Methods for performing multiplexed real-time PCR using large Stokes shift fluorescent dyes

Abstract

The present invention allows for the expansion of the multiplexing capacity of common PCR devices by using fluorogenic PCR probes made with large Stokes shift (LSS) fluorescent dyes. This approach eliminates the need for hardware in the instrument. No modifications to hardware or software components are required.

Description

[Technical field]

[0001] FIELD OF THEINVENTION The present invention relates to methods for polymerase chain reaction (PCR), and in particular to methods for performing multiplexed real-time PCR using large Stokes shift fluorescent dyes. [Background technology]

[0002] 2. Background of the Invention Polymerase chain reaction (PCR) has become a ubiquitous tool in biomedical research, disease monitoring and diagnosis. Amplification of nucleic acid sequences by PCR is described in U.S. Pat. Nos. 4,683,195, 4,683,202, and 4,965,188. PCR is now well known in the art and has been widely described in the scientific literature. See PCR Applications, ((1999) Innis et al., eds., Academic Press, San Diego); PCR Strategies, ((1995) Innis et al., eds., Academic Press, San Diego); PCR Protocols, ((1990) Innis et al., eds., Academic Press, San Diego), and PCR Technology, ((1989) Erlich, ed., Stockton Press, New York). "Real-time" PCR assays allow for simultaneous amplification and detection and / or quantification of the starting amount of target sequence. A basic TaqMan real-time PCR assay using the 5' to 3' nuclease activity of DNA polymerase is described in Holland et al., (1991) Proc. Natl. Acad. Sci. 88:7276-7280 and U.S. Patent No. 5,210,015. Real-time PCR without nuclease activity (nuclease-free assay) is described in U.S. Patent Application Publication No. 20100143901. The use of fluorescent probes in real-time PCR is described in U.S. Patent No. 5,538,848.

[0003] A typical real-time PCR protocol using fluorescent probes involves the use of a labeled probe specific to each target sequence. The probe is preferably labeled with one or more fluorescent moieties that absorb and emit light of a specific wavelength. When hybridized to a target sequence or its amplicon, the probe exhibits a detectable change in fluorescence emission as a result of probe hybridization or hydrolysis.

[0004] However, a major challenge for real-time assays remains the ability to analyze a large number of targets in a single tube. In virtually every field of medicine and diagnostics, the number of loci of interest grows rapidly. For example, multiple loci must be analyzed in forensic DNA profiling, pathogenic microbial detection, multilocus genetic disease screening, and multigene expression studies, to name a few.

[0005] For most current methods, the ability to multiplex assays is limited by the detection equipment. Specifically, the use of multiple probes in the same reaction requires the use of different fluorescent labels. Simultaneous detection of multiple probes requires that the equipment be able to distinguish the light signals emitted by each probe. Most current technologies on the market are unable to detect more than 4-7 separate wavelengths in the same reaction vessel. Thus, using one uniquely labeled probe per target, one can only detect no more than 4-7 separate targets in the same vessel. In practice, at least one target is usually a control nucleic acid. Thus, in practice, no more than 3-6 experimental targets are detected in the same tube. The use of fluorescent dyes is also limited due to the spectral bandwidth that only about 6 or 7 dyes can fit within the visible spectrum without significant overlapping interference. Thus, unless fundamental changes occur in amplification and detection strategies, the ability to multiplex assays will not keep up with clinical needs.

[0006] Further ability to multiplex real-time amplification reactions is provided by post-PCR melting assays. See US Patent Publication No. 20070072211. In melting assays, amplified nucleic acids are identified by their unique melting profile. Melting assays involve determining the melting temperature (melting point) of a double-stranded target or a duplex between a labeled probe and a target. To determine the melting temperature using a fluorescently labeled probe, as described in US Patent No. 5,871,908, the duplex between the target nucleic acid and the probe is gradually heated (or cooled) in a controlled temperature program. Dissociation of the duplex changes the distance between the interacting fluorophores or between the fluorophore and the quencher. The interacting fluorophores can be conjugated to separate probe molecules, as described in US Patent No. 6,174,670. Alternatively, one fluorophore can be conjugated to the probe and the other fluorophore can be intercalated into the nucleic acid duplex, as described in US Patent No. 5,871,908. As yet another alternative, the fluorophore may be conjugated to a single probe oligonucleotide: upon melting of the duplex, the fluorescence is quenched since the fluorophore and quencher are now brought together within the single-stranded probe.

