BR112017021135B1 - OLIGONUCLEOTIDE, SUBSTANTIALLY PURE OLIGONUCLEOTIDE PRIMER PAIR, DETECTION METHODS FOR IDENTIFYING THE PRESENCE OR ABSENCE OF PATHOGENIC LEPTOSPIRA, AND OF DIAGNOSING AN INDIVIDUAL IDUS SUSPECTED OF HAVING PATHOGENIC LEPTOSPIRA, AND, KIT - Google Patents

Abstract

OLIGONUCLEOTIDE, SUBSTANTIALLY PURE OLIGONUCLEOTIDE PRIMER PAIR, DETECTION METHODS FOR IDENTIFYING THE PRESENCE OR ABSENCE OF PATHOGENIC LEPTOSPIRA, AND OF DIAGNOSING AN INDIVIDUAL IDUS SUSPECTED OF HAVING PATHOGENIC LEPTOSPIRA, AND, KIT . The present invention provides methods and compositions for determining the presence and/or amount of pathogenic Leptospira in a test sample. In particular, substantially purified oligonucleotide primers and probes are described that can be used to qualitatively and quantitatively detect pathogenic Leptospira nucleic acid in a test sample by amplification methods. The present invention also provides primers and probes for generating and detecting control nucleic acid sequences that provide a convenient method for evaluating the internal quality control of the Leptospira probe.

Description

FIELD OF INVENTION

[001] The present invention relates, in general, to compositions and methods for detecting pathogenic Leptospira in a test sample.

BASICS OF THE INVENTION

[002] The following discussion of the fundamentals of the invention is merely provided to assist the reader in understanding the invention and is not intended to describe or constitute the art prior to the present invention.

[003] Leptospirosis is caused by a water-borne spirochete of the genus Leptospira. Until recently, Leptospira species were grouped by serological data into two species, Leptospira interrogans and the non-pathogenic L. biflexa, together totaling more than 230 serovars. More recently, sequence information has allowed Leptospira to be grouped into 16 genomospecies, including L. interrogans, biflexa, kirshneri, and borgpetersenii. Unfortunately, species cannot be neatly categorized into pathogenic and nonpathogenic because both types of serovars are present in any given genomospecies. Despite this complication, the L. interrogans serovars icterohaemorrhagiae, copenhageni, lai, australis, and autumnalis are among those most commonly found in humans, with icterohaemorrhagiae usually causing the most severe symptoms.

[004] The source of Leptospira infection is through exposure to the urine of an infected animal, although direct contact is not necessary. The infection is often seen in patients who have been in contact with contaminated bodies of water. Leptospira enters the body via cuts and abrasions or through contact with mucous membranes, with incubation lasting 2 to 20 days. The bacteria first infects the blood and then the cerebrospinal fluid (CSF), usually being undetectable in both for about the third week, after the onset of symptoms. Leptospira can be found in urine within a week of the onset of symptoms, and its presence can continue for months or years without treatment.

[005] The symptoms of leptospirosis have a wide range of severity. Infected animals are, for the most part, asymptomatic or have very mild symptoms, and do not seek medical attention. Some, however, have more severe symptoms that can lead to death. Symptoms may occur suddenly and include fever, chills, headache, body aches, abdominal pain, conjunctival suffusion, and sometimes a rash. Headaches and myalgia can be severe, and up to 25% of patients suffer from aseptic meningitis. Between 5% and 10% of all patients with leptospirosis have icteric leptospirosis, sometimes called Weil's disease, which is a more severe condition that is fatal in 5% to 15% of cases. Symptoms include those in anicteric disease and may also include jaundice, liver failure, or acute renal failure in many cases. Respiratory and cardiac involvement is also common and can lead to respiratory distress syndrome or myocarditis.

[006] The detection of Leptospira in the clinical setting is complicated. Serological studies are sometimes time-consuming and complex, and culture can take 6 to 26 weeks. Furthermore, bacteria rapidly lose viability in urine, which is the primary sample type, and culture tests provide limited utility. In contrast, real-time PCR detection is rapid, sensitive, and does not require organism viability. Samples can be frozen or mixed with preservative for transport. Although Leptospira taxonomy is complex, 16S sequence data suggest that pathogenic and non-pathogenic subspecies can be distinguished by Polymerase Chain Reaction (PCR). The described methods and compositions are designed to detect only pathogenic species. Non-pathogenic species such as L. biflexa are not detected. Additionally, the use of PCR-based methods will allow testing of blood, cerebrospinal fluid (CSF), or urine to provide an indication of the stage of infection when tested in advance.

[007] Several reports describe assays of patient samples after a nucleic acid amplification step, such as PCR (Brown et al., “Evaluation of the polymerase chain reaction for early diagnosis of leptospirosis.” J. Med. Microbiol. 43: 110-114, 1995 and Smythe et al., “A quantitative PCR (TaqMan) assay for pathogenic Leptospira spp.” BMC Infectious Diseases 2(13), 2002), but these references do not teach a method for detecting Leptospira DNA. only pathogenic. Other relevant references describe the current understanding of genotypic differences in Leptospira serovars (Levett, P.N., “Leptospirosis”. Clinical Microbiology Reviews. 14(2):296-36, 2001; Brenner et al., “Further determination of DNA relationships between serogroups and serovars in the family Leptospiraceae with a proposal for Leptospira alexanderi sp. nov., “Genetic characterization of pathogenic Leptospira species by DNA hybridization.” Int. J. Syst. 42:215-219, 1992; . J. Syst. Bacteriol. 37:407-415, 1987; Medicine 39:117-156, 1960; Kelly, “Leptospirosis”.

[008] Furthermore, despite knowledge in the art, there is currently no method for detecting only pathogenic serovars of Leptospira or an assay that is also capable of distinguishing pathogenic Leptospira from other spirochetes. The compositions and methods described herein are intended to provide such a method.

SUMMARY OF THE INVENTION

[009] The present invention provides methods and compositions for determining the presence and/or amount of pathogenic Leptospira nucleic acids in a test sample. In particular, the invention provides substantially purified oligonucleotides for qualitatively and quantitatively detecting Leptospira nucleic acids in a test sample and amplification methods are described herein. The present invention can provide a sensitive, specific method that has a wide dynamic range of detection of pathogenic Leptospira without the detection of unrelated spirochetes or non-pathogenic serovars, and that can advantageously provide both quantitative and qualitative results. The invention can be used alone, or in combination with clinical symptoms or other indicators, to diagnose an individual as having pathogenic Leptospira.

[0010] Accordingly, in one aspect, the description provides oligonucleotide primers and oligonucleotide probes used in the methods described herein to provide an assay for detecting pathogenic Leptospira. In certain embodiments, the invention provides a substantially purified oligonucleotide having a sequence selected from the group consisting of: 5'- AGTAACACGTGGGTAATCTTCCT -3' (SEQ ID NO: 1), 5'- TCTCTCGGGACCATCCAGTA -3' (SEQ ID NO: 2), and 5'- TGGGATAACTTTCCGAAAGGGAAG C -3' (SEQ ID NO: 3), wherein the oligonucleotide is linked either directly or indirectly to a detectable marker.

[0011] Direct or indirect linkage may mean that the label may be incorporated into, associated with or conjugated with the oligonucleotide, or the linkage may comprise a spacer arm of various lengths. The linkage may be by covalent or non-covalent means as long as the oligonucleotide is detectable by the means described herein and known in the art.

[0012] The detectable marker may be a fluorescent dye or the detectable marker may comprise a reporter dye and a quencher. In some embodiments, the oligonucleotide of the invention may be 5' [6~FAM] - TGGGATAACTTTCCGAAAGGGAAGC - [BHQ-1] 3'.

