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Homogeneous multiplex screening assays and kits

Homogeneous multiplex screening assays and kits description/claims

This invention relates to homogenous in vitro multiplexed assays, including particularly homogeneous amplification assays, for screening biological samples for the presence of any of a large number of nucleic acid sequences.

A variety of assay techniques are available for testing a biological sample obtained from any of a variety of sources for the presence of a nucleic acid sequence that may indicate the presence, for example, of a particular bacterium, virus or other pathogen, including a particular strain or mutant. Assays are also available for testing such samples for the presence of a nucleic acid sequence of the subject's own genomic DNA that may indicate the presence, for example, of one or another disease-related genetic mutation. Assays may include oligonucleotide probes bearing detectable labels, for example, P32 or fluorophores. Nucleic acids, either DNA or RNA, in a sample may be probed directly. Alternatively, assays may include amplification of target sequences by any of several amplification techniques, for example, PCR, NASBA or TMA. Amplification assays may be monitored in real time utilizing intercalating dyes, for example SYBR green, or fluorescently labeled probes, such as 5′ nuclease probes Livak, K. J. et al. (1995), Oligonucleotides with fluorescent Dyes at Opposite Ends Provide a Quenched Probe System Useful for Detecting PCR Product and Nucleic Acid Hybridization, PCR Meth. Appl. 4: 357-362, dual FRET probes, Espy, M. J. et al. (2002), Detection of Vaccinia Virus, Herpes Simplex Virus, Varicella-Zoster Virus, and Bacillus anthracis by LightCycler Polymerase Chain Reaction after Autoclaving: Implications for Biosafety of Bioterrorism Agents, Mayo Clin. Proc. 77: 624-628, or molecular beacon probes, Tyagi, S. and Kramer, F. R. (1996), Molecular Beacons: Probes that Fluoresce upon Hybridization, Nature Biotechnol. 14: 303-308; Tyagi, S. et al. (1998), Multicolor Molecular Beacons for Allele Discrimination, Nature Biotechnol. 16: 49-53. Real-time multiplex assays utilizing PCR amplification have been demonstrated with TaqMan dual-labeled linear probes and the 5′ nuclease detection process and, alternatively, with PCR amplification and molecular beacon probes, Tyagi. S. et al. (1998), supra; Vet, J. A. et al. (1999), Multiplex Detection of Four Pathogenic Retroviruses Using Molecular Beacons, Proc. Natl. Acad. Sci. USA 96: 6394-6399; El-Hajj, H. et al. (2001), Detection of Rifampin Resistance in Mycobacterium tuberculosis in a Single Tube with Molecular Beacons, J. Clin. Microbiol. 39: 4131-4137. Fluorescence-based multiplex assays currently are limited to about eight targets per sample by the need to minimize overlaps in emission spectra of fluorophores and, hence, are not expandable for use as highly multiplexed screening assays.

Highly multiplexed assays rely on spatial segregation of targets for signal resolution. Spatial segregation enables the use of coding schemes involving combinations of differently colored fluorophores (combinatorial coding), combinations of different amounts of each fluorophore (ratio coding), and both. One example of an assay with spatial segregation is fluorescence in situ hybridization, or FISH, for chromosomal analysis. Speicher et al. (1996), Karyotyping Human Chromosomes by Combinatorial Multi-fluor FISH, Nature Genet. 12: 368-375, for example, report the use of 27 probes combinatorially labeled using a set of six different fluorophores for analysis of chromosome spreads. Similarly, segregation of transcription sites in cell nuclei has enabled combinatorial coding utilizing multiple, singly labeled probes per site, as well as ratio coding. Singer, R. H., International (PCT) patent application WO 00/65094; Levsky, J. M. et al. (2002), Single-Cell Gene Expression Profiling, Science 297: 836-840. Another spatial-segregation probe technique is the use of multiplex probe arrays, including arrays on DNA chips. Schena, M. et al. (1995), Quantitative Monitoring of Gene Expression Patterns with a Complementary DNA Microarray, Science 20: 467-470; Gingeras, T. R. et al. (1998), Simultaneous Genotyping and Species Identification Using Hybridization Pattern Recognition Analysis of Generic Mycobacterium DNA Arrays. Genet. Res. 8: 435-448; Han et al. (2001), Quantum-Dot-Tagged Microbeads for Multiplexed Optical Coding of Biomolecules, Nature Biotechnol. 19, 631-635. Another segregation approach is the use if electrophoresis to separate ligated probe pairs of differing lengths. Tong, A. K. et al. (2001), Combinatorial Fluorescence Energy Transfer Tags for Multiplex Biological Assays, Nature Biotechnol. 19: 756-759. In that method, a variation of the oligonucleotide ligation assay (“OLA”) for SNP detection, differently labeled probes are ligated to capture probes on a target, and the hybrids are immobilized, washed, released, separated from one another by electrophoresis, and read for each probes' fluorescent code, which is a combination of colors and ratios. Array methods and electrophoretic methods remain technically complex, requiring many separate steps, including amplification, hybridization, washing and analysis.