[0007] The melting of a nucleic acid duplex is monitored by measuring the associated change in fluorescence. The change in fluorescence can be represented on a graph called a "melting profile." Different probe-target duplexes can be designed to melt (or reanneal) at different temperatures, so that each probe generates a unique melting profile. Properly designed probes will have melting temperatures that are clearly distinguishable from the melting temperatures of other probes in the same assay. Many existing software tools allow for the design of probes for same-tube multiplex assays with these objectives in mind. For example, Visual OMP™ software (DNA Software, Inc., Ann Arbor, Mich.) allows for the determination of the melting temperatures of nucleic acid duplexes under various reaction conditions.

[0008] A method of multiplex PCR using fluorescence detection and subsequent post-amplification melting assay is described in U.S. Patent No. 6,472,156. The number of targets detectable by such a method is the product of the number of detectable wavelengths and the number of distinguishable melting profiles. Thus, adding a melting assay to color detection was an advancement in the ability to detect multiple targets.

[0009] Post-amplification melting assays are most commonly used for qualitative purposes, i.e., to identify target nucleic acids (see U.S. Pat. Nos. 6,174,670; 6,427,156; and 5,871,908). It is known to differentiate the melting curve function to obtain melting peaks. Ririe et al. ("Product differentiation by analysis of DNA melting curves during the polymerase chain reaction" (1997) Anal. Biochem. 245:154-160) observed that differentiation helps resolve melting curves generated by a mixture of products. After differentiation, the melting peaks generated by each component of the mixture become easily distinguishable. It has also been known for some time that the post-amplification melting signal, i.e., melting peak, is higher in proportion to the amount of nucleic acid in the sample. For example, U.S. Pat. No. 6,245,514 teaches a post-amplification melting assay that uses a duplex intercalating dye to generate differential melting peaks, and then integrates the peaks using proprietary software. The integral provides information about the efficiency of amplification and the relative amount of amplified nucleic acid.

[0010] In practice, it would be desirable to go beyond qualitative assays and be able to quantify multiple targets in the same sample. See, for example, Sparano et al. "Development of the 21-gene assay and its application in clinical practice and clinical trials" J. Clin. Oncol. (2008) 26(5):721-728. The ability to quantify the amount of a target is useful for clinical applications such as determining the viral load in a patient's serum, measuring the expression level of a gene in response to drug therapy, or determining the molecular signature of a tumor to predict its response to treatment.

[0011] In real-time PCR assays, the signal generated by the labeled probes can be used to estimate the amount of input target nucleic acid. The higher the input, the earlier the fluorescent signal crosses a predefined threshold (Ct). Thus, by comparing samples to each other or to a control sample with a known amount of nucleic acid, the relative or absolute amount of target nucleic acid can be determined. However, existing methods are limited in their ability to simultaneously quantify multiple targets. As with the qualitative detection of multiple targets, a limiting factor is the availability of spectrally resolvable fluorophores. As explained above, state-of-the-art fluorescent labeling techniques cannot obtain distinct signals from more than six or seven separate fluorescently labeled probes in the same tube. Thus, a fundamentally different experimental approach is required to enable the amplification and detection of multiple nucleic acid targets during real-time PCR.

[0012] Commercially available fluorescence-based devices for automated polymerase chain reaction (PCR) are able to detect multiple targets in a single reaction vessel by differentiating light from different colored fluorophores (multiplexing). The choice of dyes is characterized to minimize their spectral overlap. All fluorophores in the ensemble are excited with light at or near their absorption maxima, and the emitted light (fluorescence) is detected at or near their fluorescence maxima. Individual fluorophores can be differentiated by restricting the range of excitation and emission wavelengths (bands) with optical filters. A specific combination of an excitation band and an emission band detected simultaneously defines a light channel, each of which allows the identification of one PCR target. The maximum number of achievable light channels depends on a number of interrelated factors, such as the available spectral range, excitation light intensity, fluorophore brightness, fluorophore spectral width, filter bandwidth, and detector sensitivity. State-of-the-art PCR devices with fluorescence-based detection technology use up to 4-6 optical filters per excitation and emission pathway. Thus, with standard fluorophores, 4-6 PCR targets can be differentiated. The present invention makes it possible to extend the multiplexing capacity of common PCR devices by using fluorogenic PCR probes made with large Stokes shift (LSS) fluorescent dyes, an approach that does not require modification of any hardware or software components within the instrument. Summary of the Invention

