGTTGATTCGATACC 3' (SEQ ID NO: 2) or its complement. In some embodiments, the second primer pair comprises at least one degenerate primer. In some embodiments, the second primer pair comprises a second forward primer, and the second forward primer comprises 5'TACCTACCCTTGACATACAGTG 3' (SEQ ID NO: 4) or its complement. In some embodiments, the second primer pair comprises a second reverse primer, and the second reverse primer comprises 5'CTTCCTCCGGTTTGTCAC 3' (SEQ ID NO: 5) or its complement.

[0022] In some embodiments, the method further comprises contacting the biological sample with one or more oligonucleotide probes that can specifically hybridize with amplification products or their complements. In some embodiments, the oligonucleotide probe is detectably labeled. In some embodiments, the detectable label is a fluorescent label. In some embodiments, the fluorescent label is selected from fluorescein, Cy3, Cy5, Cy5.5, tetrachloro-6-carboxyfluorescein, 2,7-dimethoxy-4,5-dichloro-6-carboxyl-fluorescein, Yakima yellow, Texas red, TYE 563, ROX, TEX 615, TYE 665, TYE 705 and hexachloro-6-carboxyfluorescein. In some embodiments, the oligonucleotide probe also includes at least one quencher. In some embodiments, the quencher is selected from TAMRA, Black Hole quencher, Deep Dark quencher, ZEN, Iowa Black FQ, Iowa Black RQ and DABCYL. In some embodiments, the oligonucleotide probe specifically hybridizes to the ssrA amplification product, and wherein the oligonucleotide probe comprises 5'TAAATATAAATGCAAACGATGAAAACTTTGC 3' (SEQ ID NO: 3) or its complement. In some embodiments, the oligonucleotide probe specifically hybridizes to the 16S rRNA amplification product, and wherein the oligonucleotide probe comprises 5'CCAGCATGTGATGGTGGGGACTCTA 3' (SEQ ID NO: 6) or its complement.

[0023] In some embodiments, the method further comprises mixing the exogenous control DNA with the biological sample. In some embodiments, the method further comprises contacting the biological sample with a third primer suitable for amplifying an exogenous control target nucleic acid, and amplifying the exogenous control target nucleic acid. In some embodiments, the exogenous control target nucleic acid comprises SEQ ID NO: 20. In some embodiments, the third primer is made up of a third forward primer comprising 5'GCTTCAGTACCTTCGGCTTG 3' (SEQ ID NO: 17) and a third reverse primer comprising 5'TTGCAGGCATCTCTGACAAC 3' (SEQ ID NO: 18). In some embodiments, the method further comprises contacting the biological sample with a third oligonucleotide probe, wherein the third oligonucleotide probe is detectably labeled and comprises 5'TGGCTCTTGGCGGTCCAGATG 3' (SEQ ID NO: 19).

[0024] In some embodiments of the methods provided herein, real-time PCR amplification is performed in a direct amplification plate in conjunction with an integrated thermal cycler.

[0025] In some embodiments of the methods provided herein, the biological sample is a bronchoalveolar lavage sample, a bronchial wash sample, a sputum sample, a nasopharyngeal (NP) aspirate or wash sample, a nasal swab, or a bacterial isolate.

[0026] In some embodiments of the methods provided herein, the therapeutic agent inhibiting Legionella pneumophila is selected from one or more agents of the following: fluoroquinolone, carbapenem, macrolide antibiotics, trimethoprim-sulfamethoxazole, Legionella pneumophila specific antibodies and Legionella pneumophila specific vaccines. In some embodiments of the methods provided herein, the fluoroquinolone is selected from ciprofloxacin, gemifloxacin, levofloxacin, norfloxacin, ofloxacin, rovafloxacin (rovafloxacin), gatifloxacin, grepafloxacin, temafloxacin, lomefloxacin, sparfloxacin, enoxacin and moxifloxacin. In some embodiments of the methods provided herein, the carbapenem is selected from imipenem, meropenem, ertapenem, doripenem, panipenem, biapenem, razupenem (PZ-601), tebipenem, lenapenem, toupenem and thienpenem (thienpenem) (thienpenem). In some embodiments of the methods provided herein, the Legionella pneumophila-specific vaccine is selected from whole cell (wP) Legionella pneumophila vaccine and acellular Legionella pneumophila vaccine. In some embodiments of the methods provided herein, the macrolide antibiotic is selected from azithromycin (Zithromax), clarithromycin (Biaxin), erythromycin (E-Mycin, Eryc, Ery-Tab, PCE, Pediazole, Ilosone) and roxithromycin. DETAILED DESCRIPTION

[0027] The present disclosure provides methods for determining whether a patient exhibiting pneumonia-like symptoms will benefit from treatment with a therapeutic agent that inhibits Legionella species and / or Legionella pneumophila. These methods are based on detecting Legionella species and / or Legionella pneumophila in a biological sample by using real-time PCR to determine the presence of ssrA and 16S rRNA target nucleic acids, respectively. In some embodiments, the method comprises: (a) providing a first primer pair suitable for amplifying an ssrA target nucleic acid; providing a second primer pair suitable for amplifying a 16S rRNA target nucleic acid; amplifying an ssrA target nucleic acid and a 16S rRNA target nucleic acid, if present; and detecting one or more amplification products produced in step (c); wherein the presence of an ssrA target nucleic acid identifies the presence of at least one Legionella species, and the presence of a 16S rRNA target nucleic acid identifies the presence of Legionella pneumophila. A kit for practicing the method is also provided. definition

[0028] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0029] As used herein, the singular forms "a", "an", and "the" include plural references unless otherwise indicated. Thus, for example, reference to an "oligonucleotide" includes a plurality of oligonucleotide molecules, and reference to a "nucleic acid" is a reference to one or more nucleic acids.

[0030] As used herein, the term "about" with respect to a number is generally considered to include numbers falling within 1%-10% in either direction (greater than or less than) of the number unless otherwise stated or otherwise obvious from the context.

[0031] As used herein, the term "amplify" or "amplification" with respect to nucleic acid sequences refers to a method of increasing the representation of a population of nucleic acid sequences in a sample. Copies of a specific target nucleic acid sequence produced in vitro in an amplification reaction are called "amplicons" or "amplification products". Amplification can be exponential or linear. The target nucleic acid can be DNA (such as, for example, genomic DNA and cDNA) or RNA. Although the exemplary methods described below involve amplification using polymerase chain reaction (PCR), a variety of other methods are known to the skilled artisan, such as isothermal methods, rolling circle methods, and the like. The skilled artisan will appreciate that these other methods can be used in place of or in conjunction with the PCR method. See, for example, Saiki, "Amplification of Genomic DNA", PCR Protocols, Innis et al., eds., Academic Press, San Diego, CA 1990, pp. 13-20; Wharam et al., Nucleic Acids Res. 29(11): E54-E54 (2001).

[0032] As used herein, "amplification mixture" is a reagent mixture for nucleic acid amplification reaction, but does not contain primers or samples. The amplification mixture includes buffer, dNTPs and DNA polymerase. The amplification mixture may also include at least one of MgCl2, KCl, nonionic and ionic detergents (including cationic detergents).

[0033] An "amplification master mix" contains an amplification mixture and primers for amplifying one or more target nucleic acids, but does not contain the sample to be amplified.

[0034] The terms "complement", "complementary" or "complementarity" as used herein with respect to polynucleotides (i.e., nucleotide sequences, such as oligonucleotides or target nucleic acids) refer to the Watson / Crick base pairing rules. The complement of a nucleic acid sequence as used herein refers to an oligonucleotide that is "antiparallel associated" when aligned with a nucleic acid sequence so that the 5' end of one sequence is paired with the 3' end of another sequence. For example, the sequence "5'-AGT-3'" is complementary to the sequence "3'-TCA-5'". Certain bases that are not normally present in naturally occurring nucleic acids may be included in the nucleic acids described herein. These bases include, for example, inosine, 7-deazaguanine, locked nucleic acids (LNA) and peptide nucleic acids (PNA). Complementarity need not be perfect; stable duplexes may contain mismatched base pairs, denatured or mismatched bases. A person skilled in the art of nucleic acid technology can determine duplex stability empirically after considering multiple variables, including, for example, the length of the oligonucleotide, the base composition and sequence of the oligonucleotide, ionic strength, and the incidence of mismatched base pairs. The complementary sequence may also be an RNA sequence complementary to a DNA sequence or its complementary sequence, and may also be a cDNA.

[0035] As used herein, the term "substantially complementary" means that two sequences hybridize under stringent hybridization conditions. It will be appreciated by the skilled artisan that substantially complementary sequences need not hybridize along their entire length. Specifically, substantially complementary sequences may comprise a continuous sequence of bases that do not hybridize to the target sequence, positioned 3' or 5' to a continuous sequence of bases that hybridizes to the target sequence under stringent hybridization conditions.

[0036] As used herein, the "cycle threshold" (Ct) of an analyte is the PCR cycle at which the fluorescent signal crosses a specified fluorescence threshold. Ct depends on the efficiency of the amplification reaction, including the starting template copy number, organism lysis, PCR amplification, hybridization or cutting of the fluorescent probe, and detection sensitivity. Ct provides a relative measure of the target nucleic acid concentration in the PCR reaction. Many factors in addition to the target nucleic acid concentration can affect the absolute value of Ct. However, the artifacts of fluorescence measurements associated with the Ct calculation from changes in the reaction mixture or instrument will result in template-independent changes in the Ct value.

[0037] As used herein, the term "detection" refers to determining the presence of a target nucleic acid in a sample. Detection does not require that the method provide 100% sensitivity and / or 100% specificity.

[0038] As used herein, the term "direct amplification" refers to a nucleic acid amplification reaction in which a target nucleic acid is amplified from a sample without prior purification, extraction, or concentration.

[0039] As used herein, the term "extraction" refers to any action taken to remove nucleic acids from other (non-nucleic acid) materials present in a sample. The term extraction includes mechanical or chemical lysis, addition of detergents or proteases, or precipitation and removal of non-nucleic acids (such as proteins).

[0040] The term "fluorophore" as used herein refers to a molecule that absorbs light of a specific wavelength (excitation frequency) and subsequently emits light of a longer wavelength (emission frequency). The term "donor fluorophore" as used herein means a fluorophore that, when in close proximity to a quencher moiety, contributes or transfers emission energy to the quencher. Due to the donation of energy to the quencher moiety, the donor fluorophore itself will emit less light at a specific emission frequency than would be emitted in the absence of the closely located quencher moiety.

[0041] As used herein, the term "hybridization" refers to the process by which two substantially complementary nucleic acid strands (at least about 65% complementary, at least about 75% or at least about 90% complementary over a stretch of at least 14 to 25 nucleotides) anneal to each other under appropriately stringent conditions to form a duplex or heteroduplex by forming hydrogen bonds between complementary base pairs. Hybridization is typically and preferably performed with probe-length nucleic acid molecules (preferably 15-100 nucleotides in length, more preferably 18-50 nucleotides in length). Nucleic acid hybridization techniques are well known in the art. See, for example, Sambrook et al., 1989, Molecular Cloning: A Laboratory Manual, 2nd edition, Cold Spring Harbor Press, Plainview, NY. Hybridization and hybridization strength (i.e., the strength of association between nucleic acids) are affected by factors such as the degree of complementarity between the nucleic acids, the stringency of the conditions involved, and the thermal melting point (T) of the hybrid formed. m ) and other factors. Those skilled in the art understand how to estimate and adjust the stringency of hybridization conditions so that sequences with at least the desired level of complementarity will stably hybridize, while sequences with lower complementarity will not hybridize. For examples of hybridization conditions and parameters, see, for example, Sambrook et al., 1989, Molecular Cloning: A Laboratory Manual, 2nd edition, Cold Spring Harbor Press, Plainview, NY; Ausubel, FM et al. 1994, Current Protocols in Molecular Biology, John Wiley & Sons, Secaucus, NJ. In some embodiments, specific hybridization occurs under stringent hybridization conditions. An oligonucleotide or polynucleotide (e.g., a probe or primer) specific for a target nucleic acid will "hybridize" with the target nucleic acid under appropriate conditions.