There is not currently available a homogeneous fluorescence hybridization assay that is suitable for use as a highly multiplexed screening assay, despite the need for such an assay. During suppressive treatment of the HIV-1 virus with protease inhibitors, for example, any of about 30 mutations is likely to proliferate over time and to require change in treatment. Hirsch, M. S. et al. (1998), Antiretroviral Drug Resistance testing in Adults with HIV Infection, JAMA 279: 1984-1991. Lacking a highly multiplexed screening assay, current practice is to sequence the virus in response to a patient's increase in viral load. Sequencing is made difficult by the fact that the arising mutant is not the only allele present. During initial diagnosis of a patient with particular symptoms, for example fever, there is available no highly multiplexed homogeneous assay to screen for an early indication of one of numerous possible infectious agents that may be the cause of the patient's symptoms.

An aspect of this invention is highly multiplexed homogeneous assays for screening samples for the presence of a target nucleic acid sequence from among at least ten, and as many as 60 or more, possible targets, utilizing conventional fluorescence detection equipment and techniques, and fluorescently labeled hybridization probes.

Another aspect of this invention is such screening assays that employ target amplification, optionally with real-time detection, that are capable of detecting small amounts of pathogens that may be found in otherwise sterile samples such as blood.

Yet another aspect of this invention is kits and oligonucleotide sets for carrying out particular screening assays according to this invention.

Assays according to this invention are highly multiplexed assays suitable for screening. By “highly multiplexed,” I mean that an assay is capable of detecting any of at least six, preferably at least 10, and in certain embodiments 30-60 and even more possible target sequences.

Assays according to this invention are homogeneous nucleic acid in vitro assays fluorescently labeled utilizing hybridization probes. By “homogeneous”, I mean that nucleic acid targets are detected in solution without segregating targets spatially and without washing away unbound hybridization probes. Fluorescence signal is obtained from the reaction mixture itself. Certain preferred embodiments include target amplification by, for example PCR or NASBA, which enables detection of small amounts of pathogens in samples. When amplification is used, the reaction vessel, for example a microcentrifuge tube, is hermetically sealed to prevent contamination of the other samples being tested at the same time, samples not yet tested, equipment and workers.

Detection in assays according to this invention is by fluorescently labeled nucleic acid hybridization probes. Fluorescent labels, when stimulated emit radiation in the visible, ultraviolet or near infrared portions of the electromagnetic spectrum. Each probe is specific to one target among the multitude of possible targets being screened. If the goal and design of an assay are to distinguish among bacterial species, one will design a probe for a species, for example against a conserved region of M. tuberculosis, or design a probe that hybridizes to non-identical sequences of multiple strains of the same species. If the goal, on the other hand, is to distinguish among strains or mutant alleles, one will design each probe against a variable region to provide the necessary distinction and make the probe strain-specific. Preferred probes for use in this invention are molecular beacon probes, which may be made mismatch tolerant or mismatch intolerant, depending on the need of a particular assay. As is known in the art, choice of target sequence and probe design are balanced to minimize both false negatives and false positives.

Multiplex capability is achieved by combinatorial coding, with or preferably without added ratio coding, utilizing a panel of distinguishable fluorophores. In a combinatorial code, each code element is identified by its unique pattern of different labels, in this case colors. The combinatorial code includes up to four different fluorophores per code element. The different fluorophores are from a panel of at least four distinguishable fluorophores, more preferably a panel of 4-8 distinguishable fluorophores. The combinatorial code may be duplex coding in which the code elements comprise two colors, optionally enlarged by additional code elements of one color. The combinatorial code may by triplex coding in which the code elements comprise three colors, optionally enlarged by additional code elements of two colors (triplex code combined with a duplex code), and optionally enlarged by code elements of one color. The combinatorial code may be quadraplex coding in which the code elements comprise four colors, optionally enlarged by additional code elements of three colors, two colors or one color, or some combination thereof.

Rather than labeling individual probe molecules with multiplex colors to give the probe the pattern assigned to it as a code element, each probe is subdivided into the required number of portions, and each portion is labeled to emit a different color signal. The portions are then mixed to create a probe that, when hybridized to its target, will include the unique color signal pattern of the code element.