[0013] In some embodiments, the invention provides a pair of substantially pure oligonucleotide primers comprising SEQ ID NO: 1 and SEQ ID NO: 2. The primers may be detectably labeled and may be used together with a detectably labeled probe. The primer pair may be suitable for amplifying the pathogenic Leptospira 16S gene or a fragment or complement thereof, including, but not limited to, SEQ ID NO: 4. The 16S gene sequences of numerous Leptospira serovars are known In the art, and in some embodiments, the invention provides primer pairs that are suitable for amplifying the 16S gene sequences of pathogenic Leptospira serovars, but that do not comprise SEQ ID NO: 1 or 2.

[0014] In one aspect, the invention provides a detection method for identifying the presence or absence of pathogenic Leptospira in a test sample, comprising detecting the presence or absence of a 16S target nucleic acid comprising at least 15 contiguous nucleotides that are at least 95% identical to SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, or a fragment or complement thereof, wherein the presence of said 16S target nucleic acid identifies the presence of pathogenic Leptospira.

[0015] In some embodiments, the detection method further comprises: (a) providing a pair of primers suitable for amplifying the target 16S nucleic acid or a fragment thereof, and providing a detectably labeled probe suitable for hybridizing to the acid 16S target nucleic acid or a fragment thereof, (b) carrying out a primer extension reaction comprising the first primer pair of step (a) under conditions suitable for producing a first reaction product when the 16S target nucleic acid is present in said sample, and (c) determine the presence or absence of pathogenic Leptospira by detecting the presence or absence of detectable probe marker.

[0016] In another aspect, the invention provides that at least one member of the primer pair used in the detection method comprises SEQ ID NO: 1 or SEQ ID NO: 2. Alternatively, at least one member of the primer pair consists of SEQ ID NO: 1 or SEQ ID NO: 2. In another aspect, the detectably labeled probe may comprise SEQ ID NO: 3 or consist of 5' [6~FAM] - TGGGATAACTTTCCGAAAGGGAAGC - [BHQ-1] 3'.

[0017] In one aspect, the invention provides a method for detecting the presence or amount of pathogenic Leptospira nucleic acids in a test sample, comprising: (a) amplifying pathogenic Leptospira nucleic acids if present in the sample using a pair of primers oligonucleotides having the sequences shown in SEQ ID NO: 1 and SEQ ID NO: 2; (b) hybridizing said amplified pathogenic Leptospira nucleic acids with an oligonucleotide probe having the sequence shown in SEQ ID NO: 3 in the presence of an enzyme that cleaves said probe when said probe hybridizes with said pathogenic Leptospira nucleic acids; and (c) detecting a signal from said probe, wherein said signal indicates the presence or amount of pathogenic Leptospira nucleic acids in said test sample.

[0018] In some embodiments, the test sample may be selected from the group consisting of serum, blood, plasma, cerebrospinal fluid, synovial fluid, and urine. In some embodiments, pathogenic Leptospira nucleic acids are extracted from the test sample prior to amplification of the nucleic acids, while in other embodiments, the test sample can be used directly. In some embodiments, the probe may comprise a reporter dye and a quencher, and in some embodiments, the reporter dye may be 6~FAM and the quencher may be BHQ-1.

[0019] In one aspect, the invention provides a method of diagnosing an individual suspected of having pathogenic Leptospira, comprising: (a) obtaining a sample from said individual suspected of having pathogenic Leptospira, (b) extracting substantially pure nucleic acids from the sample, (c) carrying out an amplification reaction in the presence of a detectably labeled probe comprising SEQ ID NO: 3 and a pair of primers comprising SEQ ID NO: 1 and SEQ ID NO: 2, wherein hybridizing the detectably labeled probe to a sequence corresponding sample nucleic acids in the presence of a polymerizing enzyme will cleave the detectable marker from the probe when the sample nucleic acids are amplified by the primer pair, (d) detecting a signal from the detectable marker of the probe, wherein said signal indicates the presence or quantity of pathogenic Leptospira nucleic acids in the sample, and (e) determine the individual suspected of having pathogenic Leptospira as having pathogenic Leptospira if the signal is detected or diagnosing the individual as not having pathogenic Leptospira if the signal is not detected.

[002] In some embodiments, the amplification reaction may comprise real-time PCR. In some embodiments, the probe may comprise a reporter dye and a quencher, and in some embodiments, the reporter dye may be 6~FAM and the quencher may be BHQ-1.

[003] In one aspect, the invention provides a kit comprising a pair of primers that specifically hybridize to a target nucleic acid comprising SEQ ID NO: 4, a fragment thereof, or a complement thereof, and a probe that specifically hybridizes to the target nucleic acid of SEQ ID NO: 4, a fragment thereof, or a complement thereof.

[004] In some embodiments of the kit, at least one member of the primer pair comprises the sequence of SEQ ID NO: 1 or 2, or a complement thereof. In some embodiments, the primer pairs consist of a first primer and a second primer, wherein the first primer comprises SEQ ID NO: 1, or a complement thereof, and the second primer comprises SEQ ID NO: 2, or a complement to it. In some embodiments, the probe may comprise SEQ ID NO: 3, or a complement thereof. In some embodiments, the detectable label on the probe comprises a reporter dye and a quencher, and in some embodiments, the reporter dye may be 6~FAM, and the quencher may be BHQ-1.

[005] In another aspect, the invention provides a kit comprising a pair of primers that specifically hybridize to a target nucleic acid comprising SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 12, SEQ ID NO: 13, or a fragment or complement thereof, and a detectably labeled probe that specifically hybridizes to the target nucleic acid comprising SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 12, SEQ ID NO: 13, or a fragment thereof, or a complement thereof. In some embodiments, the detectable marker comprises a reporter dye and a quencher, and in some embodiments, the reporter dye may be 6~FAM, and the quencher may be BHQ-1.

BRIEF DESCRIPTION OF FIGURES

[006] Figure 1 shows typical amplification graphs of the Leptospira assay described. Panel A shows high positive, low positive, and negative results in a linear view. Panel B shows high positive, low positive, and negative results in a logarithmic view.

[007] Figure 2 shows the validation results of the primer and probe sets. Set 1 is shown in Panel A, Set 2 is shown in Panel B, and Set 3 is shown in Panel C.

DETAILED DESCRIPTION OF THE INVENTION

[008] The present invention provides methods and compositions for the rapid and sensitive determination of pathogenic Leptospira nucleic acids in test samples. In particular, oligonucleotide probes and oligonucleotide primers are described that can be used in methods for quantitatively or qualitatively detecting pathogenic Leptospira nucleic acids in a sample. The present invention also provides primers and probes for generating and detecting nucleic acid sequences that provide a convenient method for evaluating the internal quality control of the described Leptospira assay.

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

[0010] As used herein, “about” means plus or minus 10%.

[0011] As used herein, the term “substantially purified” in reference to oligonucleotides does not require absolute purity. Rather, it represents an indication that the sequence is relatively purer than the sequence in its natural environment. Such oligonucleotides can be obtained by numerous methods including, for example, laboratory synthesis, restriction enzyme digestion, extraction or isolation from a sample, or PCR. A “substantially purified” oligonucleotide is preferably more than 50% pure, more preferably at least 75% pure, even more preferably at least 95% pure, and most preferably 98% pure.