[0042] As used herein, the term "individual", "patient" or "subject" can be an individual organism, a vertebrate, a mammal or a human. In a preferred embodiment, the individual, patient or subject is a human.

[0043] As used herein, the term "multiplex PCR" refers to the simultaneous generation of two or more PCR products or amplicons in the same reaction vessel. Each PCR product is initiated using a different primer pair. The multiplex reaction may also include specific probes for each product, the probes being labeled with different detectable moieties.

[0044] As used herein, "oligonucleotide" refers to a molecule having a nucleic acid base sequence on a main chain that mainly contains identical monomeric units at defined intervals. The arrangement of bases on the main chain allows it to bind to a nucleic acid having a base sequence complementary to the bases of the oligonucleotide. The most common oligonucleotide has a main chain of a phosphate sugar unit. It is possible to distinguish between oligodeoxyribonucleotides without a 2' hydroxyl and oligoribonucleotides with a 2' hydroxyl. Oligonucleotides may also include derivatives in which the hydrogen in the hydroxyl is replaced by an organic group (e.g., allyl). Oligonucleotides used as primers or probes are usually at least about 10-15 nucleotides in length, or up to about 70, 100, 110, 150 or 200 nucleotides in length, more preferably at least about 15 to 25 nucleotides in length. Oligonucleotides used as primers or probes for specific amplification or specific detection of a specific target nucleic acid are usually able to specifically hybridize with a target nucleic acid.

[0045] As used herein, "Legionella species" or "Legionella sp." refers to any microbial organism of the genus Legionella. In some embodiments, Legionella species are pathogenic and are capable of causing pneumonia, Legionnaires' disease, Pontiac fever, or a related disorder in a subject (e.g., a human).Specific pathogens include, for example, Legionella anisa, Legionella birminghamensis, Legionella bozemanii (serogroup 1), Legionella bozemanii (serogroup 2), Legionella cardiaca, Legionella cherrii, Legionella cincinnatiensis, Legionella clemsonensis, Legionella dumoffii, Legionella feeleii (serogroup 1), Legionella feeleii (serogroup 2), Legionella ), Legionella gormanii, Legionella hackeliae (serogroup 1), Legionella hackeliae (serogroup 2), Legionella jordanis, Legionella lansingensis, Legionella longbeachae (serogroup 1), Legionella longbeachae (serogroup 2), Legionella maceachernii, Legionella micdadei, Legionella oakridgensis, Legionella parisiensis, Legionella pneumophila Legionella pneumophila (Philadelphia 1), Legionella pneumophila (Knoxville 1), Legionella pneumophila (Benidorm 030E), Legionella pneumophila (France 5811), Legionella pneumophila (Allentown 1), Legionella pneumophila (OLDA), Legionella pneumophila (Oxford 4032E), Legionella pneumophila (Bellingham), Legionella pneumophila (Heysham 1), Legionella pneumophila (Camplton 1), Legionella pneumophila (Togus 1), Legionella pneumophila (Bloomington 2), Legionella pneumophila (Los Angeles), Legionella pneumophila (Portland 1), Legionella pneumophila (Dallas 1E), Legionella pneumophila (Cambridge 1), Legionella pneumophila (Chicago 1), Legionella pneumophila (Chicago 8 ), Legionella pneumophila (Concord 3), Legionella pneumophila (IN-23-G1-C2), Legionella pneumophila (Leiden 1), Legionella pneumophila (797-PA-H), Legionella pneumophila (570-CO-H), Legionella pneumophila (82A3105), Legionella pneumophila (1169-MN-H), Legionella pneumophila (Lansing 3), Legionella rubrilucens, Legionella sainthelensi (serogroup 1), Legionella sainthelensi (serogroup 2), Legionella tucsonensis and Legionella wadsworthii (L.

[0046] As used herein, a "positive control nucleic acid" or "internal positive amplification control" is a nucleic acid that is known to be present in a sample in a certain amount or level. In some embodiments, in the disclosed methods for detecting the presence of pathogenic Legionella species in a sample, the positive control nucleic acid is not naturally present in the sample, but is added to the sample prior to subjecting the reaction-sample mixture to a real-time polymerase chain reaction.

[0047] As used herein, the term "primer" refers to an oligonucleotide that can be used as a starting point for nucleic acid sequence synthesis when placed under conditions of inducing the synthesis of primer extension products complementary to the target nucleic acid chain, i.e., in the presence of different nucleotide triphosphates and polymerase in a suitable buffer ("buffer" includes pH, ionic strength, cofactors, etc.) and at a suitable temperature. One or more nucleotides in a primer can be modified, for example, by adding a methyl, biotin or digoxin moiety, a fluorescent label, or by using radioactive nucleotides. The primer sequence does not need to reflect the accurate sequence of the template. For example, a non-complementary nucleotide fragment can be attached to the 5' end of the primer, and the remainder of the primer sequence is substantially complementary to the chain. As used herein, the term primer includes all primer forms that can be synthesized, including peptide nucleic acid primers, locked nucleic acid primers, primers modified by thiophosphates, labeled primers, etc. As used herein, the term "forward primer" means a primer that anneals to the antisense strand of a double-stranded DNA (dsDNA). "Reverse primer" anneals to the positive strand of dsDNA.

[0048] Primer typically has a length of at least 10, 15, 18 or 30 nucleotides, or a length of up to about 100, 110, 125 or 200 nucleotides. In some embodiments, primers preferably have a length of about 15 to about 60 nucleotides, and most preferably a length of about 25 to about 40 nucleotides. In some embodiments, primers have a length of 15 to 35 nucleotides. For best hybridization or polymerase chain reaction amplification, there is no standard length. The optimal length for specific primer applications can be easily determined in the manner described in H.Erlich, PCR Technology, Principles and Application for DNA Amplification, (1989).

[0049] "Primer extension reaction" refers to a synthetic reaction in which an oligonucleotide primer hybridizes to a target nucleic acid and a complementary copy of the target nucleic acid is produced by polymerase-dependent 3' addition of individual complementary nucleotides. In some embodiments, the primer extension reaction is PCR.

[0050] As used herein, the term "primer pair" refers to a forward and reverse primer pair (ie, a left and right primer pair) that can be used together to amplify a given region of a target nucleic acid.

[0051] As used herein, "probe" refers to a nucleic acid that interacts with a target nucleic acid via hybridization. The probe can be fully complementary or partially complementary to the target nucleic acid sequence. The level of complementarity will generally depend on many factors based on the function of the probe. The probe can be labeled or unlabeled or modified in any of a variety of ways well known in the art. The probe can specifically hybridize with the target nucleic acid. The probe can be DNA, RNA, or RNA / DNA hybrid. The probe can be an oligonucleotide, an artificial chromosome, a fragmented artificial chromosome, a genomic nucleic acid, a fragmented genomic nucleic acid, RNA, a recombinant nucleic acid, a fragmented recombinant nucleic acid, a peptide nucleic acid (PNA), a locked nucleic acid, an oligomer of a ring heterocycle, or a conjugate of nucleic acid. The probe can contain a modified nucleobase, a modified sugar moiety, and a modified internucleotide connection. The probe can be used to detect the presence or absence of a methylated target nucleic acid. The probe is typically at least about 10, 15, 20, 25, 30, 35, 40, 50, 60, 75, 100 nucleotides or longer in length.

[0052] As used herein, a "probe element" refers to a stretch of nucleotides that (a) is associated with a primer, wherein the stretch is linked to or positioned adjacent to the primer nucleic acid sequence, and (b) specifically hybridizes to a target nucleic acid sequence to be detected under stringent conditions.

[0053] As used herein, the term "primer-probe detection system" refers to a method for real-time PCR. In some embodiments, the system is a PCR system based on Taqman and / or a PCR system based on SCORPION. In some embodiments, the primer-probe detection system comprises at least one forward primer, at least one reverse primer and at least one oligonucleotide probe. In some embodiments, the oligonucleotide probe is detectably labeled. In some embodiments, the oligonucleotide probe comprises a detectable label and a quencher portion.

[0054] As used herein, the term "quencher moiety" means a molecule in close proximity to a donor fluorophore that absorbs the emission energy generated by the donor and either dissipates the energy as heat or emits light at a wavelength longer than the donor emission wavelength. In the latter case, the quencher is considered an acceptor fluorophore. The quenching moiety can quench via proximity (i.e., collision) or by or fluorescence resonance energy transfer ("FRET"). Quenching by FRET is often used probes, while proximity quenching is used for molecular beacons and Scorpion TM Non-limiting examples of quenchers include TAMRA, Black Hole Quencher, Deep Dark Quencher, ZEN, Iowa Black FQ, Iowa Black RQ, and DABCYL.

[0055] As used herein, "reaction-sample mixture" refers to a mixture containing an amplification master mix and a sample.

[0056] As used herein, the term "sample" refers to a clinical sample obtained from a patient or an isolated microorganism. In a preferred embodiment, a sample (i.e., a "biological sample") is obtained from a biological source, such as a tissue, body fluid, or microorganism collected from a subject. Sample sources include, but are not limited to, mucus, sputum (processed or unprocessed), bronchoalveolar lavage fluid (BAL), bronchial washings (BW), blood, body fluids, cerebrospinal fluid (CSF), urine, plasma, serum, or tissue (e.g., biopsy material). Preferred sample sources include BAL, BW, and / or throat swabs or nasal washes.

[0057] The term "sensitivity" as used herein for the methods of the present technology is a measure of the ability of the method to detect preselected sequence variants in a heterogeneous sequence population. If the preselected sequence variants in a given sample are present in at least F% of the sequence in the sample, the method can detect the preselected sequence at a preselected confidence of C% and S% of the number of times, then the method has a sensitivity of S% to the variants of F%. For example, if the preselected variant sequence in a given sample is present in at least 5% of the sequence in the sample, the method can detect the preselected sequence 9 times out of 10 with a preselected confidence of 99% (F = 5%; C = 99%; S = 90%), then the method has a sensitivity of 90% to 5% of the variants. Exemplary sensitivities include at least 50%, 60%, 70%, 80%, 90%, 95%, 98% and 99%.

[0058] As used herein, the term "specificity" for oligonucleotide primers means that when the oligonucleotide is compared with the nucleic acid to be amplified, the nucleotide sequence of the primer has a sequence identity of at least 12 bases with a portion of the nucleic acid. Oligonucleotide primers that are specific to nucleic acids are primers that can hybridize with the target target and are substantially non-target nucleic acid hybridization under stringent hybridization or washing conditions. Higher levels of sequence identity are preferred and include at least 75%, at least 80%, at least 85%, at least 90%, at least 85%-95%, more preferably at least 98% sequence identity. Commercially available computer programs using algorithms well known in the art can be used to determine sequence identity with default settings. As used herein, sequences with "high sequence identity" have identical nucleotides at least at about 50% of the compared nucleotide positions, preferably at least at about 60% of the compared nucleotide positions, and more preferably at least at about 75% of the compared nucleotide positions.

[0059] As used herein, "specificity" is a measure of the ability of a method to distinguish between truly existing preselected sequence variants and sequencing artifacts or other closely related sequences. It is the ability to avoid false positive detections. False positive detections may be caused by errors introduced into the target sequence during sample preparation, sequencing errors, or inadvertent sequencing of closely related sequences (such as pseudogenes or gene family members). If applied to N 总 When the sample set is a sequence, X 真实 The sequence is a true variant and X 非真实 is a non-true variant, and the method selects at least X% of the non-true variants as non-variants, then the method has a specificity of X%. For example, if when applied to a sample set of 1,000 sequences, of which 500 sequences are true variants and 500 are non-true variants, the method selects 90% of the 500 non-true variant sequences as non-variants, then the method has a specificity of 90%. Exemplary specificities include at least 50%, 60%, 70%, 80%, 90%, 95%, 98%, and 99%.