[0013] The present invention relates to the use of fluorescent dyes with a large Stokes shift (LSS) to increase the number of targets that can be simultaneously detected in a single reaction vessel during polymerase chain reaction (PCR) (multiplexing), as defined in the appended claims. LSS dyes have been used in molecular imaging, cell imaging, tissue imaging, and as control reference dyes in PCR reactions, but not for the specific purpose of expanding the multiplexing capabilities of PCR instruments designed for fluorescent signal detection in real-time PCR.

[0014] Accordingly, in one aspect, the present invention provides a method for detecting at least two target nucleic acid sequences in a sample, comprising: (a) reacting, in a single reaction vessel, the sample suspected of containing the at least two target nucleic acid sequences with: i. a first pair of oligonucleotide primers having nucleotide sequences complementary to each strand of a first target nucleic acid sequence, and a second pair of oligonucleotide primers having nucleotide sequences complementary to each strand of a second target nucleic acid sequence; ii. a first oligonucleotide probe comprising a nucleotide sequence at least partially complementary to the first target nucleic acid sequence and annealing within the first target nucleic acid sequence bounded by the first pair of oligonucleotide primers, the first oligonucleotide probe being labeled with a large Stokes shift (LSS) fluorescent dye capable of generating a detectable signal and a first quencher moiety capable of quenching the detectable signal generated by the LSS fluorescent dye, the LSS fluorescent dye being cleaved from the first quencher moiety by a nuclease-sensitive cleavage site. a first oligonucleotide probe, the first oligonucleotide probe being separated from the first oligonucleotide probe; and iii a second oligonucleotide probe comprising a nucleotide sequence at least partially complementary to the second target nucleic acid sequence and annealing within the second target nucleic acid sequence bounded by the second pair of oligonucleotide primers, the second oligonucleotide probe being labeled with a small Stokes shift (SSS) fluorescent dye capable of generating a detectable signal and a second quencher moiety capable of quenching the detectable signal generated by the SSS fluorescent dye, the SSS fluorescent dye being separated from the second quencher moiety by a nuclease-sensitive cleavage site, the SSS fluorescent dye having an absorption peak maximum significantly different from an absorption peak maximum of the LSS fluorescent dye on the first oligonucleotide probe and an emission peak maximum similar to an emission peak maximum of the LSS fluorescent dye on the first oligonucleotide probe, the significant difference being at least 80 nanometers in wavelength;(b) amplifying the first and second target nucleic acid sequences by polymerase chain reaction (PCR) using a nucleic acid polymerase having 5' to 3' nuclease activity, whereby during the extension step of each PCR cycle, the 5' to 3' nuclease activity of the nucleic acid polymerase enables cleavage and separation of the LSS fluorescent dye from the first quenching moiety on the first oligonucleotide probe and cleavage and separation of the SSS fluorescent dye from the second quenching moiety on the second oligonucleotide probe; (c) determining the wavelength of the absorption peak maximum of the LSS fluorescent dye or its (d) measuring a detectable signal from the LSS fluorescent dye by excitation at or near the wavelength of the absorption peak of the SSS fluorescent dye; (d) repeating steps (b) and (c) for a number of PCR cycles to produce a desired amount of amplification product from the first target nucleic acid sequence and the second target nucleic acid sequence; (e) detecting the presence of the first target nucleic acid sequence from the signal detected from the LSS fluorescent dye, and detecting the presence of the second target nucleic acid sequence from the signal detected from the SSS fluorescent dye;

[0015] In one embodiment, the SSS fluorescent dye on the second oligonucleotide probe has an emission peak maximum significantly different from the emission peak maximum of the LSS fluorescent dye on the first oligonucleotide probe and an absorption peak maximum similar to the absorption peak maximum of the LSS fluorescent dye on the first oligonucleotide probe, the significant difference being at least 80 nanometers in wavelength. In another embodiment, the difference between the absorption peak maximum of the LSS fluorescent dye and the absorption peak maximum of the SSS fluorescent dye is greater than 80 nanometers in wavelength. In yet another embodiment, the difference is greater than 100 nanometers in wavelength. In a further embodiment, the difference between the emission peak maximum of the LSS fluorescent dye and the emission peak maximum of the SSS fluorescent dye is greater than 80 nanometers in wavelength. In yet another embodiment, the difference is greater than 100 nanometers in wavelength.