[0012] As used herein, the term “oligonucleotides” refers to a short polymer composed of deoxyribonucleotides, ribonucleotides or any combination thereof. These oligonucleotides are at least 5 nucleotides in length, preferably 10 to 70 nucleotides in length, with 15 to 26 nucleotides being more common. In certain embodiments, the oligonucleotides are joined to each other with a detectable label or are linked to a detectable label.

[0013] Oligonucleotides used as primers or probes to specifically amplify (i.e., amplify a specific target nucleic acid sequence) or specifically detect (i.e., detect a specific target nucleic acid sequence) a target nucleic acid are , in general, capable of specifically hybridizing with the target nucleic acid.

[0014] As used herein, the term “suitable for amplifying”, when referring to the oligonucleotide primer or pairs of oligonucleotide primers, means primers that specifically hybridize to a target nucleic acid and are capable of providing an initiation site for a primer extension reaction in which a complementary copy of the target nucleic acid is synthesized.

[0015] As used herein, the term “hybridize (hybridization)” refers to the process in which two complementary nucleic acid strands anneal to each other under appropriate stringent conditions. Hybridizations are typically and preferably conducted with probe length nucleic acid molecules, preferably 10 to 100 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”, Second Edition, Cold Spring Harbor Press, Plainview, NY. Those skilled in the art understand how to estimate and adjust the stringency of hybridization conditions such that sequences having at least a desirable level of complementarity will hybridize stably, while those having lower complementarity will not hybridize stably. For examples of hybridization conditions and parameters, see, e.g., Sambrook, et al., 1989, “Molecular Cloning: A Laboratory Manual,” Second Edition, Cold Spring Harbor Press, Plainview, NY; Ausubel, F. M. et al. 1994, “Current Protocols in Molecular Biology”. John Wiley & Sons, Secaucus, N.J.

[0016] The term “stringent hybridization conditions”, as used herein, refers to hybridization conditions at least as stringent as the following: hybridization in 50% formamide, 5X SSC, 50 mM NaH2PO4, pH 6.8, SDS 0.5%, sonicated salmon sperm DNA 0.1 mg/mL, and 5X Denhart's solution at 42°C overnight; washing with 2X SSC, 0.1% SDS at 45°C; and washing with 0.2X SSC, 0.1% SDS at 45°C. In another example, stringent hybridization conditions should not permit the hybridization of two nucleic acids that differ along a stretch of 20 contiguous nucleotides by more than two bases.

[0017] The terms “target nucleic acid” or “target sequence”, as used herein, refer to a sequence that includes a segment of nucleotides of interest to be amplified and/or detected. Copies of the target sequence that are generated during the amplification reaction are called amplification products or amplicons. Target nucleic acids may be composed of segments of a chromosome, a complete gene with or without an intergenic sequence, segments or portions of a gene with or without an intergenic sequence, or nucleic acid sequence whose probes are designed or whose primers are designed. Target nucleic acids may include a wild-type sequence(s), a mutation, deletion or duplication, tandem repeat regions, a gene of interest, a region of a gene of interest, or any upstream or downstream region thereof. . Target nucleic acids may represent alternative sequences or alleles of a specific gene. Target nucleic acids can be derived from genomic DNA, cDNA, or RNA. As used herein the target nucleic acid may be DNA or RNA extracted from a cell or a nucleic acid copied or amplified from said DNA or RNA, or may include extracted nucleic acids further converted using a bisulfite reaction.

[0018] As used herein, the term “Leptospira nucleic acids” refers to DNA and/or RNA comprising a contiguous sequence of a Leptospira genome, or the complement thereof. Leptospira nucleic acids may be Leptospira genomic DNA, Leptospira messenger RNA, or the complement of these sources, obtained by any method including obtaining the nucleic acid from a biological source, in vitro nucleic acid synthesis, or amplification of the nucleic acid. by any method known in the art.

[0019] The terms “amplification” or “amplify”, as used herein, include methods for copying a target nucleic acid, thereby increasing the number of copies of a selected nucleic acid sequence. Amplification can be exponential or linear. A target nucleic acid can be either DNA or RNA. Sequences amplified in this way form an “amplicon” or “amplification product”. Although the exemplary methods described below generally refer to amplification using the polymerase chain reaction (PCR), numerous other methods are known in the art for amplification of nucleic acids (e.g., isothermal methods, rolling circle, etc.). The skilled artisan will understand that these other methods can be used either in place of, or in conjunction with, the PCR methods. See, for example, Saiki, “Amplification of Genomic DNA” in PCR Protocols, Innis et al., Eds., Academic Press, San Diego, CA 1990, pp 13-20; Wharam, et al., Nucleic Acids Res. 2001 Jun 1;29(11):E54-E54; Hafner, et al., Biotechniques 2001 Apr;30(4):852-6, 858, 860; Zhong, et al., Biotechniques 2001 Apr;30(4):852-6, 858, 860.

[0020] The term “complement”, “complementary”, or “complementarity”, as used herein, with reference to polynucleotides (i.e., a sequence of nucleotides such as an oligonucleotide or a target nucleic acid) refers to the rules of standard Watson/Crick pairing. The complement of a nucleic acid sequence such that the 5' end of one sequence is paired with the 3' end of the other sequence is in "antiparallel association". For example, the sequence “5‘-A-G-T-3‘” is complementary to the sequence “3‘-T-C-A-5‘”. Certain bases not commonly found in natural nucleic acids may be included in the nucleic acids described herein; these include, for example, inosine, 7-deazaguanine, Locked Nucleic Acids (LNA), and Peptide Nucleic Acids (PNA). Complementarity does not have to be perfect; Stable duplexes may contain mismatched base pairs, degenerate bases, or unpaired bases. Those skilled in the art of nucleic acid technology can determine the stability of the duplex empirically by considering numerous variables including, for example, the length of the oligonucleotide, the base composition and sequence of the oligonucleotide, the ionic strength and the incidence of pairs. of mismatched bases. A complementary sequence may also be an RNA sequence complementary to the DNA sequence or its complementary sequence, and may also be a cDNA. The term "substantially complementary", as used herein, means that two sequences specifically hybridize to each other (defined above). The skilled artisan will understand that substantially complementary sequences need not hybridize over their entire length.

[0021] As used herein, the term “sample”, “test sample”, or “biological sample”, refers to any liquid or solid material believed to comprise Leptospira nucleic acids. In preferred embodiments, a test sample is obtained from a biological source, such as cultured cells or a tissue or fluid sample from an animal, more preferably, a human. Preferred samples of the invention include, but are not limited to, plasma, serum, whole blood, blood cells, lymphatic fluid, cerebrospinal fluid, synovial fluid, urine, saliva, and skin or other organs (e.g., biopsy material ). The term “patient sample”, as used herein, may also refer to a tissue sample obtained from a human seeking diagnosis or treatment of a disease related to a Leptospira infection. Each of these terms can be used interchangeably.

[0022] The term “detectable label”, as used herein, refers to a composition or portion that is detectable by spectroscopic, photochemical, biochemical, immunochemical, electromagnetic, radiochemical, or chemical means such as fluorescence, chemfluorescence, or chemiluminescence, or any other appropriate means. Preferred detectable labels are fluorescent dye molecules such as fluorescein, phycoerythrin, CY3, CY5, allophycocyanin, Texas Red, peridinin-chlorophyll, cyanine, FAM, 6~FAM, JOE, TAMRA, tandem conjugates such as phycoerythrin-CY5, and the like. These examples do not mean that they are limiting.