[0060] As used herein, the term "stringent hybridization conditions" refers to hybridization conditions that are at least as stringent as the following: hybridization overnight at 42° C. in 50% formamide, 5× SSC, 50 mM NaH2PO4 (pH 6.8), 0.5% SDS, 0.1 mg / mL sonicated salmon sperm DNA, and 5× Denhart's solution; washing at 45° C. with 2× SSC, 0.1% SDS; and washing at 45° C. with 0.2× SSC, 0.1% SDS. In another example, stringent hybridization conditions should not allow hybridization of two nucleic acids that differ by more than two bases over a stretch of 20 consecutive nucleotides.

[0061] As used herein, “ The term "PCR detection system" refers to a method for real-time PCR. In this method, amplification master mix includes a Probe. The probe contains donor and quencher fluorophores on either end of the probe that are close enough to each other that the quencher absorbs the fluorescence of the donor. However, when the probe hybridizes to the amplified segment, the 5'-exonuclease activity of the Taq polymerase cleaves the probe, allowing the donor fluorophore to emit fluorescence that can be detected.

[0062] As used herein, the term "target nucleic acid" or "target sequence" refers to a target nucleic acid sequence to be detected and / or quantified in a sample to be analyzed. The target nucleic acid can be composed of a chromosome segment, a complete gene with or without an intergenic sequence, a segment or part of a gene with or without an intergenic sequence, or a nucleic acid sequence for which a probe or primer is designed. The target nucleic acid can include one or more wild-type sequences, mutations, deletions, insertions or duplications, tandem repeat elements, a target gene, a target gene region, or any upstream or downstream region of the region. The target nucleic acid can represent an alternative sequence or allele of a specific gene. The target nucleic acid can be derived from genomic DNA, cDNA, or RNA. Biological sample collection and preparation

[0063] The methods and compositions of the present technology can be used to detect pathogenic Legionella species by determining target nucleic acid sequences corresponding to ssrA genes and 16S rRNA genes in biological samples obtained from subjects. Samples for pathogenic Legionella species detection can also include cultures of bacterial isolates grown on appropriate culture media to form colonies, wherein the cultures are prepared from biological samples obtained from subjects.

[0064] Methods disclosed herein can be used to detect and quantify pathogenic Legionella species in biological samples derived from sterile and / or non-sterile sites."Sterile sites" include body fluids such as whole blood, plasma, cell-free plasma, urine, cerebrospinal fluid, synovial fluid, pleural fluid, pericardial fluid, intraocular fluid, tissue biopsy or endotracheal aspirates. As used herein, "cell-free plasma" refers to plasma containing less than 1% cells by volume. "Non-sterile sites" include sputum, feces, skin swabs, inguinal swabs, nasal swabs and pharyngeal swabs. In some embodiments, biological samples include nasopharyngeal (NP) aspirates or swabs or nasal washes. In other embodiments, biological samples include cultures of isolated bacteria grown on appropriate culture media to form colonies. Samples can also include bacterial isolates.

[0065] In some embodiments, the sample is transported or stored in a sterile vial containing VCM or M4 medium. VCM medium contains Hank's balanced salts, bovine serum albumin, L-cysteine, gelatin, sucrose, L-glutamic acid, HEPES buffer, vancomycin, amphotericin B, colistin, and optionally phenol red. M4 medium contains gelatin, vancomycin, amphotericin B, and colistin.

[0066] The biological sample may be suspected of containing pathogenic Legionella species and / or nucleic acid of one or more pathogenic Legionella species. Additionally, the biological sample may be obtained from a subject suspected of being infected with one or more pathogenic Legionella species. The biological sample may be contacted with an amplification master mix for a microfluidic / microelectronic centrifugal platform.

[0067] In some embodiments, the disclosed method adopts untreated biological sample, thereby produces direct, simplified sample to result process.In other embodiments, if used for the separated nucleic acid (DNA or RNA) purified from biological sample according to any method well known to those skilled in the art, the detection method disclosed herein will be effective.If necessary, the sample can be collected or concentrated by centrifugation etc. The cells of the sample can be cracked such as by enzyme treatment, hot surfactant treatment, ultrasonic treatment or its combination.Alternatively, a commercially available nucleic acid extraction kit can be used to process the biological sample.

[0068] In some embodiments, before contacting with the biological sample, there are one or more primer pairs in the amplification master mixture, and the amplification master mixture also includes DNA polymerase, dNTP and PCR buffer. The amplification of ssrA gene and 16S rRNA gene preferably occurs in a multiplex form. Alternatively, the individual PCR reaction for each target sequence can also be used. The biological sample can be contacted with the one or more primer pairs and / or with the amplification master mixture in a direct amplification plate to form a reaction-sample mixture. For example, the biological sample can be contacted with the amplification master mixture in a direct amplification plate (such as the direct amplification plate sold by FocusDiagnostics, Inc. (Cypress, California, USA)), as a part of SIMPLEXA direct real-time PCR assay, with 3M TM The direct amplification plate is a thin, circular plate containing a plurality of designated areas, each of which contains a well for receiving an amplification master mix and an associated well for receiving an unprocessed patient sample. Upon or after the addition of the amplification master mix and sample, a sample-reaction mixture is generated in the direct amplification plate.

[0069] In some embodiments, the biological sample is isolated from a subject. In some embodiments, the subject is a mammal. In some embodiments, the mammal is a cow, horse, pig, cat, dog, mouse, monkey, rat or human. In some embodiments, the subject is a human. In specific embodiments, the subject is a human patient with one or more pneumonia-like symptoms. Real-time PCR

[0070] Amplification of the target nucleic acid can be detected by any of a variety of methods well known in the art, such as gel electrophoresis, column chromatography, hybridization with probes, sequencing, melting curve analysis, or "real-time" detection.

[0071] For real-time detection, primers and / or probes can be detectably labeled to allow detection of differences in fluorescence when the primers are incorporated or when the probes, for example, hybridize and amplify in an instrument capable of monitoring changes in fluorescence during the reaction. Real-time detection methods for nucleic acid amplification are well known and include, for example, System, Scorpion TM Primer systems and the use of intercalating dyes for double-stranded nucleic acids.

[0072] In real-time quantitative PCR, the accumulation of amplification product is measured continuously in both standard dilutions of target DNA and in samples containing unknown amounts of target DNA. The initial template concentration in the standard sample is compared to the PCR product required to produce a specific threshold concentration of product. TM The standard curve is constructed by correlating the target PCR results with the target PCR results after the same Ct in the test samples. TM Product accumulates, which allows interpolation of target DNA concentration from the standard curve.

[0073] In some embodiments, amplified nucleic acid is detected by hybridization with a specific probe. Amplified fragments can be detected using a probe oligonucleotide complementary to a part of the target sequence amplified. In certain embodiments, hybridization can be detected in real time. In an alternative embodiment, hybridization is not detected in real time. The amplified nucleic acid in each target sequence can be detected simultaneously (i.e. in the same reaction vessel, such as multiplex PCR) or detected separately (i.e. in a separate reaction vessel). In certain embodiments, multiple target nucleic acids are detected simultaneously using two or more gene-specific oligonucleotide probes that can distinguish marks (e.g., via different detectable parts, such as color), wherein a probe hybridizes with the first target sequence, and another probe hybridizes with the second target sequence.

[0074] In some embodiments, different primer pairs are labeled with different distinguishable detectable moieties. Thus, for example, HEX and FAM fluorescent dyes can be present on different primer pairs in multiplex PCR and associate with the resulting amplicon. In other embodiments, a forward primer is labeled with a detectable moiety, while a reverse primer is labeled with a different detectable moiety, such as using a FAM dye for a forward primer and a HEX dye for a reverse primer. Using different detectable moieties can be used to distinguish amplified products that are identical in length or very similar in length.

[0075] For sequence-modified nucleic acids, targets can be independently selected from the top strand or the bottom strand. Thus, all targets to be detected can comprise a combination of top strand, bottom strand, or top strand and bottom strand targets.

[0076] A common method for real-time PCR uses fluorescent probes, such as Probes, Molecular Beacons, and Scorpion Primer-Probes. Real-time PCR quantifies the initial amount of template with greater specificity, sensitivity, and reproducibility than other forms of quantitative PCR that detect the amount of the final amplified product. Real-time PCR does not detect the size of the amplicon. Scorpion TM and The probes employed in the technology are based on the principle of fluorescence quenching and involve a donor fluorophore and a quencher moiety.

[0077] Real-time PCR is performed using any suitable instrument capable of detecting accumulation of PCR amplification products. Most commonly, the instrument is capable of detecting fluorescence from one or more fluorescent labels. For example, an instrument (e.g., ABI Real-Time PCR System Real-time detection on a sequence detector monitors fluorescence during each PCR cycle and calculates the measured value or Rn value of the reporter signal. The threshold cycle or Ct value is the cycle at which the fluorescence intersects the threshold. The threshold can be determined by the sequence detection system software or manually.

[0078] In some embodiments, the probes employed are detectably labeled, and detection is accomplished by detecting the probe label for each amplification product.A quencher may further be associated with the detectable label, which prevents detection of the label prior to amplification of the probe's target. Probes are examples of such probes.

[0079] The probe (Heid et al., Genome Res. 6:986-994, 1996) uses the fluorescent 5' exonuclease activity of Taq polymerase to measure the amount of a target sequence in a DNA sample. The probe is an oligonucleotide containing a donor fluorophore, usually at or near the 5' base, and a quencher moiety, typically at or near the 3' base. The quencher moiety can be a dye, such as TAMRA; or it can be a non-fluorescent molecule, such as 4-(4-dimethylaminophenylazo)benzoic acid (DABCYL). See Tyagi et al., 16 Nature Biotechnology 49-53 (1998). When irradiated, the excited fluorescent donor transfers energy to the nearby quencher moiety by FRET rather than fluorescence. Therefore, the close proximity of the donor and the quencher prevents the donor fluorescence from being emitted when the probe is intact.

[0080] The probe is designed to anneal to the internal region of the PCR product. When the probe is bound to the template thereon, its 5' exonuclease activity cuts the probe. This stops the activity of the quencher (no FRET), and the donor fluorophore begins to emit fluorescence, which increases in proportion to the probe cleavage rate in each cycle. The accumulation of PCR products is detected by monitoring the increase in reporter dye fluorescence. If the quencher is an acceptor fluorophore, the accumulation of PCR products can be detected by monitoring the reduction in the fluorescence of the acceptor fluorophore.

[0081] In certain embodiments, a dual-function primer-probe detection system (e.g., Scorpion TM Real-time PCR is performed using a single molecule (Scorpion primer). In the case of the Scorpion primer, sequence-specific priming and PCR product detection are achieved using a single molecule. The Scorpion primer maintains a stem-loop configuration in an unhybridized state. The fluorophore is attached to the 5' end and is quenched by the portion coupled to the 3' end, but in some embodiments, this arrangement can be switched. The 3' portion of the stem and / or loop also contains a sequence that is complementary to the extension product of the primer and is connected to the 5' end of the specific primer via a non-amplifiable monomer. After the primer portion is extended, the specific probe sequence is able to bind to its complement within the extended amplicon, thereby opening the hairpin loop. This prevents fluorescence from being quenched, and a signal is observed. TM The primer portion of the primer amplifies the specific target, thereby generating an extension product. TM Binding of the probe element of the primer to the extension product causes the separation of the fluorophore from the quencher, thus generating a fluorescent signal.

[0082] In some embodiments, the probes employed in the disclosed methods comprise or consist of short fluorescently labeled DNA sequences designed to detect DNA sequence segments with genetic variations, such as the probes disclosed in French et al., Mol Cell Probes, 5(6):363-74 (2001) (incorporated herein by reference in its entirety). is an example of this type of probe.