[0016] In one embodiment, the LSS fluorescent dye is selected from the group consisting of ALEXA FLUOR 430, ATTO 430LS, ATTO 490LS, ATTO 390LS, CASCADE YELLOW, CF350, CHROMEO 494, CYTO 500 LSS, CYTO 510 LSS, CYTO 514 LSS, CYTO 520 LSS, DAPOXYL, DY 480XL, DY 481XL, DY 485XL, DY 510XL, DY 511XL, DY 520XL, The LSS fluorescent dye is selected from the group consisting of DY 521XL, DY 601XL, DY 350XL, DY 360XL, DY 370XL, DY 375XL, DY 380XL, DY 395XL, DY 396XL, DYLIGHT 515-LS, DYLIGHT 485-LS, DYLIGHT 510-LS, DYLIGHT 521-LS, FURA 2, INDO 1, KROME ORANGE, LUB 04, LUCIFER YELLOW, NBD X, NILE RED, PULSAR 650, PYMPO, STAR 440SXP, STAR 470SXP, STAR 520SXP, VIOGREEN, CF 350, SETAU 405, and PACIFIC ORANGE. In a further embodiment, the LSS fluorescent dye is DY396XL. In yet a further embodiment, the LSS fluorescent dye is CHROMEO 494. In another embodiment, the LSS fluorescent dye is ATTO 490LS.

[0017] In one embodiment, the LSS fluorescent dye has a fluorescent signal intensity that remains stable at temperatures up to 100° C. In one embodiment, the LSS fluorescent dye is ATTO 490LS. In another embodiment, the first oligonucleotide probe, the second oligonucleotide probe, or both the first and second oligonucleotide probes are tagged probes compatible with TAGS technology.

[0018] In another embodiment, the method of the invention is carried out in a single reaction vessel that is a capillary, the capillary having (i) a proximal end having an opening through which a sample can be introduced; (ii) a distal end; and (iii) at least a first segment containing at least one nucleic acid extraction reagent, a second segment distal to the first segment and containing a wash reagent, and a third segment distal to the second segment and containing one or more amplification reagents, each of the segments (A) defined by a capillary; and (B) at least partially fluidly isolated by a fluid-tight seal formed by joining opposing wall portions of the capillary to one another, such that (1) the seal is disrupted by application of fluid pressure to the segment partially fluidly isolated by the seal; and (2) the seal is disrupted by application of fluid pressure to the segment partially fluidly isolated by the seal. (C) at least a first segment, a second segment, and a third segment that are expandable to receive a volume of fluid expelled from another segment and compressible to be substantially fluid-free when compressed; (iv) a cap for closing the opening, the capillary including a chamber in fluid communication with the capillary, allowing free escape of gas but retaining all liquid volume and infectious agents within the tube; (v) a rigid frame in which the proximal and distal ends of the capillary are held; and (vi) an integral capillary tensioning mechanism or attachment of the capillary to the frame that pulls the capillary to a sufficient tension to facilitate compression and flattening of the capillary. [Brief description of the drawings]