[0023] The term “fluorochrome”, as used herein, refers to a molecule that absorbs a quantum of electromagnetic radiation at one wavelength, and emits, in response, one or more photons at a different wavelength, typically longer. In preferred embodiments, a fluorochrome may be a member of a pair of physically linked fluorochromes that exhibit fluorescence energy transfer. A pair of fluorescence energy transfer fluorochromes can be excited by a quantum of electromagnetic radiation at a wavelength at which the donor fluorochrome is excited; however, the fluorescence of the donor fluorochrome, which would be expected in the absence of the acceptor fluorochrome, is quenched at least in part, and the emission of the acceptor fluorochrome at an emission wavelength is absorbed.

[0024] In particularly preferred embodiments, a fluorochrome is a member of a physically linked “molecular beacon” pair. In these embodiments, the molecular beacon pair may be excited by a quantum of electromagnetic radiation at a wavelength at which a first fluorochrome member of the pair is excited; however, the fluorescence of the first fluorochrome, which would be assumed in the absence of the second fluorochrome, is at least partially suppressed. However, unlike energy transfer pairs, emission from the acceptor fluorochrome at an emission wavelength is not observed. Thus, these markers comprise a pair of dyes, the first of which is called a “reporter”, and the second of which is called a “suppressor”. When the two dyes are held in close proximity, such as at the ends of a nucleic acid probe, the quenching portion prevents detection of a fluorescent signal from the reporter portion. However, when the two dyes are separated, the fluorescent signal from the reporter portion becomes detectable.

[0025] As used herein, “Scorpion® primer” or “Scorpion® probe” refers to an oligonucleotide having a 3' primer with an extended 5' probe tail having a hairpin structure that has a pair of fluorophore/quencher. Optionally, the Scorpion® primer/probe additionally contains an amplification blocker (e.g., hexethylene glycol (“HEG”) that separates the probe portion from the primer portion.

[0026] As used herein, the term “Scorpion® detection system” refers to a method for real-time PCR. This method uses a bifunctional molecule (called here a “Scorpion®”), which contains a PCR primer element covalently linked, by a polymerase blocking group, to a probe element. Additionally, each Scorpion® molecule contains a fluorophore that interacts with a quencher to reduce background fluorescence.

[0027] As used herein, the term “detection”, used in the context of detecting a signal from a detectable marker to indicate the presence of a target nucleic acid in the sample, does not require that the method provide 100% sensitivity and /or 100% specificity. As is well known, “sensitivity” is the probability that a test will be positive, assuming that the person has a target nucleic acid sequence, while “specificity” is the probability that a test will be negative, assuming that the person does not have the target nucleic acid sequence. A sensitivity of at least 50% is preferred, although sensitivities of at least 60%, at least 70%, at least 80%, at least 90% and at least 99% are obviously more preferred. A specificity of at least 50% is preferred, although specificities of at least 60%, at least 70%, at least 80%, at least 90% and at least 99% are clearly more preferred. Detection also covers assays with false positives and false negatives. False negative rates can be 1%, 5%, 10%, 15%, 20% or even higher. False positive rates can be 1%, 5%, 10%, 15%, 20% or even higher.

[0028] As used herein “detection via TaqMan® PCR” refers to a method for real-time PCR. In this method, a TaqMan® probe that hybridizes to the amplified nucleic acid segment is included in the PCR reaction mixture. The TaqMan® probe comprises a reporter dye and a quencher fluorophore at each end of the probe and in sufficiently close proximity to each other so that fluorescence from the reporter is captured by the quencher. However, when the probe hybridizes with the amplified segment, the 5' exonuclease activity of Taq polymerase cleaves the probe, thus allowing the reporter fluorophore to emit fluorescence that can be detected.

[0029] A “fragment” in the context of a gene fragment refers to a sequence of nucleotide residues that is at least about 20 nucleotides, at least about 25 nucleotides, at least about 30 nucleotides, at least about 40 nucleotides, at least about 50 nucleotides, or at least about 100 nucleotides. The fragment is typically smaller than about 400 nucleotides, smaller than about 300 nucleotides, smaller than about 250 nucleotides, smaller than about 200 nucleotides, or smaller than 150 nucleotides. In certain embodiments, the fragments can be used in various hybridization procedures or microarray procedures to identify specific pathogens.

[0030] By “isolated”, when referring to a nucleic acid (e.g., an oligonucleotide) we mean a nucleic acid that is part of a substantial portion of the genome in which it occurs in nature. For example, any nucleic acid that has been produced synthetically (e.g., by serial condensation of bases) is considered to be isolated. Likewise, nucleic acids that are recombinantly expressed, produced by a primer extension reaction (e.g., PCR), or otherwise excised from a genome, are also considered to be isolated.

[0031] The term “ligand”, as used herein, refers to one or more chemical bonds or a chemical group used to link one moiety to another, serving as a divalent bridge, where it provides a group between the two chemical moieties. .

Leptospira Assay:

[0032] The compositions and methods described herein comprise primers and probes for the amplification and detection of a Leptospira target DNA sequence. The described DNA primers hybridize to flanking target regions within the 16S ribosomal RNA gene of pathogenic Leptospira strains such as L. interrogans and L. kirchneri. The DNA primers used in this assay do not hybridize to the 16S gene of non-pathogenic strains such as L. biflexa, or to strains of intermediate pathogenicity such as L. illini. The compositions and methods described here will also allow the detection of pathogenic strains only common in specific regions of the world, L. santarosai, and L. weilii.

[0033] In some embodiments, the methods comprise obtaining a biological sample from an individual, extracting DNA from the sample, and performing a detection assay on the extracted DNA in which the DNA is contacted with a detectably labeled probe and primers that are specific for the 16S ribosomal RNA gene from pathogenic strains of Leptospira.

Sample collection and preparation:

[0034] Samples may comprise samples of blood, plasma, urine, saliva, cerebrospinal fluid (CSF), tissue, or other commonly used types of biological sample.

[0035] In some embodiments, urine can be collected in a sterile plastic container with a leak-proof lid and then frozen at -20°C to -70°C. Repeated freezing and thawing should be avoided. Urine specimens can be transported frozen, or alternatively, immediately transferred into a dedicated urine transport tube, such as an Aptima® Urine Specimen Transport Tube.

[0036] In some embodiments, the FCE can be collected in a sterile plastic container with a leak-proof lid. Alternatively, blood can be collected in sterile tubes, preferably containing EDTA or another anticoagulant. These samples can be stored and transported while refrigerated at 2°C to 8°C.

Nucleic acid isolation and extraction:

[0037] Nucleic acid (DNA or RNA) can be isolated from a sample according to any methods well known in the art. If necessary, the sample can be collected or concentrated by centrifugation and the like. Sample cells may be subjected to lysis, such as by treatments with enzymes, heat, surfactants, ultrasonication, or a combination thereof. Lysis treatment is performed for the purpose of obtaining a sufficient quantity of pathogen-derived nucleic acids, if present in the sample, to detect using polymerase chain reaction. DNA extraction methods may include, but are not limited to, ethanol precipitation, organic extraction such as phenol-chloroform extraction, salting separation, density gradients in cesium chloride, anion exchange methods, silica-based methods including commercially available column kits, and automated high-throughput purification systems.

[0038] In one embodiment, DNA extraction can be performed using a “MagNA Pure LC” automated nucleic acid extraction system or a similar automated nucleic acid extraction system. Numerous commercial kits also produce suitable DNA, including, but not limited to, “QIAamp™ mini blood kit”, “Agencourt Genfind™”, “Roche Cobas® Roche MagNA Pure®” or phenol-chloroform extraction using “Eppendorf Phase Lock Gels®”.