[0083] In some embodiments of the method, at least one primer or at least one probe of each primer pair in the amplification reaction comprises a detectable moiety. Alternatively, the detectable moiety can be on a probe attached to a primer, such as with a primer-probe. In some embodiments, the detectable moiety or label is a fluorophore. Suitable fluorescent moieties include, but are not limited to, the following fluorophores: 4-acetamido-4'-isothiocyanatostilbene-2,2'disulfonic acid, acridine and derivatives (acridine, acridine isothiocyanate), Alexa Fluor (Alexa Fluor). 350. Alexa 488. Alexa 546. Alexa 555. Alexa 568. Alexa 594. Alexa 647 (Molecular Probes)), 5-(2'-aminoethyl)aminonaphthalene-1-sulfonic acid (EDANS), 4-amino-N-[3-vinylsulfonyl)phenyl]naphthalimide-3,5-disulfonate (Firefly Yellow VS), N-(4-anilino-1-naphthyl)maleimide, o-aminobenzamide, R-6G, 530 / 550, FL, Bright Yellow, Cal Fluor Red (CFR610), coumarin and derivatives (coumarin, 7-amino-4-methylcoumarin (AMC, coumarin 120), 7-amino-4-trifluoromethylcoumarin (coumarin 151)), Tetrachlorotetrabromofluorescein sodium (cyanosine), 4',6-diamidino-2-phenylindole (DAPI), 5',5"-dibromopyrogallol-sulfonphthalein (bromopyrogallol red), 7-diethylamino-3-(4'-isothiocyanatophenyl)-4-methylcoumarin, diethylenetriamine pentaacetate, 4,4'-diisothiocyanatodihydro-stilbene-2,2'-disulfonic acid, 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid, 5-[dimethylamino]naphthalene-1-sulfonyl chloride (DNS, dansyl chloride), 4-(4'-dimethylaminophenylazo)benzoic acid (DABCYL), 4-dimethylaminophenylazophenyl-4'-isothiocyanate (DABITC), EclipseTM (Epoch Biosciences Inc.), eosin and derivatives (eosin, eosin isothiocyanate), erythrosine and derivatives (erythrosine B, erythrosine isothiocyanate), ethidium, fluorescein and derivatives (5-carboxyfluorescein (FAM), 5-(4,6-dichlorotriazin-2-yl)aminofluorescein (DTAF), 2',7'-dimethoxy-4'5'-dichloro-6-carboxyfluorescein (JOE), fluorescein, fluorescein isothiocyanate (FITC), hexachloro-6-carboxyfluorescein (HEX), QFITC ​​(XRITC), tetrachlorofluorescein (TET)), fluorescamine, IR144, IR1446, lanthanide phosphors, malachite green isothiocyanate, 4-methylumbelliferone, o-cresolphthalein, nitrotyrosine, pararosaniline, phenol red, B-phycoerythrin, R-phycoerythrin, allophycocyanin, o-phthalaldehyde, Oregon Propidium iodide, pyrene and derivatives (pyrene, pyrene butyrate, 1-pyrene butyric acid succinimidyl ester), 7. 9. twenty one, 35(Molecular Probes), Reactive Red 4( Brilliant Red 3B-A), rhodamine and derivatives (6-carboxy-X-rhodamine (ROX), 6-carboxyrhodamine (R6G), lissamine rhodamine B sulfonyl chloride, rhodamine (Rhod), rhodamine B, rhodamine 123, rhodamine green, rhodamine X isothiocyanate, sulforhodamine B, sulforhodamine 101, sulfonyl chloride derivative of sulforhodamine 101 (Texas Red), N,N,N',N'-tetramethyl-6-carboxyrhodamine (TAMRA), tetramethylrhodamine, tetramethylrhodamine isothiocyanate (TRITC)), riboflavin, rhodamine, terbium chelate derivatives, Quasar as well as

[0084] The appropriate quencher is selected based on the fluorescence spectrum of the particular fluorophore. Useful quenchers include, for example, BlackHole TM Quenchers BHQ-1, BHQ 2 and BHQ-3 (Biosearch Technologies, Inc.) and ATTO series quenchers (ATTO 540Q, ATTO 580Q and ATTO 612Q; Atto-Tec GmbH).

[0085] In some embodiments of the method, the reaction-sample mixture is subjected to real-time polymerase chain reaction (PCR) conditions, under which each target nucleic acid present in the biological sample is amplified, and the one or more amplified products are detected and measured. In some embodiments, the biological sample is directly loaded into a direct amplification plate without performing a separate front-end specimen preparation, and then the target analyte is detected and distinguished by real-time PCR in the same plate. In certain embodiments, amplification is performed in a direct amplification plate (8-well plate from Focus Diagnostics, Inc.). In some embodiments, SIMPLEXA is used to directly determine the real-time PCR amplification in the direct amplification plate, and an integrated thermal cycler (such as the 3M sold by 3M (St. Paul, Minnesota, USA) is used. TM Integrated cycler) for detection. 3M TM An integrated cycler can receive direct amplification plates and can perform multiple assays on each plate. This device can heat at >5°C / sec and cool at >4°C / sec. Cycling parameters can vary, depending on the length of the amplification product to be extended. In certain embodiments, an internal positive amplification control (IPC) can be included in the sample, utilizing oligonucleotide primers, probes, and / or primer-probes. Alternative methods for detecting target nucleic acids

[0086] Alternatively, the detection of the target nucleic acid can be performed by measuring the reaction endpoint. In the endpoint detection, the one or more amplicons can be detected by first size-separating the amplicons and then detecting the size-separated amplicons. The separation of amplicons of different sizes can be accomplished by gel electrophoresis, column chromatography, capillary electrophoresis or other separation methods known in the art.

[0087] Detectable labels can be incorporated into nucleic acids, associated with nucleic acids or conjugated with nucleic acids. Labels can be attached by spacer arms of varying lengths to reduce potential steric hindrance or effects on other useful or desired properties. See, for example, Mansfield, 9 Mol. Cell. Probes 145-156 (1995). Detectable labels can be incorporated into nucleic acids by covalent or non-covalent means (e.g., by transcription, such as random primer labeling using Klenow polymerase, or nick translation, or amplification or equivalent means as known in the art). For example, nucleotide bases are conjugated to detectable moieties (e.g., fluorescent dyes) and then incorporated into nucleic acids during nucleic acid synthesis or amplification.

[0088] Other examples of useful labels to aid in the detection of target nucleic acids include radioisotopes (e.g. 32 P. 35 S. 3 H. 14 C. 125 I. 131 I), electron-dense reagents (e.g., gold), enzymes (e.g., horseradish peroxidase, β-galactosidase, luciferase, alkaline phosphatase), colorimetric labels (e.g., colloidal gold), magnetic labels (e.g., Dynabeads TM ), biotin, digoxigenin, or haptens and proteins for which antisera or monoclonal antibodies are available. Other labels include ligands or oligonucleotides that can form complexes with corresponding receptors or oligonucleotide complements, respectively. The label can be directly incorporated into the nucleic acid to be detected, or the label can be attached to a probe (e.g., an oligonucleotide) or an antibody that hybridizes or binds to the nucleic acid to be detected.

[0089] In other embodiments, fluorescent nucleotide analogs can be used to label nucleic acids, see, for example, Jameson, Methods. Enzymol. 278: 363-390 (1997); Zhu, Nucl. Acids Res. 22: 3418-3422 (1994). U.S. Patent Nos. 5,652,099 and 6,268,132 also describe nucleoside analogs for incorporation into nucleic acids (e.g., DNA and / or RNA) or oligonucleotides to produce fluorescent oligonucleotides via enzyme or chemical synthesis. U.S. Patent No. 5,135,717 describes phthalocyanine and tetrabenzotriazaporphyrin reagents used as fluorescent labels.

[0090] In some embodiments, detectably labeled probes can be used in hybridization assays, including but not limited to Northern blots, Southern blots, microarrays, spot or slot blots, and in situ hybridization assays (such as fluorescent in situ hybridization (FISH)) to detect target nucleic acid sequences in biological samples. Certain embodiments may employ hybridization methods for measuring the expression of polynucleotide gene products (such as mRNA). Methods for performing polynucleotide hybridization assays have been fully developed in the art. Hybridization assay procedures and conditions will vary depending on the application and are selected according to known general binding methods, including those mentioned in the following documents: Maniatis et al. Molecular Cloning: A Laboratory Manual (2nd Edition Cold Spring Harbor, NY, 1989); Berger and Kimmel Methods in Enzymology, Vol. 152, Guide to Molecular Cloning Techniques (Academic Press, Inc., San Diego, Calif, 1987); Young and Davis, PNAS.80: 1194 (1983). The present invention provides a screening assay for Legionella

[0091] In various embodiments of the present disclosure, primers and probes are used in the methods described herein to amplify and detect target nucleic acid sequences of pathogenic Legionella species. In certain embodiments, the target nucleic acid may include ssrA genes from all Legionella species and 16S rRNA genes from Legionella pneumophila. In addition, primers may also be used to amplify one or more control nucleic acid sequences.

[0092] The primers and probes of the present technology are used in the methods described herein to amplify and detect a target nucleic acid corresponding to the ssrA gene comprising SEQ ID NO: 7 and a target nucleic acid corresponding to the 16S rRNA gene comprising SEQ ID NO: 8. In one embodiment, the method involves using primer pairs specific for the ssrA and 16S rRNA genes. The target nucleic acids described herein can be detected individually or in multiplex format using individual labels for each target.

[0093] Specific primers, probes, and primer-probes for amplifying and detecting all or fragments of a marker gene specific for Legionella pneumophila include those directed to sequences present in Legionella pneumophila but not in other Legionella species. Detection of Legionella pneumophila-specific genes helps distinguish samples containing Legionella pneumophila from samples that may contain another pathogenic species of Legionella.

[0094] A suitable marker gene is the 16S rRNA gene (see, e.g., GenBank Accession No. NC_002942.5), and is shown below. AACTGAAGAGTTTGATCCTGGCTCAGATTGAACGCTGGCGGCATGCTTAACACATGCAAGTCGAACGGCAGCATTGTCTAGCTTGCTAGACAGATGGCGAGTGGCGAACGGGTGAGTAACGCGTAGGAATATGCCTTGAAGAGGGGGACAACTTGGGGAAACTCAAGCTAATACCGCATAATGTCTGAGGACGAAAGCTGGGGACCTTCGGGCCTGGCGCTTTAAGATTAGCCTGCGTCCGATTAGCTAGTTGGTGGGGTAAGGGCCTACCAAGGCGACGATCGGTAGCTGGTCTGAGAGGATGACCAGCCACACTGGAACTGAGACACGGTCCAGACTCCTACGGGAGGCAGCAGTGGGGAATATTGGACAATGGGGGCAACCCTGATCCAGCAATGCCGCGTGTGTGAAGAAGGCCTGAGGGTTGTAAAGCACTTTCAGTGGGGAGGAGGGTTGATAGGTTAAGAGCTGATTAACTGGACGTTACCCACAGAAGAAGCACCGGCTAACTCCGTGCCAGCAGCCGCGGTAATACGGAGGGTGCGAGCGTTAATCGGAATTACTGGGCGTAAAGGGTGCGTAGGTGGTTGATTAAGTTATCTGTGAAATTCCTGGGCTTAACCTGGGACGGTCAGATAATACTGGTTGACTCGAGTATGGGAGAGGGTAGTGGAATTTCCGGTGTAGCGGTGAAATGCGTAGAGATCGGAAGGAACACCAGTGGCGAAGGCGGCTACCTGGCCTAATACTGACACTGAGGCACGAAAGCGTGGGGAGCAAACAGGATTAGATACCCTGGTAGTCCACGCTGTAAACGATGTCAACTAGCTGTTGGTTATATGAAAATAATTAGTGGCGCAGCAAACGCGATAAGTTGACCGCCTGGGGAGTACGGTCGCAAGATTAAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGATGCAACGCGAAGAACCT TACCTACCCTTGACATACAGTGAATTTTGCAGAGATGCATTAGTGCCTTCGGGAACACTGATACAGGTGCTGCAT GGCTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGTAACGAGCGCAACCCTTATCCTTAGTTGCCAGC ATGTGATGGTGGGGACTCTAAGGAGACTGCCGGTGACAAACCGGAGGAAG GCGGGGATGACGTCAAGTCATCATGGCCCTTACGGGTAGGGCTACACACGTGCTACAATGCCCGATACAGAGGGCGGCGAAGGGGCGACCTGGAGCAAATCCTTAAAAGTCGGTCGTAGTCCGGATTGGAGTCTGCAACTCGACTCCA TGAAGTCGGAATCGCTAGTAATCGCGAATCAGCATGTCGCGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCATGGGAGTGGGTTGCACCAGAAGTAGATAGTCTAACCTTCGGGGGGACGTTTACCACGGTGT(SEQ ID NO:8)

[0095] The nucleic acid sequences of the 16S rRNA amplicons generated using the methods disclosed herein are underlined.