[0019] [Figure 1]Channel assignment matrix for a 6-color PCR instrument. Conceptual diagram of how the conventional light channels of a 6-color PCR instrument can be expanded by new combinations of excitation and emission filters resulting in 21 possible detection channels. The central wavelengths of the excitation and emission filters are shown in nanometers. The excitation and emission descriptors are UV (u), blue (b), green (g), amber (a), red (r), IR, (i). A light channel is formed by combining one excitation filter with one emission filter. For example, the two-letter channel descriptor for excitation in the UV and emission in the green is "ug". The channels highlighted in light grey correspond to conventional channels accessible with standard short Stokes shift (SSS) fluorophores. The white fields correspond to channels accessible with currently available LSS dyes. The channels highlighted in dark grey are accessible by resonant electron transfer (RET) probes. [Diagram 2] Absorption (left) and emission spectra (right) for a 6-color PCR instrument (cobas® Liat® Analyzer). Spectra of four standard SSS fluorescent dyes and one LSS dye (Chromeo 494) are shown. Spectra were normalized to arbitrary absorption and fluorescence units (AU / FU). Wavelength regions covered by optical filters are shown as horizontal lines. Chromeo 494 (dashed line), which has excitation / emission maxima at 494 nm / 628 nm, can be excited with blue light and detected with amber and / or red emission filters (ba and / or br channels). [Diagram 3] Absorption (left) and emission spectra (right) of a 6-color PCR instrument (cobas® Liat® analyzer). Spectra of four standard SSS fluorescent dyes and one LSS dye (Dy396XL) are shown. The spectra were normalized to arbitrary absorption and fluorescence units (AU / FU). The wavelength regions covered by the optical filters are shown as horizontal lines. Dy396XL (dashed line), which has excitation / emission maxima at 392 nm / 572 nm, can be excited with UV light and detected with a green emission filter (ug channel). [Figure 4] Real-time PCR growth curves of six TaqMan probes in the cobas® Liat® CT / NG / TV / MG test with target input at 3x LoD level. Multiplexing in the cobas® Liat® analyzer was achieved by combining four SSS dyes with two LSS dyes (Chromeo494, Dy395XL). Growth curve baselines were normalized to 0 for comparison. [Diagram 5] Statistical analysis of MGPB probes labeled with Dy395XL or Dy396XL in a 5-channel cobas®Liat® CT / NG / MG study. Due to baseline differences between MG-Dy395XL and MG-Dy396XL probes, the performance (Ct, amplitude, and Kexp) of the probe with the higher baseline was normalized according to the baseline ratio. [Figure 6] Statistical analysis of MGPC probes labeled with Dy395XL or Dy396XL in a 5-channel cobas®Liat® CT / NG / MG study. Due to baseline differences between MG-Dy395XL and MG-Dy396XL probes, the performance (Ct, amplitude, and Kexp) of the probe with the higher baseline was normalized according to the baseline ratio. [Figure 7A] 7A-7C are front and side views of an exemplary embodiment of a sample tube including a capillary tube for the cobas® Liat® analyzer. [Figure 7B] 7A and 7B are cross-sectional views of sample tubes placed inside the analyzer, according to an exemplary embodiment of the present invention. [Figure 8A] Another exemplary embodiment of a sample tube including a capillary tube for the cobas® Liat® analyzer. [Figure 8B] Another exemplary embodiment of a sample tube including a capillary tube for the cobas® Liat® analyzer. FIG. 8B is a perspective view of the sample tube. [Figure 9]Optical channel allocation matrix for a 5-color PCR instrument. Conceptual diagram of how the conventional optical channels of a 5-color PCR instrument can be expanded by new combinations of excitation and emission filters. The central wavelengths of the excitation and emission filters are shown in nanometers. The optical channels on the diagonal field (light grey) are accessible with standard short Stokes shift (SSS) fluorophores. The dark grey fields correspond to optical channels accessed with LSS dyes. In one embodiment, the target was detected with LSS dyes by excitation at 495 nm and detection at 645 nm (ATTO 490LS dye). In another embodiment, the target was detected with LSS dyes by excitation at 435 nm and detection at 580 nm (RLS dye). The white fields correspond to additional channels conceptually accessible with LSS dyes or resonant electron transfer (RET) probes. [Figure 10] Absorption (left) and emission spectra (right) for 5-color PCR instruments (LightCycler® and cobas® x800 analyzer). Spectra of 5 standard SSS fluorescent dyes and one LSS dye, ATTO 490LS, that can be detected by excitation at 495 nm and detection at 645 nm are shown. Spectra were normalized to arbitrary absorption and emission units (AU / FU). Wavelength regions covered by optical filters are shown as horizontal lines. [Figure 11] Absorption (left) and emission spectra (right) for 5-color PCR instruments (LightCycler® and cobas® x800 analyzer). Spectra of 5 standard fluorescent dyes and one LSS dye (RLS) that can be detected by excitation at 435 nm and detection at 580 nm are shown. Spectra were normalized to arbitrary absorbance and fluorescence units (AU / FU). The wavelength ranges covered by the optical filters are shown as horizontal lines. [Figure 12]Real-time PCR growth curves demonstrating compatibility of extended optical multiplexing based on LSS dyes with temperature-based multiplexing. A notable example shows individual detection of three targets in the ATTO 490LS optical channel (495 nm / 645 nm) across three thermal channels (TCs). TC1 is based on ATTO 490LS and a standard TaqMan probe with a fluorescence readout at 58°C. TC2 and TC3 are based on a tagged probe design with ATTO 490LS that generates a target fluorescent signal at 80°C and 91°C, respectively. Target concentration was 1000 cp / reaction and all samples targeted were present. PCR growth curves for each thermal channel where a positive PCR signal is expected are marked with an asterisk. In the higher thermal channels, the slight upward or downward slope in the absence of target is caused by the non-optimized thermal compensation coefficient of the fluorescent dyes. Data were generated on a LightCycler® 480 analyzer. [Figure 13] Real-time PCR growth curves demonstrating the compatibility of extended optical multiplexing based on LSS dyes with temperature-based multiplexing. A notable example shows the individual detection of three targets in the RLS optical channel (435 nm / 580 nm) across three thermal channels (TCs). TC1 is based on a standard TaqMan probe with RLS dyes and a fluorescence readout at 58°C. TC2 and TC3 are based on a tagged probe design with RLS dyes that generate target fluorescent signals at 80°C and 91°C, respectively. The concentration of each target was 1000 cp / reaction, except for the no template control (NTC). In the higher thermal channels, the slight upward or downward slope in the absence of target is caused by the non-optimized thermal compensation coefficients of the fluorescent dyes. Data were generated on a LightCycler® 480 analyzer. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0020] Detailed Description of the Invention definition The term "sample" as used herein includes any specimen or culture (e.g., microbial culture) that contains nucleic acid. The term "sample" is also meant to include both biological and environmental samples. Samples can include specimens of synthetic origin. Biological samples include whole blood, serum, plasma, umbilical cord blood, chorionic villi, amniotic fluid, cerebrospinal fluid, spinal fluid, lavage fluid (e.g., bronchoalveolar epithelium, stomach, peritoneal, duct, ear, arthroscopic), biopsy samples, urine, feces, sputum, saliva, nasal mucus, prostatic fluid, semen, lymph, bile, tears, sweat, breast milk, breast fluid, embryonic cells, and fetal cells. In a preferred embodiment, the biological sample is blood, more preferably plasma. As used herein, the term "blood" encompasses whole blood or any fraction of blood, such as serum and plasma, as generally defined. Plasma refers to the fraction of whole blood resulting from centrifugation of blood treated with an anticoagulant. Serum refers to the aqueous portion of bodily fluids remaining after a blood sample has clotted. Environmental samples include environmental materials such as surface matter, soil, water, and industrial samples, as well as samples obtained from food and dairy processing equipment, devices, utensils, fixtures, utensils, disposable and non-disposable items. These examples should not be construed as limiting the types of samples applicable to the present invention.