Specific probes and specific primers for Leptospira:

[0039] In various embodiments of the present invention, oligonucleotide primers and oligonucleotide probes can be used in the methods described herein to amplify and detect pathogenic Leptospira target sequences. In certain embodiments, the target nucleic acids may include the 16S ribosomal RNA gene from pathogenic strains of Leptospira such as L. interrogans and L. kirchneri. Additionally, a second set of primers can also be used to amplify one or more control nucleic acid sequences. The target nucleic acids described herein can be detected individually or in a multiplex format, using individual markers for each target.

[0040] The skilled artisan is able to design and prepare primers that are suitable for amplifying a target sequence considering this description. The length of amplification primers for use in the present invention depends on several factors including the identity of the nucleotide sequence and the ambient temperature at which these nucleic acids are hybridized or used during in vitro nucleic acid amplification. The considerations necessary for determining a preferred length for an amplification primer of a specific sequence identity are well known to the person of ordinary skill in the art.

[0041] In a preferred embodiment, the Leptospira-specific primers are 5'- AGTAACACGTGGGTAATCTTCCT -3' (SEQ ID: 1) and 5'- TCTCTCGGGACCATCCAGTA -3' (SEQ ID: 2), although the skilled artisan will understand that other probes can be used. Alternative primers may be 70, 75, 80, 85, 90, 95, 96, 97, 98, or 99% identical to SEQ ID NO: 1 or SEQ ID NO: 2. In other embodiments, the primers may be designed so that bind to, and are suitable for amplifying, a target sequence of the Leptospira 16S gene. For example, primers can be designed to amplify SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, or fragments thereof. or add-ons.

[0042] Primers can be between 10 and 30 nucleotides in length. For example, the primers can be 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides in length. One skilled in the art will understand that the precise length and composition of the primer are dependent on the sample and assay conditions. As described herein, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, or fragments or complements thereof provide the target sequences suitable for designing primers capable of detecting pathogenic Leptospira. In some embodiments, primers comprising 10 to 30 nucleotides of SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, or their fragments or complements can be used for the detection of pathogenic Leptospira. In other embodiments, primers that are at least 90%, at least 95%, or at least 99% identical to sequences comprising 10 to 30 nucleotides of SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6 , SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, or fragments or complements thereof can be used for the detection of pathogenic Leptospira. SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6 SEQ ID NO: 11, SEQ ID NO: 12, and SEQ ID NO: 13 are shown in Table 1. Table 1 - 16S Sequences

[0043] Additional primers that amplify a target nucleic acid sequence can be designed using, for example, a computer program such as OLIGO (Molecular Biology Insights, Inc., Cascade, CO). Important attributes when designing oligonucleotides to be used as amplification primers include, but are not limited to, an amplification product of appropriate size to facilitate detection (e.g., by electrophoresis or real-time PCR), similar melting temperatures for the members of a primer pair, and the length of each primer (i.e., primers need to be long enough to anneal for sequence specificity and to initiate synthesis, but not so long that fidelity is reduced during oligonucleotide synthesis). Typically, oligonucleotide primers are 15 to 35 nucleotides in length.

[0044] In some embodiments, a mixture of primers having degeneracy at one or more nucleotide positions may be provided. Degenerate primers are used in PCR where there is variability in the target sequence, that is, the sequence information is ambiguous. Typically, degenerate primers will exhibit variability at no more than about 4 nucleotide positions, at no more than about 3 nucleotide positions, preferably at no more than about 2 nucleotide positions, and most preferably at no more. than about 1 nucleotide position.

[0045] The design of oligonucleotides to be used as hybridization probes can be carried out in a similar way to the design of primers. As with oligonucleotide primers, oligonucleotide probes typically have similar melting temperatures, and the length of each probe needs to be sufficient for sequence-specific hybridization to occur, but not so long that fidelity is reduced during synthesis. Oligonucleotide probes are generally 15 to 60 nucleotides in length.

[0046] In a preferred embodiment, the Leptospira-specific probe is 5' [6~FAM] - TGGGATAACTTTCCGAAAGGGAAGC - [BHQ1] 3 (SEQ ID NO: 3), although the skilled artisan will understand that other probes can be used. Alternative hybridization probes may be 70, 75, 80, 85, 90, 95, 96, 97, 98, or 99% identical to SEQ ID NO: 3. In other embodiments, probes may be designed such that they hybridize with, and are suitable for detecting a target sequence of the Leptospira 16S gene. For example, probes can be designed to hybridize to SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, or fragments thereof. or add-ons.

[0047] Probes can be between 10 and 30 nucleotides in length. For example, probes can be 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides in length. One skilled in the art will understand that the precise length and precise composition of a probe will depend on the sample and assay conditions. As described herein, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, or fragments or complements thereof provide target sequences suitable for designing probes capable of detecting pathogenic Leptospira. In some embodiments, a probe comprising 10 to 30 nucleotides of SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, or their fragments or complements can be used for the detection of pathogenic Leptospira. In other embodiments, a probe that is at least 90%, at least 95%, or at least 99% identical to sequences comprising 10 to 30 nucleotides of SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6 , SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, or fragments or complements thereof can be used for the detection of pathogenic Leptospira. SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 11, SEQ ID NO: 12, and SEQ ID NO: 13 are shown in Table 1 above.

[0048] Additional probes that can detect a target nucleic acid sequence can be designed using, for example, a computer program such as OLIGO (Molecular Biology Insights, Inc., Cascade, CO). Important attributes when designing oligonucleotides to be used as probes include, but are not limited to, a target sequence of appropriate size to facilitate detection (e.g., by electrophoresis or real-time PCR), similar or higher melting temperatures. high compared to reaction primers (if used in an amplification reaction such as RT-PCR), and the probe length (i.e., the probe needs to be long enough to anneal according to sequence specificity, but not so long that fidelity is reduced). Typically, oligonucleotide probes are 5 to 40 nucleotides in length.

Amplification and detection of a target sequence:

[0049] Nucleic acid samples or isolated nucleic acids can be amplified by various methods known to the skilled artisan. Preferably, PCR is used to amplify nucleic acids of interest. Briefly, in PCR, two primer sequences are prepared that are complementary to regions on opposite complementary strands of a target sequence. Excess deoxynucleotide triphosphates (dNTPs) are added to a reaction mixture together with a DNA polymerase, e.g. Taq polymerase.

[0050] If the target sequence is present in a sample, the primers will bind to the sequence and the polymerase will cause extension of the primers along the target sequence by the addition of nucleotides. By increasing or lowering the temperature of the reaction mixture, the extended initiators will dissociate from the label to form reaction products, the excess initiators will bind to the label and the reaction products and the process is repeated, thus generating the reaction products. amplification. Cyclization parameters can be varied depending on the length of the amplification products to be extended. An internal positive amplification control (IPC) can be included in the sample using oligonucleotide primers and/or oligonucleotide probes. IPC can be used to monitor both the conversion process and any subsequent amplification.

[0051] In some embodiments, the PCR reaction may comprise thermocyclization of 35, 40, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, or up to 60 cycles. The denaturation temperature may be in the range of about 95°C to 105°C or be about 95°C, and the elongation temperature may be in the range of about 50°C to 75°C or be about of 60°C. Cyclization can be adjusted so that the denaturation temperature is maintained for 1 second to 3 minutes. One skilled in the art will know that these parameters can be optimized depending on, among other things, the samples and primers being used.

[0052] Real-time PCR is performed using any suitable instrument capable of detecting the accumulation of the PCR amplification product. Most commonly, the instrument is capable of detecting fluorescence from one or more fluorescent markers. For example, real-time detection on the instrument (e.g. an “ABI Real-Time PCR System 7500® Sequence Detector”) monitors fluorescence and calculates the measured reporter signal, or Rn value, during each cycle of PCR. The threshold cycle, or Ct value, is the cycle in which the fluorescence intercepts the threshold value. The threshold value can be determined manually or by the computer program of the sequence detection system.