[0096] In some embodiments, the 16S rRNA target nucleic acid comprises 5′TACCTACCCTT GACATACAGTGAATTTTGCAGAGATGCATTAGTGCCTTCGGGAACACTGATACAGGTGCTGCATGGCTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGTAACGAGCGCAACCCTTATCCTTAGTTGCCAGCATGTGATGGTGGGGACTCTAAGGAGACTGCCGGTGACAAACCGGAGGAAG 3′ (SEQ ID NO: 9) or a fragment thereof.

[0097] Exemplary primers and labeled probe sequences for amplification and detection of 16S rRNA elements include:

[0098] The ssrA gene encodes a tmRNA binding protein and is present in all Legionella species. A non-limiting exemplary nucleotide sequence of ssrA is provided under GenBank Accession No. AE017354.1 (bp 3213808 to 3214278) and is shown below. The nucleic acid sequence of the ssrA amplicon generated using the methods disclosed herein is underlined: ATGTGGGCAGAAGTATTATCCAGCGATGCCTTTGCGCCGCTTTGAGGAG GAAGGTATTTTCAACCCCAACTGGACATGACTTTTTAAAATCCATTC TGGAGGTAGGCGGCTCAAGAAAAGCAGCCGATGCTTTTGTTGAATTCA GAGGAAGACCCGCGACGATTGATGCCTTGCTGCGCCATAACGGGATTT TATAAAACAGGACTTGCGCCCCCCAATCCTCTCGGTAAAACATTTTTT GCCTTGACCTTGGGGTTTTCCGTAAGTCCTGAAAATATATTCGGTATGCT GGCGGGATTTTGCTTACATGCCGGCATTTATGTTATAATTAAAGTGTAC AGAATGGGGGGCGACCTGGCT TCGACGTGGGTTGCAAAACCGGAAGT GCATGCCGAGAAGGAGATCTCTCGTAAATAAGACTCAATTAAATATAAA TGCAAACGATGAAAACTTTGCTGGTGGGGAAGCTATCGCTGCCTAATA AGCACTTTAGTTAAACCATCACTGTGTACTGGCCAATAAACCCAGTATC CCGTTCGACCGAGCCCGCTTATCGGTATCGAATCAACGGTCATA AGAGA TAAGCTAGCGTCCTAATCTATCCCGGGTTATGGCGCGAAACTCAGGGAA TCGCTGTGTATCATCCTGCCCGTCGGAGGAGCCACAGTTAAATTCAAA AGACAAGGCTATGCATGTAGAGCTAAAGGCAGAGGACTTGCGGACGC GGGTTCGATTCCCGCCGCCTCCACCAATTCATTATCCGATACAGTCCAAT ACCGGGTCTTTCCCAAATACCTGAATCTTCTACACATCTTGTTTATTCCA AACAAACATGATCAAATCACCTCTTTTTGAGGTATGTATGGACTTAGCA GTTGAAGATACTACAGCATGGTCGGAAGCTATTTTTGGTTCAGTTGCTT TAGGGGATAAACGACTTACTCGTCGGTTAATTCAAATAGGCAAACAATT ATCATCGACGCCTGGTGGTTCTCTTTCAGGAAGTTGTGGAGGGCAGGA TGCGCTTATAGAAGGTAGTTATCGTTTTTTACGAAACAAACGAGTCACAGCGAATCAAATTGCAGAGGGTGGTT(SEQ ID NO:7)

[0099] In some embodiments, the ssrA target nucleic acid comprises 5'TCGACGTGGGTTGC AAAACCGGAAGTGCATGCCGAGAAGGAGATCTCTCGTAAATAAGACTCAATTAAATATAAATGCAAACGATGAAAACTTTGCTGGTGGGGAAGCTATCGCTGCCTAATAAGCACTTTAGTTAAACCATCACTGTGTACTGGCCAATAAACCCAGTATCCCGTTCGACCGAGCCCGCTTATCGGTATCGAATCAACGGTCATA 3' (SEQ ID NO: 10) or a fragment thereof.

[0100] Exemplary primers and labeled probe sequences for amplification and detection of ssrA target sequences include:

[0101] In some embodiments, the ssrA detection assay does not detect bacteria other than Legionella species. For example, in some embodiments, the ssrA assay comprises PCR primers and probes that are specific for Legionella species and do not amplify nucleic acids derived from one or more of: Bacillus cereus, Chlamydophilapneumoniae, Haemophilus influenzae, Klebsiella pneumonia, RSV B, Mycoplasma pneumonia, Streptococcus pneumoniae, Staphylococcus aureus, Moraxella catarrhalis, Influenza A, Influenza B, Pseudomonas sp., and Enterobacter sp.

[0102] Qualitative detection and differentiation of Legionella species and Legionella pneumophila using the disclosed method can utilize primer pairs, labeled probes, and real-time PCR to amplify and detect ssrA and 16SrRNA genes on a direct amplification plate with an integrated cycler system. Using this method, target genomic DNA is specifically amplified and simultaneously detected in the same reaction by fluorescently labeled probes.

[0103] In some embodiments, one or more primers and / or probes used herein are degenerate primers or probes, meaning a mixture of oligonucleotide sequences in which some positions contain multiple possible bases, thereby obtaining a population of primers with similar sequences that cover all possible nucleotide combinations of a given protein sequence. Degenerate nucleotides are designated by R. A non-limiting example of a degenerate primer is SEQ ID NO: 1.

[0104] In some embodiments, a positive control comprising a nucleic acid derived from a Legionella pneumophila stock organism is used for screening assays. For example, suitable control DNA can be obtained from Microbiologics (Cat. No. 0211P). In some embodiments, the positive control is diluted in a buffer (such as TE buffer or water). In some embodiments, the dilution in the buffer is about 1:1,000, 1:10,000, 1:100,000, 1:1,000,000 or 1:10,000,000.

[0105] In some embodiments, an amplification or extraction control comprising exogenous nucleic acid is used for screening assays. In some embodiments, the amplification or extraction control sample does not contain nucleic acids derived from Legionella species. For example, suitable control DNA can be obtained from Diasorin (Cat. No. 151599). In some embodiments, the amplification or extraction control is diluted in a buffer (such as TE buffer or water). In some embodiments, the dilution in the buffer is about 1:1,000, 1:10,000, 1:100,000, 1:1,000,000 or 1:10,000,000. In some embodiments, before amplifying the target nucleic acid, the amplification or extraction control sample is mixed with the biological sample.

[0106] In some embodiments, the control nucleic acid sequence comprises 5′TAACCCCGCGATAAAGACAGAAGATTATGCATACGAGATCAAAGGAGCCGGCCTTTTCTCTAGAGATCTCTTATTTTCCTTGAAGTCACCTGTTTATGTTAAAGCAGGTGAGCAGGTATACATTCAGTACGATCTGAACAAAAGCAATGCAGAACTTGCTCTCGACTATGGTTTTGTGGAATCAAACCCTAAACGGAACTCATATACTTTAACAATAGAGATACCAGAATCAGACCCATTCTTTGGGGATAAGTTGGATATTGCTGAGAGTAACAAGATGGGTGAGACCGGATACTTTGACATAGTAGACGGCCAGACTCTTCCCGCTGGTAT GCT TCAGTACCTTCGGCTTGTGGCTCTTGGCGGTCCAGATGCTTCTTATTAGAATCTATCTTCAATAACACCATATGG GGTCATCTTGAATTGCCTGTAAGTCGTACAAACGAGGAACTCATATGCCGTGTTGTCAGAGATGCCTGCAA ATCTGCTCTGTCTGGTTTTGATACGACCATTGAAGAGGATGAGAAGCTTCTGGACAAAGGAAAGCTTGAGCCTAGGTTGGAAATGGCTCTCAAG 3' (SEQ ID NO: 13), and a control nucleic acid sequence was amplified using a forward primer comprising 5'GCTTCAGTACCTTCGGCTTG 3' (SEQ ID NO: 17), a reverse primer comprising 5'TTGCAGGCATCTCTGACAAC 3' (SEQ ID NO: 18), and a detectably labeled nucleic acid probe comprising 5'TGGCTCTTGGCGGTCCAGATG 3' (SEQ ID NO: 19).

[0107] In some embodiments, the control target nucleic acid comprises 5'GCTTCAGTACCTTCGGCTTGTGGCTCTTGGCGGTCCAGATGCTTTCTTATTAGAATCTATCTTCAATAACACCATATGGGGTCATCTTGAATTGCCTGTAAGTCGTACAAACGAGGAACTCATATGCCGTGTTGTCAGAGATGCCTGCAA 3' (SEQ ID NO: 20).

[0108] In some embodiments, a negative control that does not contain nucleic acid derived from Legionella spp. is used in the screening assay. Non-limiting examples of suitable negative controls include nuclease-free water, sterile nuclease-free water, and TE buffer.

[0109] Thus, in some aspects, provided herein are methods for detecting the presence of at least one Legionella species in a biological sample, the methods comprising, consisting of, or consisting essentially of: (a) providing a first primer pair suitable for amplifying a ssrA target nucleic acid; providing a second primer pair suitable for amplifying a 16S rRNA target nucleic acid; amplifying the ssrA target nucleic acid and the 16S rRNA target nucleic acid, if present; and detecting one or more amplification products produced in step (c); wherein the presence of the ssrA target nucleic acid identifies the presence of at least one Legionella species, and the presence of the 16S rRNA target nucleic acid identifies the presence of Legionella pneumophila. Treatment of Legionella infection

[0110] Disclosed herein are methods for determining whether a patient exhibiting pneumonia-like symptoms would benefit from treatment with a therapeutic agent that inhibits Legionella spp. and / or Legionella pneumophila.

[0111] Thus, provided herein are methods for selecting a subject exhibiting pneumonia-like symptoms for treatment with a therapeutic agent that inhibits Legionella pneumophila, the method comprising, consisting of, or consisting essentially of: (a) contacting a sample isolated from the subject with a first primer pair suitable for amplifying a ssrA target nucleic acid; (b) contacting the sample with a second primer pair suitable for amplifying a 16S rRNA target nucleic acid; (c) amplifying the ssrA target nucleic acid and the 16S rRNA target nucleic acid, if present; and (d) detecting one or more amplification products produced in step (c); and (e) if an amplification product of the 16S rRNA target nucleic acid is detected, selecting the subject for treatment with a therapeutic agent that inhibits Legionella pneumophila.

[0112] Also provided herein are methods of treating a subject having a Legionella pneumophila infection, the methods comprising administering to a subject selected by a method comprising, consisting of, or consisting essentially of: (a) contacting a sample isolated from the subject with a first primer pair suitable for amplifying an ssrA target nucleic acid; (b) contacting the sample with a second primer pair suitable for amplifying a 16S rRNA target nucleic acid; (c) amplifying the ssrA target nucleic acid and the 16S rRNA target nucleic acid, if present; and (d) detecting one or more amplification products produced in step (c); and (e) if an amplification product of the 16S rRNA target nucleic acid is detected, selecting the subject for treatment with the Legionella pneumophila inhibitory therapeutic.