For the preferred highly quenched probes such as molecular beacon probes, each portion will emit a single color. For less preferred probes labeled with two different interacting fluorophores, such as 5′-nuclease probes, one option is to utilize a common short-wavelength fluorophore (which I call the “harvester” fluorophore), vary the long-wavelength fluorophores (which I call the “emitter” fluorophores), and detect the signal change in the latter. In that case, each portion will emit a signal in a different color. Another option is the reverse, namely, to use a common emitter fluorophore, vary the harvester fluorophores, and detect the signal change in the latter. In that case, each portion will also emit a signal in a different color, but for purposes of coding it is the harvester that becomes the detected emitter. However, a third option is to vary both fluorophores to permit detection of a ratio change in their emissions, so that the signal from one portion might be the ration a/b, the ratio from a second portion might be the ratio c/d, and the ratio from a third portion might be e/f, where a, b, c, d, e and f are distinguishable fluorophores. In this case the long-wavelength fluorophores are varied and can be considered to characterize the signal one obtains from any given probe portion. For ease of understanding, descriptions of combinatorial codes in this application and in the figures generally describe embodiments in which one detects for each probe portion the emission of a particular color, which can be characterized as the emitter fluorophore. It is to be understood that the teachings also apply to embodiments utilizing ratios, and persons skilled in the art will be able to apply the teachings to those embodiments.

If one begins with a panel of eight different fluorophores, for example, and chooses to subdivide each probe into four portions and to label each of the four portions with a different color, the codebook can include as many as 70 different combinations; if one begins with the same-sized panel of fluorophores but subdivides each probe into three portions and labels each of the three portions with a different color, the codebook can include as many as 56 different combinations; and if one begins with the same-sized panel of fluorophores but subdivides each probe into two portions and labels each of the two portions with a different color, the codebook can include as many as 28 different combinations. Four-color, three-color and two-color codes may be combined with each other and even with a one-color code, but my most preferred embodiments do not include such combinations. In ratio coding differing amounts of the same label are used for differentiation. Combinatorial codes according to this invention may be augmented with ratio coding, preferably integral ratio coding, that is, ratios of whole numbers such as 2:1, 3:1, 4:1 or 5:1, and vice versa. It is preferred that portions of molecular beacon probes be labeled with a non-fluorescent quencher and one signature-color fluorophore, most preferably without added ratio coding for reasons that will be explained.

As stated above, assays according to this invention are homogeneous detection assays. There is no segregation of targets. There is no separation of unbound probes from probes bound to their targets. Consequently, in assays according to this invention there must be a detectable signal change indicative of a probe hybridizing to its target. Preferred molecular beacon probes are highly quenched when free-floating at the detection temperature in the assay but change conformation and fluoresce when hybridized to their respective targets. Another type of probe whose fluorescence is restored by hybridization to its target and that is generally suitable for use in assays according to this invention is a “yin-yang” probe, a bimolecular probe labeled with a fluorophore on a target complementary strand and a quencher on a competing strand. Li, Q. et al. (2002), A New Class of Homogeneous Nucleic Acid Probes Based on Specific Displacement Hybridization, Nucleic Acids Res. 30: e5. Other probe-assay schemes result in a fluorescent signal change indicative of probes hybridizing to targets. In the 5′-nuclease detection process, end-labeled linear (or random-coil) probes with two different fluorophores interact by fluorescence resonance energy transfer (FRET) when free-floating but are cleaved during primer extension in a polymerase chain reaction (PCR) amplification, resulting in a signal change (emission from the shorter wavelength fluorophore increases, emission from the longer wavelength fluorophore decreases, and the ratio of the two changes accordingly). Livak, K. J. et al. (1995), supra. Another system, so-called LightCycler probes, works in the opposite manner. Each probe is a pair of fluorescently labeled linear oligonucleotides that hybridize adjacently on a target and interact by FRET, thereby producing a signal change (emission from the shorter wavelength fluorophore decreases, emission from the longer wavelength fluorophore increases, and the ratio of the two changes accordingly). Espy, M. J. et al. (2002), supra. For FRET systems, I refer to the shorter wavelength fluorophore as a “harvester” and the longer wavelength fluorophore as an “emitter”. However, because the background resulting from FRET quenching is considerably higher than contact-mediated quenching that is commonly used in molecular beacon probes and yin-yang probes (Tyagi et al. (1998), supra; Marras, S. A. E. et al. (2002), Efficiencies of Fluorescence Resonance Energy Transfer and Contact-Mediated Quenching in Oligonucleotide Probes, Nucleic Acids Res. 30: e122), FRET quenching is not preferred for screening assays according to this invention. Assays according to this invention preferably employ probes that are quenched by contact-mediated quenching, such as molecular beacon probes or yin-yang probes. My most preferred probes are molecular beacon probes that include a non-fluorescent quencher. These probes are most preferred, not only because of their low background signal, but also because of their ease of design for a multiplex system. Bonnet, G. et al. (1998), Thermodynamic Basis of the Enhanced Specificity of Structured DNA Probes, Proc. Natl. Acad. Sci. USA 96: 6171-6176.