[0053] Nucleic acid amplification can be detected by any of numerous methods well known in the art such as gel electrophoresis, column chromatography, hybridization with a probe, sequencing, melting curve analysis, or “real-time” detection. . For real-time detection, primers and/or probes can be detectably labeled to allow for differences in fluorescence when the primers become incorporated or when the probes are hybridized, for example, and amplified in an instrument capable of monitoring the change in fluorescence during the reaction. Real-time detection methods for nucleic acid amplification are well known and include, for example, the TaqMan® system, the Scorpion®™ primer system, and the use of intercalating dyes for double-stranded nucleic acid.

[0054] In some embodiments, the amplified nucleic acids are detected by hybridization with a specific probe. Probe oligonucleotides complementary to a portion of the amplified target sequence can be used to detect amplified fragments. Hybridization can be detected in real time or in non-real time. The amplified nucleic acids for each of the target sequences can be detected simultaneously (i.e., in the same reaction vessel) or individually (i.e., in separate reaction vessels). For sequence-modified nucleic acids, the target can be independently selected from the top strand or the bottom strand. In this way, all targets to be detected may comprise the top strand, the bottom strand, or a combination of the top strand and bottom strand targets.

[0055] In some embodiments, Leptospira-specific primers can be used in a polymerase chain reaction to amplify and detect pathogenic Leptospira. The PCR reaction may comprise sterile nuclease-free water, forward and reverse primers, Leptospira-specific probe(s), internal control primers, and a “master mix” comprising a DNA polymerase, dNTPs, salts, buffering agent. reaction, and magnesium. Forward and reverse primers can be used at a reaction concentration in the range of 100 nM to 1.5 μM, or more particularly 250-950 nM, 350-850 nM, 450-750 nM, 550-650 nM. In some embodiments, the forward and reverse primers can be used at a reaction concentration of 450, 460, 470, 480, 490, 500, 510, 520, 530, 540, 550, 560, 570, 580, 590, 600, 610, 620, 630, 640, 650, 670, 680, 690, 700, 710, 720, 730, 740, 750, 760, 770, 780, 790, 800, 810, 820, 830, 840, or 850 nm. Leptospira-specific probes can be used at a reaction concentration in the range of 1-500 nM, or more particularly, 25-400 nM, 50-300 nM, or 75-200 nM. In some embodiments, the Leptospira-specific probe can be used at a reaction concentration of 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, or 250 nM.

[0056] In some embodiments, the standard PCR mixture may contain a DNA polymerase, salts, magnesium, dNTPs, and reaction buffering agent, among other necessary and optional constituents.

[0057] TaqMan® probes (Heid, et al., Genome Res 6: 986-994, 1996) use the fluorogenic 5' exonuclease activity of Taq-polymerase to measure the amount of target sequences in test samples. TaqMan® probes are oligonucleotides that contain a reporter dye typically at or near the 5' base, and a quencher portion typically at or near the 3' base. The quencher moiety may be a dye, such as TAMRA, a Black Hole™ quencher, or may 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 reporter transfers energy to the neighboring quencher moiety by FRET (Fluorescence Resonance Energy Transfer) rather than by fluorescence. In this way, the close proximity between the reporter and the quencher prevents fluorescence emission from the donor while the probe is intact.

[0058] TaqMan® probes are designed to anneal with an internal region of a PCR product. When the polymerase replicates a template to which a TaqMan® probe is bound, its 5' exonuclease activity cleaves the probe. This stops the activity of the quencher (non-FRET) and the reporter fluorophore begins to emit fluorescence that increases with each cycle proportional to the probe cleavage rate. Accumulation of PCR product is detected by monitoring the increase in fluorescence of the reporter dye (note that primers may not be labeled). If the quencher is an acceptor fluorophore, then accumulation of PCR product can be detected by monitoring the decrease in fluorescence of the acceptor fluorophore.

[0059] Some embodiments of the described compositions and methods comprise a specific probe for the Leptospira amplicon. The probe used for detection of the Leptospira amplicon can be optionally labeled with a reporter dye and a quencher as with a TaqMan® probe. For example, an exemplary probe may comprise a FAM reporter dye at its 5' end and a BHQ-1 quencher at its 3' end. Those skilled in the art will know that this is just one example of numerous combinations of reporter and quencher dye that can be used in such an assay. Useful reporter dyes include, but are not limited to, BODIPY FL, FAM, 6~FAM, VIC, 4-acetamido-4'-isothiocyanatostilbene-2,2'disulfonic acid, acridine and derivatives (acridine, acridine isothiocyanate) Alexa Fluor® 350, Alexa Fluor® 488, Alexa Fluor® 546, Alexa Fluor® 555, Alexa Fluor® 568, Alexa Fluor® 594, Alexa Fluor® 647 (Molecular Probes), 5-(2'-aminoethyl)aminonaphthalene-1 acid -sulfonic acid (EDANS), 4-amino-N-[3-vinylsulfonyl)phenyl]naphthalimide-3,5-disulfonate (Lucifer Yellow VS), N-(4-anilino-1-naphthyl) maleimide, anthranilamide, Green “Oregon Green 488”, Rhodamine Green (“Rhodamine Green”), Green “Oregon Green 514”, TET, Gold “Cal Gold”, BODIPY R6G, Yellow “Yakima Yellow”, JOE, HEX, Orange “ Cal Orange”, BODIPY TMR-X, Quasar-570/Cy3, TAMRA, Red “Rhodamine Red-X”, Rhodamine Red (“Rhodamine Red”), BODIPY 581/591, Cy3.5, ROX, Red “Cal Red” , Texas Red (“Texas Red”), BODIPY TR-X, BODIPY 630/665-X, Pulsar-650, Quasar-670/Cy5, and Cy5.5.

[0060] Suitable quenchers are selected based on the fluorescence spectrum of the specific fluorophore. Useful suppressors include, for example, the Black Hole™ suppressors BHQ-1, BHQ-2, and BHQ-3 (Biosearch Technologies, Inc.), and the ATTO series of suppressors (ATTO 540Q, ATTO 580Q, and ATTO 612Q; Atto-Tec GmbH). Other useful suppressors include, but are not limited to, dabcyl, QSY 35, Eclipse, QSY 7, QSY 9, ElleSupressor, Black “Iowa Black”, QSY21, Qxl suppressors, Black “Iowa Black FQ”, Black “Iowa Black RQ” , and IRDye QC-1.

[0061] An individual skilled in the art will understand that certain pairs of reporter and quencher dyes are preferred. For example, in one embodiment, the reporter/suppressor pair is 6~FAM/BHQ1. In another embodiment, the reporter may be Quasar-670, Cal Red, Quasar-570, or TAMRA and the suppressor may be BHQ-1, BHQ-2, or BHQ-3.

[0062] When the reporter dye and the quencher are in close proximity (that is, they are present in an intact oligonucleotide probe) the reporter fluorescence is quenched. However, when the oligonucleotide probe is elongated in the described assay, the 5'-3' nuclease activity of DNA polymerase will cleave the probe if it is specifically bound to the target sequence between the forward and reverse primer sites. This releases the reporter dye and the quencher, and upon excitation, the fluorescent signal produced by the reporter dye is no longer suppressed. This results in an increase in fluorescence that can be detected.