[0113] In some embodiments of the methods provided herein, the first primer pair comprises at least one degenerate primer. In some embodiments, the first primer pair comprises a first forward primer, and the first forward primer comprises 5'TCGACGTGGGTTGCRAAACG 3' (SEQ ID NO: 1) or its complement. According to the method according to any one of the preceding claims, the first primer pair comprises a first reverse primer, and the first reverse primer comprises 5'TATGACCGTTGATTCG ATACC 3' (SEQ ID NO: 2) or its complement. In some embodiments, the second primer pair comprises at least one degenerate primer. In some embodiments, the second primer pair comprises a second forward primer, and the second forward primer comprises 5'TACCTACCCTTGACATACAGTG 3' (SEQ ID NO: 4) or its complement. In some embodiments, the second primer pair comprises a second reverse primer, and the second reverse primer comprises 5'CTTCCTCCGGTTTGTCAC3' (SEQ ID NO: 5) or its complement.

[0114] In some embodiments, the method also includes contacting the biological sample with one or more oligonucleotide probes that can specifically hybridize with the amplification product or its complement. In some embodiments, the oligonucleotide probe is detectably labeled. In some embodiments, the detectable label is a fluorescent label. In some embodiments, the fluorescent label is selected from fluorescein, Cy3, Cy5, Cy5.5, tetrachloro-6-carboxyfluorescein, 2,7-dimethoxy-4,5-dichloro-6-carboxyl-fluorescein, Yakima yellow, Texas red, TYE 563, ROX, TEX 615, TYE 665, TYE 705 and hexachloro-6-carboxyfluorescein. In some embodiments, the oligonucleotide probe also includes at least one quencher. In some embodiments, the quencher is selected from TAMRA, Black Hole quencher, Deep Dark quencher, ZEN, Iowa Black FQ, Iowa Black RQ and DABCYL. In some embodiments, the oligonucleotide probe specifically hybridizes to the ssrA amplification product, and wherein the oligonucleotide probe comprises 5'TAAATATAAATGCAAACGATGAAAACTTTGC 3' (SEQ ID NO: 3) or its complement. In some embodiments, the oligonucleotide probe specifically hybridizes to the 16S rRNA amplification product, and wherein the oligonucleotide probe comprises 5'CCAGCATGTGATGGTGGGGACTCTA 3' (SEQ ID NO: 6) or its complement.

[0115] In some embodiments, the method further comprises mixing the exogenous control DNA with the biological sample. In some embodiments, the method further comprises contacting the biological sample with a third primer suitable for amplifying the exogenous control target nucleic acid, and amplifying the exogenous control target nucleic acid. In some embodiments, the exogenous control target nucleic acid comprises SEQ ID NO: 20. In some embodiments, the third primer is composed of a third forward primer comprising 5'GCTTCAGTACCTTCGGCTTG 3' (SEQ ID NO: 17) and a third reverse primer comprising 5'TTGCAGGCATCTCTGACAAC 3' (SEQ ID NO: 18). In some embodiments, the method further comprises contacting the biological sample with a third oligonucleotide probe, wherein the third oligonucleotide probe is detectably labeled and comprises 5'TGGCTCTTGGCGGTCCAGATG 3' (SEQ ID NO: 19).

[0116] In some embodiments of the methods provided herein, real-time PCR amplification is performed in a direct amplification plate in conjunction with an integrated thermal cycler.

[0117] In some embodiments of the methods provided herein, the biological sample is a bronchoalveolar lavage sample, a bronchial wash sample, a sputum sample, a nasopharyngeal (NP) aspirate or wash sample, a nasal swab, or a bacterial isolate.

[0118] Examples of therapeutic agents that inhibit Legionella species and / or Legionella pneumophila include fluoroquinolones, carbapenems, trimethoprim-sulfamethoxazole (e.g., Bactrim, Septra), and Legionella pneumophila-specific antibodies. In some embodiments, fluoroquinolones are selected from ciprofloxacin, gemifloxacin, levofloxacin, norfloxacin, ofloxacin, rovafloxacin, gatifloxacin, grepafloxacin, temafloxacin, lomefloxacin, sparfloxacin, enoxacin, and moxifloxacin. In certain embodiments, carbapenems are selected from imipenem, meropenem, ertapenem, doripenem, panipenem, biapenem, lazupenem (PZ-601), tebipenem, lenapenem, tomopenem, and siempenem (thienomycin).

[0119] Examples of therapeutic agents that inhibit Legionella species and / or Legionella pneumophila include whole cell (wP) Legionella species and / or Legionella pneumophila vaccines, acellular Legionella species and / or Legionella pneumophila vaccines, trimethoprim-sulfamethoxazole (e.g., sulfamethoxazole, co-trimoxazole), telithromycin, and macrolide antibiotics. In some embodiments, the macrolide antibiotic is selected from azithromycin (Zithromax), clarithromycin (Biasi), erythromycin (E-Mycin, Eryc, Ery-Tab, PCE, erythromycin ethylsuccinate, elosone) and roxithromycin.

[0120] Examples of additional therapeutic agents that inhibit Legionella species and / or Legionella pneumophila include trimethoprim-sulfamethoxazole (e.g., sulfamethoxazole, co-trimoxazole), ciprofloxacin, gemifloxacin, levofloxacin, norfloxacin, ofloxacin, rovafloxacin, gatifloxacin, grepafloxacin, temafloxacin, lomefloxacin, sparfloxacin, enoxacin, and moxifloxacin.

[0121] Pneumonia-like symptoms include, but are not limited to, chest pain, confusion, altered mental awareness, cough, sputum, fatigue, fever, sweating, shivering, chills, subnormal body temperature, nausea, vomiting, diarrhea, shortness of breath, lung inflammation, and fluid in the lungs.

[0122] In some embodiments, the subject is a mammal. In some embodiments, the mammal is a cow, horse, pig, cat, dog, mouse, monkey, rat or human. In some embodiments, the subject is a human. In specific embodiments, the subject is a human patient with one or more pneumonia-like symptoms. Interpretation of results

[0123] After performing real-time PCR on the sample-reaction mixture and detecting and measuring the fluorescent signals associated with the amplified ssrA, 16S rRNA and control target sequences, the method of the present technology also provides an algorithm for determining the presence of one or more related pathogenic Legionella species, which provides a final result by matching the cycle threshold (Ct) from the amplified target nucleic acid sequence.

[0124] In some embodiments, for a reaction comprising 40 cycles, a positive Ct is a Ct of less than or equal to about 35, about 36, about 37, about 38, about 39, or about 40. In some embodiments, for a reaction comprising 40 cycles, a negative Ct is a Ct of greater than about 35, about 36, about 37, or about 38, about 39, or about 40. In some embodiments, for a reaction comprising 45 cycles, a positive Ct is a Ct of less than or equal to about 40, about 41, about 42, about 43, about 44, or about 45. In some embodiments, for a reaction comprising 45 cycles, a negative Ct is a Ct of greater than or equal to about 40, about 41, about 42, about 43, or about 44, or about 45. In some embodiments, for a reaction comprising 50 cycles, a positive Ct is a Ct of less than or equal to about 45, about 46, about 47, about 48, about 49, or about 50. In some embodiments, a negative Ct is a Ct of greater than about 45, about 46, about 47, about 48, or about 49, or a Ct of about 50 for a reaction comprising 50 cycles.

[0125] The Legionella species algorithm states: Negative control Positive Control

[0126] Therefore, the presence or absence of pathogenic Legionella species in a biological sample can be determined based on: Kits and Compositions

[0127] The present disclosure also provides kits for detecting a target nucleic acid sequence corresponding to a pathogenic Legionella species.

[0128] Thus, provided herein is a kit for detecting the presence of at least one Legionella species in a biological sample, the kit comprising, consisting of, or consisting essentially of: (a) a first primer pair that amplifies an ssrA target nucleic acid; (b) a second primer pair that amplifies a 16S rRNA target nucleic acid; (c) a first oligonucleotide probe that is capable of specifically hybridizing to a segment of the ssrA target nucleic acid; and (d) a second oligonucleotide probe that is capable of specifically hybridizing to a segment of the 16S rRNA target nucleic acid; wherein the first oligonucleotide probe and the second oligonucleotide probe are detectably labeled.

[0129] In some embodiments of the kits provided herein, the kits further comprise a third primer pair for amplifying a control target nucleic acid. In some embodiments, the first primer pair is capable of specifically hybridizing to a ssrA target nucleic acid comprising nucleotides at least 85%-95% identical to SEQ ID NO:7 or its complement. In some embodiments, the second primer pair is capable of specifically hybridizing to a 16S rRNA target nucleic acid comprising nucleotides at least 85%-95% identical to SEQ ID NO:8 or its complement.

[0130] In some embodiments of the kit provided herein, the first primer pair comprises at least one degenerate primer. In some embodiments, the first primer pair comprises a first forward primer, and the first forward primer comprises 5'TCGACGTGGGTTGCRAAACG 3' (SEQ ID NO: 1) or its complement. In some embodiments, the first primer pair comprises a first reverse primer, and the first reverse primer comprises 5'TATGACCGTTGATTCGATACC 3' (SEQ ID NO: 2) or its complement. In some embodiments, the second primer pair comprises at least one degenerate primer. In some embodiments, the second primer pair comprises a second forward primer, and the second forward primer comprises 5'TACCTACCCTTGACATACAGTG 3' (SEQ ID NO: 4) or its complement. In some embodiments, the second primer pair comprises a second reverse primer, and the second reverse primer comprises 5'CTTCCTCCGGTTTGTCAC 3' (SEQ ID NO: 5) or its complement. In some embodiments, the first nucleic acid probe comprises 5'TAAATATAAATGCAAACGATGAAAACTTTGC 3' (SEQ ID NO: 3) or its complement. In some embodiments, the second nucleic acid probe comprises 5'CAACCAGCCGCTGCTGACGGTC 3' (SEQ ID NO: 9) or its complement.

[0131] In some embodiments of the kit provided herein, detectable label is a fluorescent label. In some embodiments, the fluorescent label is selected from fluorescein, Cy3, Cy5, Cy5.5, tetrachloro-6-carboxyfluorescein, 2,7-dimethoxy-4,5-dichloro-6-carboxyl-fluorescein, Yakiwa Yellow, Texas Red, TYE 563, ROX, TEX 615, TYE 665, TYE 705 and hexachloro-6-carboxyfluorescein. In some embodiments, at least one oligonucleotide probe also includes at least one quencher. In some embodiments, the oligonucleotide probe includes two quenchers. In some embodiments, the quencher is selected from TAMRA, Black Hole quencher, Deep Dark quencher, ZEN, Iowa Black FQ, Iowa Black RQ and DABCYL.

[0132] The kits of the present technology comprise at least two oligonucleotides that can be used as primers or primer-probes for amplifying ssrA and 16SrRNA target nucleic acid sequences to determine the presence of pathogenic Legionella species in a biological sample.

[0133] In some embodiments, the kit comprises a liquid culture medium containing at least one target-specific nucleic acid probe at a concentration of 250 nM or lower. Using such a kit, probes are provided in a desired amount to carry out reliable multiple detection reactions according to the technology of the present invention. In some embodiments, the target-specific nucleic acid probe is detectably labeled.

[0134] In some embodiments, the kit further comprises a buffer required for performing an assay or reaction (such as amplifying and / or detecting a target nucleic acid sequence corresponding to a pathogenic Legionella species), an enzyme having polymerase activity, an enzyme having polymerase activity and lacking 5'→3' exonuclease activity or both 5'→3' and 3'→5' exonuclease activities, an enzyme cofactor (such as magnesium or manganese), a salt, a chain-extending nucleotide (such as a deoxynucleoside triphosphate (dNTP), a modified dNTP, a nuclease-resistant dNTP, or a labeled dNTP).