[0021] The term "target" or "target nucleic acid" as used herein is intended to mean any molecule whose presence is to be detected or measured, or whose function, interaction or properties are to be studied. Thus, a target includes essentially any molecule for which a detectable probe (e.g., an oligonucleotide probe) or assay exists or can be produced by one of skill in the art. For example, a target can be a biomolecule, such as a nucleic acid molecule, a polypeptide, a lipid or a carbohydrate, that can bind to or otherwise contact a detectable probe (e.g., an antibody), and a detectable probe also includes a nucleic acid that can be detected by the methods of the present invention. As used herein, a "detectable probe" refers to any molecule or agent that can hybridize or anneal to a target biomolecule of interest and allows for the specific detection of the target biomolecule described herein. In one aspect of the present invention, the target is a nucleic acid and the detectable probe is an oligonucleotide. The terms "nucleic acid" and "nucleic acid molecule" can be used interchangeably throughout this disclosure. The above terms refer to oligonucleotides, oligos, polynucleotides, deoxyribonucleotides (DNA), genomic DNA, mitochondrial DNA (mtDNA), complementary DNA (cDNA), bacterial DNA, viral DNA, viral RNA, RNA, message RNA (mRNA), transfer RNA (tRNA), ribosomal RNA (rRNA), siRNA, catalytic RNA, clones, plasmids, M13, P1, cosmids, bacterial artificial chromosomes (BAC), yeast artificial chromosomes (YAC), amplified nucleic acids, amplicons, PCR products and other types of amplified nucleic acids, RNA / DNA hybrids, and polyamide nucleic acids (PNAs), all of which may be in either single-stranded or double-stranded form, and unless otherwise limited, include known analogs of natural nucleotides that can perform functions similar to naturally occurring nucleotides, as well as combinations and / or mixtures thereof. Thus, the term "nucleotide" refers to both natural and modified / non-natural nucleotides, including nucleoside tri-, di-, and monophosphates, and monophosphate monomers present in polynucleic acids or oligonucleotides.The nucleotide may also be a ribonucleotide, a 2'-deoxynucleotide, or a 2',3'-deoxynucleotide, as well as a myriad of other nucleotide mimetics well known in the art. Mimetics include chain-terminating nucleotides such as 3'-O-methyl, halogenated bases or sugar substitutions, non-sugar, alternative sugar structures including alkyl ring structures, alternative bases including inosine, deaza modifications, chi-, and / or psi-, linker modifications, mass label modifications, phosphodiester modifications or substitutions including phosphorothioates, methylphosphonates, boranophosphates, amides, esters, ethers, and / or cleavable bonds such as photocleavable nitrophenyl moieties, at the base or completely internucleotide substitutions.