[0063] In some embodiments of the described compositions and described methods, the Black Hole™ suppressor can be used to reduce background noise. In a preferred embodiment, the Leptospira-specific probe comprises a reporter dye and a quencher so that it can be detected after amplification of the target sequence.

[0064] Additional detectable markers include, but are not limited to, radioisotopes (e.g., P, S, H, C, I, I), electron-dense reagents (e.g., gold), enzymes (e.g., horseradish peroxidase, beta-galactosidase, luciferase, alkaline phosphatase), colorimetric tags (e.g., colloidal gold), magnetic tags (e.g., Dynabeads™), biotin, dioxygenin, or haptens and proteins for which antisera or monoclonal antibodies are available. Other labels include ligands or oligonucleotides capable of forming a complex with the corresponding receptor or oligonucleotide complement, respectively.

[0065] The label can be directly incorporated into the nucleic acid to be detected, or it can be linked to a probe (for example, an oligonucleotide) or an antibody that hybridizes with or binds to the nucleic acid to be detected. A label can be linked either directly or indirectly to probes or primers, and a label can be linked covalently or non-covalently. A label can be linked by spacer arms of various lengths to reduce steric hindrance or influence other useful or desired properties. See, for example, Mansfield, 9 Mol. Cell. Probes 145-156 (1995). Detectable markers can be incorporated into nucleic acids by covalent or non-covalent means, for example, by transcription, such as random primer labeling using Klenow polymerase, or “nick translation”. -3'-phosphodiester] on a strand of unlabeled double-stranded DNA molecule), or amplification, or equivalent as is known in the art. For example, a nucleotide base is conjugated to a detectable moiety, such as a fluorescent dye, and then incorporated into nucleic acids during nucleic acid synthesis or amplification.

[0066] Alternatively, the detectable markers can be part of a Scorpion® detection system. With Scorpion® detection systems, detection of sequence-specific DNA synthesis and PCR product priming is performed using a single molecule. The Scorpion® probe maintains a stem-loop configuration in the unhybridized state. The fluorophore is quenched by a portion coupled to the 5' end, although in suitable embodiments, the fluorophore is attached to the 5' end and is quenched by a portion coupled to the 3' end. The 3' portion of the stem also contains a sequence that is complementary to the primer extension product. This sequence is linked to the end of the Scorpion® probe via a non-amplifiable monomer. After extension using the Scorpion® primer, the specific probe sequence is able to bind to its complement within the extended amplicon, thus opening the hairpin loop. This prevents the fluorescence from being quenched, and a signal is observed. A specific target is amplified by the reverse primer and the primer portion of the Scorpion® probe, resulting in an extension product. A fluorescent signal is generated due to the separation of the fluorophore from the quencher resulting from the binding of the Scorpion® probe element to the extension product.

Internal controls:

[0067] To ensure the absence of non-specific PCR inhibitors in a sample, an internal positive amplification control (IPC) can be included with each specimen. Probe and positive control primers can be added to create a multiplex reaction with target and sample primers. The IPC amplicon can be detected with a labeled probe and a reporter dye attached to the 5' end of the probe. A sample may be interpreted as negative if analysis of the internal positive control indicates that DNA amplification occurred in the reaction tube but there was no detection of the Leptospira-specific probe.

EXAMPLES Example 1. Sample preparation and DNA extraction

[0068] A 250 μL aliquot of “Leptospira Positive Control” control was removed and thawed at room temperature. 200 μL of sterile water was used for a negative control. 1 positive control and 1 negative control were performed for each batch of extractions. 200 μL of control or specimen was pipetted into a given well of the sample cartridge. Refrigerated or frozen urine samples mixed with urine transport medium were allowed to warm to room temperature (18°C to 26°C) before loading into the sample cartridge.

[0069] Positive Internal Control DNA was added to a lysis buffer before extraction, and 200 μL was added to each well containing a sample.

Example 2. DNA amplification and detection

[0070] DNA amplification was performed using an “ABI 7500 Real-Time PCR System”. Before amplification, samples were combined with a standard mixture.

[0071] The amplification graphs of the controls were examined. The graphs showed a sigmoidal curve with a distinct exponential growth phase followed by a plateau phase.

[0072] Sample amplifications were also examined. Negative plots were also visible as a line near the bottom portion of the plot due to poor resolution at lower fluorescence values.

[0073] An example of typical curves for the Leptospira assay is shown in Figures 1A and 1B. The left turn is typical of a high positive result. The right turn is typical of a low positive result. The horizontal line is the threshold. All positive results cross this line. The cycle in which a given graph crosses the threshold line is called the Threshold Cycle (Ct). Negative graphs do not have a sigmoidal shape, and/or do not cross the threshold line. Data can be viewed in a linear format, as in Figure 1A, or in a log format, as in Figure 1B. The decision as to which format should be used to view amplification graphs is left to the discretion of the technician, however each graph has advantages. Positive results may be more intuitively obvious in linear format because only positive plots are distinguishable above background noise in this format. The log view, however, allows better discernment of all differences between the graph lines, especially at low fluorescence.

[0074] If the amplification graphs of the controls do not show curves, this may be an indication that there has been contamination, improper preparation of the standard mixture or controls, or degradation of the fluorescent probe. The IPC must amplify, and must be within the specified Ct range for the IPC result to be valid. If the IPC results are outside the specified Ct range and targets are not detected, then the target results may not be valid.

[0075] Examination of the clinical samples was carried out after the controls were examined and shown to have the correct results. Amplification plots were examined for each sample. When the amplification graph showed an exponential increase, the amplification curve was considered a positive, valid result.

Example 3: Validation of primers and probes to distinguish pathogenic Leptospira from non-pathogenic Leptospira

[0076] Leptospira weillii (ATCC no. 43285) is a non-interrogans pathogen that may have problems when detected in the Leptospira assay. Multiple primer sets were tested to determine detection effectiveness.

[0077] For validation, thawed ATCC bacteria were diluted 10 times and frozen in 250 μL or 1 mL aliquots. The thawed samples had their DNA extracted. Samples were estimated to have approximately 108 cells/mL. DNAs from serial dilutions of the bacteria were used to determine the detection limits of the various primer pairs.

[0078] Three different sets of primer pairs were used for validation tests. Set 1 consisted of primers F76 and R148, Set 2 consisted of primers F71 and R148, and Set 3 consisted of primers FSmythe and RSmythe. The sequences of these primers can be found in Table 2. For Sets 1 and 2, the P101 probe was used to detect amplification of the target sequence, and for Set 3, PSmythe was used to detect amplification of the target sequence. Table 2 - Primer and probe sequences

[0079] 100 μM of each primer in a given set and 10 μM of the corresponding probe were combined in a reaction mixture comprising the standard mix, Taq-polymerase, Leptospira weillii DNA, and water. Thermocyclization was performed using an “ABI 7500 Real-Time PCR System”.

[0080] The real-time amplification detection results are found in Table 3. Table 3 - Primer set detection results

[0081] The cyclization result data can be seen in Figure 2. The bold horizontal lines represent the threshold cycle value.

[0082] The PCR efficiency of Set 1 was 97.8%. The PCR efficiency of the Set was 99.0%. The PCR efficiency of Set 3 was 91.3%. Although the primer sets differ in terms of Ct scores, Sets 1 and 2 had significantly higher PCR efficiencies than Set 3. This validation indicates that Sets 1 and 2 are more suitable for reliable detection of pathogenic Leptospira. Additionally, Sets 1 and 2 had higher detection limits compared to Set 3, indicating that Set 3 would likely have problems detecting some Leptospira species.

[0083] Although the invention has been described and exemplified in sufficient detail for those skilled in the art to prepare and use it, various alternatives, modifications, and improvements should be evident without departing from the spirit and scope of the invention.