[0135] In one embodiment, the kit of the present technology also includes a positive control nucleic acid sequence and a negative control nucleic acid sequence to ensure the integrity of the assay during the experimental run. The kit may also contain a tool for comparing the number of copies of one or more of ssrA and 16S rRNA in a biological sample with a reference nucleic acid sample (e.g., a sample with a known number of copies of one or more of ssrA and 16S rRNA). The kit may also include instructions for use, software for automated analysis, containers, packaging (such as packaging intended for commercial sale), etc.

[0136] The test kit may also include one or more of the following: washing buffer and / or reagents, hybridization buffer and / or reagents, labeling buffer and / or reagents and detection means. The buffer and / or reagents are usually optimized for the specific amplification / detection techniques intended to use the test kit. The protocol for the different steps of the procedure using these buffers and reagents may also be included in the test kit.

[0137] The test kit may additionally include an assay definition scan card and / or instructions for using the oligonucleotide in the assay, such as printed instructions or electronic instructions. In some embodiments, the test kit includes an amplification reaction mixture or an amplification master mix. The reagents included in the test kit may be contained in one or more containers (such as vials).

[0138] Primers, probes and / or primer-probes specific for amplification and detection of DNA internal controls can be included in the amplification master mix as target primer pairs to monitor potential PCR inhibition. The reagents required for amplification and detection of the target and internal controls can be formulated as an all-in-one amplification master mix, which can be provided in the kit as a single reaction aliquot.

[0139] In one aspect, provided herein is a composition comprising, consisting of or consisting essentially of a detectably labeled oligonucleotide probe, the oligonucleotide probe comprising 5'TAAATATAAATGCAAACGATGAAAACTTTGC 3' (SEQ ID NO: 3). In some embodiments, the detectable label is a fluorescent label. In some embodiments, the fluorescent label is selected from fluorescein, Cy3, Cy5, Cy5.5, tetrachloro-6-carboxyfluorescein, 2,7-dimethoxy-4,5-dichloro-6-carboxyfluorescein, Yakima Yellow, Texas Red, TYE 563, ROX, TEX 615, TYE 665, TYE 705 and hexachloro-6-carboxyfluorescein. In some embodiments, the oligonucleotide probe also includes at least one quencher. In some embodiments, the quencher is selected from TAMRA, Black Hole quencher, Deep Dark quencher, ZEN, Iowa Black FQ, Iowa Black RQ and DABCYL.

[0140] In another aspect, provided herein is a composition comprising a detectably labeled oligonucleotide probe comprising 5'TAAATATAAATGCAAACGATGAAA ACTTTGC 3' (SEQ ID NO: 3). In some embodiments, the detectable label is a fluorescent label. In some embodiments, the fluorescent label is selected from fluorescein, Cy3, Cy5, Cy5.5, tetrachloro-6-carboxyfluorescein, 2,7-dimethoxy-4,5-dichloro-6-carboxyfluorescein, Yakima Yellow, Texas Red, TYE563, ROX, TEX 615, TYE 665, TYE 705 and hexachloro-6-carboxyfluorescein. In some embodiments, the oligonucleotide probe also includes at least one quencher. In some embodiments, the quencher is selected from TAMRA, Black Hole quencher, Deep Dark quencher, ZEN, Iowa Black FQ, Iowa Black RQ and DABCYL. Example Example 1: Detection of pathogenic Legionella species using real-time PCR

[0141] Bronchoalveolar lavage fluid samples were collected from patients. An internal positive control DNA target was added to the external lysis buffer prior to extraction. DNA was extracted using the MagNA Pure 96 instrument using the "DNA / Virus NA SV 2.0" kit. The elution volume was set to 50.

[0142] A Legionella PCR master mix containing the following reagents was generated and stored at -10°C to -90°C:

[0143] Real-time PCR was performed using the following conditions: i) preheating samples at 50°C for 120 seconds, 1 cycle; ii) activating the polymerase at 95°C for 10 minutes, 1 cycle; and iii) denaturing at 95°C for 15 seconds and annealing at 60°C for 35 seconds, 40 cycles.

[0144] The target genomic DNA is specifically amplified and simultaneously detected in the same reaction by a fluorescently labeled probe. The Ct is detected and the results are analyzed according to the following algorithm: Example 2: Cross-reactivity of multiplex assays for Legionella.

[0145] For cross-reactivity determination, control nasal swab specimens will be spiked with one of the test organisms listed below (n=5 per organism). Bacillus cereus Chlamydophila pneumoniae Haemophilus influenzae Klebsiella pneumoniae RSV B Mycoplasma pneumoniae Streptococcus pneumoniae Staphylococcus aureus Moraxella catarrhalis Influenza A virus Influenza B virus Pseudomonas species Enterobacter species

[0146] Each sample will be subjected to the Legionella multiplex assay. In each case, a Ct value of ≤ 40 is interpreted as a positive result for Legionella cross-reactivity, a Ct value of ≤ 40 is interpreted as a positive result for Legionella cross-reactivity, and a Ct value of ≤ 40 is interpreted as a positive result for Legionella cross-reactivity.

[0147] It is expected that no cross-reactivity will be observed with any of the above-mentioned microbial species. Equivalent

[0148] The technology of the present invention is not limited to the specific embodiments described in this application, and the embodiments are intended to be a single description of the individual aspects of the technology of the present invention. As those skilled in the art should be aware, many modifications and changes can be made to the technology of the present invention without departing from the spirit and scope of the technology of the present invention. It should be clear to those skilled in the art based on the foregoing description that, in addition to the methods and equipment listed herein, the functionally equivalent methods and equipment within the scope of the technology of the present invention. Such modifications and changes are intended to fall within the scope of the technology of the present invention. It should be understood that the technology of the present invention is not limited to specific methods, reagents, compounds, compositions or biological systems, and they can certainly change. It should also be understood that the terms used herein are only used for the purpose of describing specific embodiments and are not intended to be restrictive.

[0149] The terms "comprising", "including", "containing", etc. should be understood in a broad and non-restrictive manner. Additionally, the terms and expressions employed herein have been used as terms of description rather than limitation, and it is not intended that any equivalents of the features shown and described or portions thereof be excluded by the use of such terms and expressions, but it should be recognized that various modifications are possible within the scope of the present disclosure as claimed.

[0150] In addition, where features or aspects of the disclosure are described in terms of Markush groups, those skilled in the art will recognize that the disclosure is also thereby described in terms of any individual member or subgroup of members of the Markush group.

[0151] As will be understood by those skilled in the art, for any and all purposes, particularly with respect to providing a written description, all ranges disclosed herein also encompass any and all possible sub-ranges and combinations of sub-ranges thereof. Any listed range can be easily identified as fully describing the same range and enabling the same range to be decomposed into at least equal halves, thirds, quarters, fifths, tenths, etc. As a non-limiting example, each range discussed herein can be easily decomposed into lower thirds, middle thirds, and upper thirds, etc. As will be equally understood by those skilled in the art, all words such as "up to", "at least", "greater than", "less than", etc. include the numbers and relate to the ranges that can subsequently be decomposed into sub-ranges as discussed above. Finally, as will be understood by those skilled in the art, the range includes each individual member. Therefore, for example, a group having 1-3 cells refers to a group having 1, 2, or 3 cells. Similarly, a group having 1-5 cells refers to a group having 1, 2, 3, 4, or 5 cells, etc.

[0152] All patents, patent applications, provisional applications, and publications mentioned or cited herein are incorporated by reference in their entirety, including all figures and tables, to the extent they are not inconsistent with the explicit teachings of this specification.