Assays according to this invention include, but are not limited to, embodiments that employ target amplification. Several exponential amplification techniques are well known. Among them are the polymerase chain reaction (PCR), which includes thermal cycling, and isothermal amplification schemes such as nucleic acid sequence-based amplification (NASBA), strand-displacement amplification (SDA), transcription-mediated amplification (TMA), rolling-circle amplification (RCA), and ramification amplification methodology (RAM). Amplification assay embodiments may utilize end-point detection or real-time detection. It is preferred to perform amplification in a sealed environment, for example sealed tubes, in the presence of the hybridization probes to minimize the possibility of cross contamination between samples that results from opening sample tubes.

While not limited by sample source, assays according to this invention advantageously may utilize human or animal samples sometimes referred to as being normally sterile, such as blood, tissue or spinal fluid. Such samples are advantageous as compared, for example, to sputum, stool or environmental samples, because they will contain fewer extraneous DNA sequences that could potentially interfere with amplification and detection. It is known that at least small amounts of many pathogens can be found in a patient's blood, even in septic syndromes resulting from bacterial infections. It is preferred to utilize normally sterile sources rather than non-sterile sources where a particular screening assay permits a choice.

Certain amplification assays according to this invention may advantageously take advantage of primers that amplify all or a number of nucleic acid targets being screened. When screening for mutations, it may be that numerous possible mutations occur in a particular variable nucleic acid sequence of interest, and one pair of primers flanking that region can be used to amplify all such possible mutations. Certain of these so-called “universal primers” bind to conserved regions of multiple species and are used for amplifying numerous targets, for example genes of various bacteria. See, for example, Kox, L. F. F. et al. (1995), PCR Assay Based on DNA Coding for 16S rRNA for Detection and Identification of Mycobacteria in Clinical Samples. J. Clin. Microbiol. 33: 3225-3233; Iwen, P. C. et al. (1995), Evaluation of Nucleic Acid-Based Test (PACE 2C) for Simultaneous detection of Chlamydia trachomatis and Neisseria gonorrhoeae in Endocervical Specimens, J. Clin. Microbiol. 33: 2587-2591; Yang, S. et al. (2002), Quantitative Multiprobe PCR Assay for Simultaneous Detection and Identification to Species Level of Bacterial Pathogens, J. Clin. Microbiol. 40: 3449-3454; Schonhuber, W. et al. (2001), Utilization of mRNA Sequences for Bacterial Identification, BMC Microbiol. 1: 2; Wong, R. S. and Chow, A. W. (2002), Identification of Enteric Pathogens by Heat Shock Protein 60 kDa (HSP 60) Gene Sequences, FEMS Microbiol. Lett. 206: 107-113. One pair, more likely two or three pairs, of primers may suffice for particular screening assays.

This invention includes assay kits for performing particular multiplex screening assays according to this invention. Kits include at least the intended complement of detection probes. We refer to collections of oligonucleotides as “oligonucleotide sets.” When the assay is an amplification assay, for example a PCR assay, the oligonucleotide sets preferably also include the primers needed for amplification. They may also include any amplifiable control sequence. Kits may include additional assay reagents such as enzymes, amplification buffer and, if desired, sample preparation reagents for isolation of nucleic acids; that is, up to all reagents needed for performing the assay, from sample preparation through detection.

Fluorescence-based screening assays of this invention are generally adaptable to detection instruments, which may include thermal cycling capability. Instruments may use a single-wavelength source or a multi-wavelength source, for example a white light combined with selectable filters, multiple laser sources or multiple light-emitting diode sources. Detection instruments vary in their ability to distinguish emissions from multiple fluorophores, which needs to be taken into account when selecting fluorophore panels for a particular assay. In preferred embodiments of assays according to this invention, emissions from fluorophores for each probe are balanced in intensity such that intensities from the subdivided portions are closely spaced, which aids identification. Fluorophores vary in intensity, target sequences vary in accessibility for probe hybridization, and instruments may vary in their responses to different wavelengths. Relative amounts of portions may be varied to achieve balance. Where possible, it is preferred that all portions for all probes be balanced to further aid in identification. The results of these assays can be automatically decoded and interpreted by computer programs in real time, enabling the assays to be highly accurate, and to be carried out in an automated, high-throughput fashion in clinical diagnostic settings.

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