[0084] An individual skilled in the art easily recognizes that the present invention is well adapted to achieve the objectives and to obtain the aforementioned purposes and advantages, and also those inherent thereto. Modifications thereto and other uses will occur to those skilled in the art. These modifications are included within the spirit of the invention and are defined by the scope of the claims.

[0085] It will be readily apparent to a person skilled in the art that various substitutions and modifications can be made to the invention described herein without deviating from the scope and spirit of the invention.

[0086] All patents and publications mentioned in the specification are indicators of the levels of those people commonly skilled in the technique to which the invention belongs. All patents and publications are incorporated herein by reference to the same extent as if each individual publication were specifically and individually designated for incorporation by reference.

[0087] The invention illustratively described herein can be adequately practiced in the absence of any element or elements, any limitation or any limitations that are not specifically described herein. Thus, for example, in each occurrence here, any of the terms “comprising”, “consisting essentially of” and “consisting of” may be replaced by either of the other two terms. The terms and expressions that have been used are used as terms of description and not of limitation, and it is not intended that, in the use of such terms and expressions, there will be exclusion of any equivalents of the attributes shown and described or portions thereof, but it is recognized that various modifications are possible within the scope of the claimed invention. Accordingly, it should be understood that although the present invention has been specifically described by preferred embodiments and optional attributes, those skilled in the art may resort to modifications and variations of the concepts described herein, and that such modifications and variations are considered within the scope of this invention. invention as defined by the attached claims.

[0088] Non-limiting examples are also presented in the following claims.

Claims (21)

1. Substantially purified oligonucleotide, characterized by the fact that the oligonucleotide sequence consists of: (a) 5'- AGT AAC ACG TGG GTA ATC TTC CT -3' (SEQ ID NO: 1), (b) 5'- TCTCTCGGGACCATCCAGTA -3' (SEQ ID NO: 2), or (c) 5'- TGGGATAACTTTCCGAAAGGGAAG C -3' (SEQ ID NO: 3), and wherein the oligonucleotide is linked directly or indirectly to a detectable tag and wherein the tag detectable is a fluorescent dye or a reporter dye and a quencher. 2. Oligonucleotide according to claim 1, characterized by the fact that it is 5' [6~FAM] - TGGGATAACTTTCCGAAAGGGAAGC - [BHQ-1] 3'. 3. Substantially pure oligonucleotide primer pair, characterized in that the first member of the primer pair consists of SEQ ID NO: 1, wherein the second member of the primer pair consists of the sequence of SEQ ID NO: 2, and in that each member of the primer pair hybridizes specifically to the target sequence of SEQ ID NO: 4 or a complement thereof. 4. Detection method for identifying the presence or absence of pathogenic Leptospira in a sample, characterized by the fact that it comprises detecting the presence or absence of a 16S target nucleic acid comprising at least 15 contiguous nucleotides of SEQ ID NO: 4, or a fragment or complement thereof, by: (a) providing a primer pair suitable for amplifying the 16S target nucleic acid or a fragment thereof, and providing a detectably labeled probe suitable for hybridizing to the 16S target nucleic acid or a fragment thereof, wherein the first member of the primer pair consists of the sequence of SEQ ID NO: 1, wherein the second member of the primer pair consists of the sequence of SEQ ID NO: 2, and wherein the detectably labeled probe comprises the sequence of SEQ ID NO: 3; (b) carrying out a primer extension reaction comprising the first primer pair of step (a) under conditions suitable for producing a first reaction product when the 16S target nucleic acid is present in said sample; and (c) determining the presence or absence of pathogenic Leptospira by detecting the presence or absence of the probe detectable marker, wherein the presence of the 16S target nucleic acid identifies the presence of pathogenic Leptospira. 5. Method according to claim 4, characterized by the fact that the detectably labeled probe consists of 5' [6~FAM] - TGGGATAACTTTCCGAAAGGGAAGC - [BHQ-1] 3'. 6. Method of detecting the presence or quantity of pathogenic Leptospira nucleic acids in a test sample, characterized in that it comprises: (a) amplifying pathogenic Leptospira nucleic acids if present in the sample using a pair of oligonucleotide primers, wherein the first member of the primer pair consists of the sequence of SEQ ID NO: 1, wherein the second member of the primer pair consists of the sequence of SEQ ID NO: 2; (b) hybridizing said amplified pathogenic Leptospira nucleic acids with an oligonucleotide probe comprising the sequence of SEQ ID NO: 3 in the presence of an enzyme that cleaves said probe when said probe hybridizes with said pathogenic Leptospira nucleic acids; and (c) detecting a signal from said probe, wherein said signal indicates the presence or amount of pathogenic Leptospira nucleic acids in said test sample. 7. The method of claim 6, wherein said test sample is selected from the group consisting of serum, blood, plasma, cerebrospinal fluid, synovial fluid, and urine. 8. Method according to claim 6 or 7, characterized by the fact that said pathogenic Leptospira nucleic acids are extracted from said sample before amplification step (a). 9. Method according to claim 6, characterized in that the probe comprises a reporter dye and a quencher. 10. Method according to claim 9, characterized by the fact that the reporter dye is 6~FAM. 11. Method according to claim 9 or 10, characterized by the fact that the quencher is BHQ-1. 12. Method of diagnosing an individual suspected of having pathogenic Leptospira, characterized by the fact that it comprises: (a) obtaining a sample from said individual suspected of having pathogenic Leptospira, (b) extracting substantially pure nucleic acids from the sample, (c) performing an amplification reaction in the presence of a detectably labeled probe comprising the sequence of SEQ ID NO: 3 and a pair of primers, wherein the first member of the primer pair consists of the sequence of SEQ ID NO: 1, wherein the second member of the primer pair consists of the sequence of SEQ ID NO: 2, wherein hybridization of the detectably labeled probe to a corresponding sequence of sample nucleic acids in the presence of a polymerizing enzyme will cleave detectable labels from the probe when the sample nucleic acids are amplified by the primer pair, (d) detecting a probe signal, wherein said signal indicates the presence or absence of pathogenic Leptospira nucleic acids in the sample, and (e) determining the individual suspected of having pathogenic Leptospira as having pathogenic Leptospira if the signal is detected or diagnose the individual as not having pathogenic Leptospira if the signal is not detected. 13. Method according to claim 12, characterized by the fact that the amplification reaction comprises real-time PCR. 14. Method according to claim 12 or 13, characterized in that the probe comprises a reporter dye and a quencher. 15. Method according to claim 14, characterized by the fact that the reporter dye is 6~FAM. 16. Method according to claim 14 or 15, characterized by the fact that the quencher is BHQ-1. 17. Kit, characterized in that it comprises: a pair of primers that specifically hybridize with a nucleic acid having a target 16S sequence of SEQ ID NO: 4 or a complement thereof and a detectably labeled probe that specifically hybridizes with a nucleic acid having the sequence SEQ ID NO: 4 or a complement thereof, wherein the first member of the primer pair consists of the sequence SEQ ID NO: 1, or a complement thereof, and wherein the second member of the primer pair consists following SEQ ID NO: 2, or a complement thereof. 18. Kit according to claim 17, characterized by the fact that the probe comprises the sequence of SEQ ID NO: 3, or a complement thereof. 19. Kit according to claim 17 or 18, characterized in that the detectable marker comprises a reporter dye and a quencher. 20. Kit according to claim 19, characterized by the fact that the reporter dye is 6~FAM. 21. Kit according to claim 19 or 20, characterized by the fact that the quencher is BHQ-1.