[0153] The present disclosure relates to the following embodiments. 1. A method for detecting the presence of at least one Legionella species in a biological sample, the method comprising: (a) providing a first primer pair suitable for amplifying a ssrA target nucleic acid; (b) providing a second primer pair suitable for amplifying a 16S rRNA target nucleic acid; (c) amplifying the ssrA target nucleic acid and the 16S rRNA target nucleic acid, if present; and (d) detecting one or more amplification products produced in step (c); wherein the presence of the ssrA target nucleic acid identifies the presence of at least one Legionella species, and the presence of the 16S rRNA target nucleic acid identifies the presence of Legionella pneumophila. 2. A method according to claim 1, wherein the first primer pair comprises at least one degenerate primer. 3. The method according to item 1 or 2, wherein the first primer pair comprises a first forward primer comprising 5'TCGACGTGGGTTGCRAAACG 3' (SEQ ID NO: 1) or its complement. 4. The method according to any one of the preceding items, wherein the first primer pair comprises a first reverse primer comprising 5'TATGACCGTTGATTCGATACC 3' (SEQ ID NO: 2) or its complement. 5. The method according to any one of the preceding items, wherein the second primer pair comprises at least one degenerate primer. 6. The method according to any of the preceding items, wherein the second primer pair comprises a second forward primer comprising 5'TACCTACCCTTGACATACAGTG 3' (SEQ ID NO: 4) or its complement. 7. The method according to any of the preceding items, wherein the second primer pair comprises a second reverse primer comprising 5'CTTCCTCCGGTTTGTCAC 3' (SEQ ID NO: 5) or its complement. 8. The method according to any of the preceding items, further comprising contacting the biological sample with one or more oligonucleotide probes capable of specifically hybridizing to the amplification product or its complement. 9. A method according to claim 8, wherein the oligonucleotide probe is detectably labeled. 10. A method according to claim 9, wherein the detectable label is a fluorescent label. 11. A method according to claim 10, wherein the fluorescent label is selected from fluorescein, Cy3, Cy5, Cy5.5, tetrachloro-6-carboxyfluorescein, 2,7-dimethoxy-4,5-dichloro-6-carboxyfluorescein, Yakima Yellow, Texas Red, TYE 563, ROX, TEX 615, TYE 665, TYE 705 and hexachloro-6-carboxyfluorescein. 12. A method according to item 10, wherein the oligonucleotide probe further comprises at least one quencher. 13. A method according to item 12, wherein the quencher is selected from TAMRA, Black Hole quencher, DeepDark quencher, ZEN, Iowa Black FQ, Iowa Black RQ and DABCYL. 14. A method according to any one of items 8 to 13, wherein the oligonucleotide probe specifically hybridizes to a ssrA amplification product, and wherein the oligonucleotide probe comprises 5'TAAATATAAATGCAAACGATGAAAACTTTGC 3' (SEQ ID NO: 3) or its complement. 15. The method according to any one of items 8 to 14, wherein the oligonucleotide probe specifically hybridizes to a 16S rRNA amplification product, and wherein the oligonucleotide probe comprises 5'CCAGCATGTGATGGTGGGGACTCTA 3' (SEQ ID NO: 6) or its complement. 16. The method according to any one of items 1-15, further comprising mixing exogenous control DNA with the biological sample. 17. The method according to item 16 further comprises contacting the biological sample with a third primer pair suitable for amplifying an exogenous control target nucleic acid, and amplifying the exogenous control target nucleic acid. 18. A method according to item 17, wherein the exogenous control target nucleic acid comprises SEQ ID NO:20. 19. The method according to item 18, wherein the third primer pair consists of a third forward primer comprising 5'GCTTCAGTACCTTCGGCTTG 3' (SEQ ID NO: 17) and a third reverse primer comprising 5'TTGCAGGCATCTCTGACAAC 3' (SEQ ID NO: 18). 20. The method according to item 17 or 18, further comprising contacting the biological sample with a third oligonucleotide probe, wherein the third oligonucleotide probe is detectably labeled and comprises 5'TGGCTCTTGGCGGTCCAGATG 3' (SEQ ID NO: 19). 21. A method according to any one of items 1-20, wherein real-time PCR amplification is performed in a direct amplification plate in cooperation with an integrated thermal cycler. 22. A method according to any one of items 1-21, wherein the biological sample is a bronchoalveolar lavage fluid sample, a bronchial wash sample, a sputum sample, a nasopharyngeal (NP) aspirate or wash sample, a nasal swab, or a bacterial isolate. 23. A kit for detecting the presence of at least one Legionella species in a biological sample, the kit comprising: (a) a first primer pair for amplifying a ssrA target nucleic acid; (b) a second primer pair for amplifying a 16S rRNA target nucleic acid; (c) a first oligonucleotide probe capable of specifically hybridizing to a segment of the ssrA target nucleic acid; and (d) a second oligonucleotide probe capable of specifically hybridizing to a segment of the 16S rRNA target nucleic acid; Wherein the first oligonucleotide probe and the second oligonucleotide probe are detectably labeled. 24. A kit according to item 23, further comprising a third primer pair for amplifying a control target nucleic acid. 25. A kit according to item 23 or 24, wherein the first primer pair is capable of specifically hybridizing to a ssrA target nucleic acid comprising nucleotides at least 85%-95% identical to SEQ ID NO: 1 or its complement. 26. A kit according to any one of items 23-25, wherein the second primer pair is capable of specifically hybridizing to a 16S rRNA target nucleic acid comprising nucleotides that are at least 85%-95% identical to SEQ ID NO: 2 or a complement thereof. 27. A kit according to any one of items 23-26, wherein the first primer pair comprises at least one degenerate primer. 28. The kit according to any one of items 23 to 27, wherein the first primer pair comprises a first forward primer comprising 5'TCGACGTGGGTTGCRAAACG 3' (SEQ ID NO: 1) or its complement. 29. The kit according to any one of items 23 to 28, wherein the first primer pair comprises a first reverse primer comprising 5'TATGACCGTTGATTCGATACC 3' (SEQ ID NO: 2) or a complement thereof. 30. A kit according to any one of items 23-29, wherein the second primer pair comprises at least one degenerate primer. 31. The kit according to any one of items 23 to 30, wherein the second primer pair comprises a second forward primer comprising 5'TACCTACCCTTGACATACAGTG 3' (SEQ ID NO: 4) or its complement. 32. The kit according to any one of items 23-31, wherein the second primer pair comprises a second reverse primer comprising 5'CTTCCTCCGGTTTGTCAC 3' (SEQ ID NO: 5) or its complement. 33. The kit according to any one of items 23-32, wherein the first nucleic acid probe comprises 5'TAAATATAAATGCAAACGATGAAAACTTTGC 3' (SEQ ID NO: 3) or its complement. 34. The kit according to any one of items 23-33, wherein the second nucleic acid probe comprises 5'CAACCAGCCGCTGCTGACGGTC 3' (SEQ ID NO: 9) or its complement. 35. A kit according to any one of items 23-34, wherein the detectable label is a fluorescent label. 36. A kit according to claim 35, wherein the fluorescent label is selected from fluorescein, Cy3, Cy5, Cy5.5, tetrachloro-6-carboxyfluorescein, 2,7-dimethoxy-4,5-dichloro-6-carboxyfluorescein, Yakima Yellow, Texas Red, TYE563, ROX, TEX 615, TYE 665, TYE 705 and hexachloro-6-carboxyfluorescein. 37. A kit according to any one of items 23-36, wherein at least one oligonucleotide probe further comprises at least one quencher. 38. A kit according to claim 37, wherein the quencher is selected from TAMRA, Black Hole quencher, DeepDark quencher, ZEN, Iowa Black FQ, Iowa Black RQ and DABCYL. 39. A composition comprising a detectably labeled oligonucleotide probe comprising 5'TAAATATAAATGCAAACGATGAAAACTTTGC 3' (SEQ ID NO: 3). 40. A composition according to item 39, wherein the detectable label is a fluorescent label. 41. A composition according to item 40, wherein the fluorescent label is selected from fluorescein, Cy3, Cy5, Cy5.5, tetrachloro-6-carboxyfluorescein, 2,7-dimethoxy-4,5-dichloro-6-carboxyfluorescein, Yakima Yellow, Texas Red, TYE563, ROX, TEX 615, TYE 665, TYE 705 and hexachloro-6-carboxyfluorescein. 42. A composition according to any one of items 39-41, wherein the oligonucleotide probe further comprises at least one quencher. 43. A kit according to item 42, wherein the quencher is selected from TAMRA, Black Hole quencher, DeepDark quencher, ZEN, Iowa Black FQ, Iowa Black RQ and DABCYL. 44. A method for selecting a subject exhibiting pneumonia-like symptoms for treatment with a therapeutic agent that inhibits Legionella pneumophila, the method comprising: (a) contacting a sample isolated from the subject with a first primer pair suitable for amplifying a ssrA target nucleic acid; (b) contacting the sample with a second primer pair suitable for amplifying a 16S rRNA target nucleic acid; (c) amplifying the ssrA target nucleic acid and the 16S rRNA target nucleic acid, if present; and (d) detecting one or more amplification products produced in step (c); and (e) if the amplification product of the 16S rRNA target nucleic acid is detected, selecting the subject for treatment with a therapeutic agent that inhibits Legionella pneumophila. 45. A method of treating a subject having a Legionella pneumophila infection, the method comprising administering to a subject selected by the method of item 44 a therapeutic agent that inhibits Legionella pneumophila. 46. ​​A method according to item 44 or 45, wherein the first primer pair comprises at least one degenerate primer. 47. A method according to any one of items 44-46, wherein the first primer pair comprises a first forward primer comprising 5'TCGACGTGGGTTGCRAAACG 3' (SEQ ID NO: 10) or its complement. 48. The method according to any one of items 44-47, wherein the first primer pair comprises a first reverse primer comprising 5'TATGACCGTTGATTCGATACC 3' (SEQ ID NO: 2) or its complement. 49. A method according to any one of items 44-48, wherein the second primer pair comprises at least one degenerate primer. 50. The method according to any one of items 44-49, wherein the second primer pair comprises a second forward primer comprising 5'TACCTACCCTTGACATACAGTG 3' (SEQ ID NO: 4) or its complement. 51. A method according to any one of items 44-50, wherein the second primer pair comprises a second reverse primer, the second reverse primer comprising 5'CTTCCTCCGGTTTGTCAC 3' (SEQ ID NO: 5) or its complement. 52. The method according to any one of items 44-51, further comprising contacting the biological sample with one or more oligonucleotide probes capable of specifically hybridizing to the amplification product or its complement. 53. A method according to claim 52, wherein the oligonucleotide probe is detectably labeled. 54. A method according to claim 53, wherein the detectable marker is a fluorescent marker. 55. A method according to claim 54, wherein the fluorescent label is selected from fluorescein, Cy3, Cy5, Cy5.5, tetrachloro-6-carboxyfluorescein, 2,7-dimethoxy-4,5-dichloro-6-carboxyfluorescein, Yakima Yellow, Texas Red, TYE 563, ROX, TEX 615, TYE 665, TYE 705 and hexachloro-6-carboxyfluorescein. 56. A method according to any one of items 52-55, wherein the oligonucleotide probe further comprises at least one quencher. 57. A method according to claim 56, wherein the quencher is selected from TAMRA, Black Hole quencher, DeepDark quencher, ZEN, Iowa Black FQ, Iowa Black RQ and DABCYL. 58. A method according to any one of items 52-57, wherein the oligonucleotide probe specifically hybridizes to a ssrA amplification product, and wherein the oligonucleotide probe comprises 5'TAAATATAAATGCAAACGATGAAAACTTTGC 3' (SEQ ID NO: 3) or its complement. 59. A method according to any one of items 52-58, wherein the oligonucleotide probe specifically hybridizes to a 16S rRNA amplification product, and wherein the oligonucleotide probe comprises 5'CCAGCATGTGATGGTGGGGACTCTA 3' (SEQ ID NO: 6) or its complement. 60. The method according to any one of items 44-59, further comprising mixing exogenous control DNA with the biological sample. 61. The method according to item 60 further comprises contacting the biological sample with a third primer pair suitable for amplifying an exogenous control target nucleic acid, and amplifying the exogenous control target nucleic acid. 62. A method according to item 61, wherein the exogenous control target nucleic acid comprises SEQ ID NO:20. 63. A method according to item 62, wherein the third primer pair consists of a third forward primer comprising 5'GCTTCAGTACCTTCGGCTTG 3' (SEQ ID NO: 17) and a third reverse primer comprising 5'TTGCAGGCATCTCTGACAAC 3' (SEQ ID NO: 18). 64. The method according to item 61 or 62, further comprising contacting the biological sample with a third oligonucleotide probe, wherein the third oligonucleotide probe is detectably labeled and comprises 5'TGGCTCTTGGCGGTCCAGATG 3' (SEQ ID NO: 19). 65. A method according to any one of items 44-64, wherein real-time PCR amplification is performed in a direct amplification plate in cooperation with an integrated thermal cycler. 66. A method according to any one of items 44-65, wherein the biological sample is a bronchoalveolar lavage fluid sample, a bronchial wash sample, a sputum sample, a nasopharyngeal (NP) aspirate or wash sample, a nasal swab, or a bacterial isolate. 67. The method of any one of items 44-66, wherein the therapeutic agent that inhibits Legionella pneumophila is one or more agents selected from the group consisting of fluoroquinolones, carbapenems, macrolide antibiotics, trimethoprim-sulfamethoxazole, Legionella pneumophila-specific antibodies, and Legionella pneumophila-specific vaccines. 68. The method of claim 67, wherein the fluoroquinolone is selected from the group consisting of ciprofloxacin, gemifloxacin, levofloxacin, norfloxacin, ofloxacin, rovafloxacin, gatifloxacin, grepafloxacin, temafloxacin, lomefloxacin, sparfloxacin, enoxacin and moxifloxacin. 69. The method of claim 67, wherein the carbapenem is selected from imipenem, meropenem, ertapenem, doripenem, panipenem, biapenem, lazupenem (PZ-601), tabipenem, lenapenem, tomopenem and cerembenem (thienomycin). 70. The method according to item 67, wherein the Legionella pneumophila-specific vaccine is selected from the group consisting of whole cell (wP) Legionella pneumophila vaccines and acellular Legionella pneumophila vaccines. 71. A method according to claim 67, wherein the macrolide antibiotic is selected from azithromycin (Zithromax), clarithromycin (Biasi), erythromycin (E-Mycin, Eryc, Ery-Tab, PCE, erythromycin ethylsuccinate, ilosone) and roxithromycin.

Claims

1. A method for detecting the presence of at least one Legionella species in a biological sample, the method comprising: (a) providing a first primer pair suitable for amplifying a ssrA target nucleic acid; (b) providing a second primer pair suitable for amplifying a 16S rRNA target nucleic acid; (c) amplifying the ssrA target nucleic acid and the 16S rRNA target nucleic acid, if present; and (d) detecting one or more amplification products produced in step (c); wherein the presence of the ssrA target nucleic acid identifies the presence of at least one Legionella species, and the presence of the 16S rRNA target nucleic acid identifies the presence of Legionella pneumophila.

2. The method of claim 1, wherein the first primer pair comprises at least one degenerate primer. 3 . The method according to claim 1 , wherein the first primer pair comprises a first forward primer comprising 5′TCGACGTGGGTTGCRAAACG 3′ (SEQ ID NO: 1) or a complement thereof.

4. The method according to any one of the preceding claims, wherein the first primer pair comprises a first reverse primer comprising 5'TATGACCGTTGATTCGATACC 3' (SEQ ID NO: 2) or its complement.

5. The method according to any one of the preceding claims, wherein the second primer pair comprises at least one degenerate primer.

6. The method according to any one of the preceding claims, wherein the second primer pair comprises a second forward primer comprising 5'TACCTACCCTTGACATACAGTG 3' (SEQ ID NO: 4) or its complement.

7. The method according to any one of the preceding claims, wherein the second primer pair comprises a second reverse primer comprising 5'CTTCCTCCGGTTTGTCAC 3' (SEQ ID NO: 5) or its complement.

8. The method according to any one of the preceding claims, further comprising contacting the biological sample with one or more oligonucleotide probes capable of specifically hybridizing to the amplification product or its complement.

9. The method of claim 8, wherein the oligonucleotide probe is detectably labeled.

10. The method of claim 9, wherein the detectable label is a fluorescent label.

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