[0022] The presence or absence of a target can be measured quantitatively or qualitatively. Targets can be in a variety of different forms, including, for example, simple or complex mixtures or in substantially purified form. For example, a target can be part of a sample containing other components, or it can be the only or major component of a sample. Thus, a target can be a component of a whole cell or tissue, a cell or tissue extract, a fractionated lysate thereof, or a substantially purified molecule. Additionally, a target can have either a known or unknown sequence or structure.

[0023] The term "amplification reaction" refers to any in vitro means for producing multiple copies of a target sequence of nucleic acid.

[0024] "Amplification" refers to the process of subjecting a solution to conditions sufficient to allow amplification. Components of an amplification reaction include, but are not limited to, for example, primers, polynucleotide templates, polymerase, nucleotides, dNTPs, etc. The term "amplification" typically refers to the "exponential" increase of target nucleic acid. However, as used herein, "amplification" can also refer to a linear increase in the number of selected target sequences of nucleic acid, but is different from a one-off single primer extension process.

[0025] "Polymerase chain reaction" or "PCR" refers to a method in which specific segments or subsequences of a target double-stranded DNA are amplified in a geometric progression. PCR is well known to those of skill in the art, see, e.g., U.S. Patent Nos. 4,683,195 and 4,683,202; and PCR Protocols: A Guide to Methods and Applications, Innis et al., eds, 1990.

[0026] As used herein, "oligonucleotide" refers to a linear oligomer of natural or modified nucleoside monomers linked by phosphodiester bonds or analogs thereof. Oligonucleotides include deoxyribonucleosides, ribonucleosides, their anomeric forms, peptide nucleic acids (PNAs), and the like, capable of specifically binding to a target nucleic acid. Typically, the monomers are linked by phosphodiester bonds or analogs thereof to form oligonucleotides ranging in size from a few monomer units (e.g., 3-4) to tens of monomer units (e.g., 40-60). When an oligonucleotide is represented by a sequence of letters such as "ATGCCTG," it will be understood that the nucleotides are in 5'-3' order from left to right, with "A" representing deoxyadenosine, "C" representing deoxycytidine, "G" representing deoxyguanosine, "T" representing deoxythymidine, and "U" representing the ribonucleoside uridine, unless otherwise specified. Typically, oligonucleotides contain the four naturally occurring deoxynucleotides, but they can also contain ribonucleosides or non-natural nucleotide analogs, as described above. If an enzyme has specific oligonucleotide or polynucleotide substrate requirements for activity (e.g., single-stranded DNA, RNA / DNA duplexes, etc.), the selection of the appropriate composition of the oligonucleotide or polynucleotide substrate is well within the knowledge of one of ordinary skill in the art.

[0027] As used herein, "oligonucleotide primer" or simply "primer" refers to a polynucleotide sequence that hybridizes to a sequence on a target nucleic acid template and facilitates detection of an oligonucleotide probe. In an amplification embodiment of the invention, the oligonucleotide primer serves as a starting point for nucleic acid synthesis. In a non-amplification embodiment, the oligonucleotide primer can be used to create a structure that can be cleaved by a cleavage agent. The length of the primer can vary and is often less than 50 nucleotides long, for example 12-25 nucleotides long. The length and sequence of the primer for use in PCR can be designed based on principles known to those of skill in the art.

[0028] As used herein, the term "oligonucleotide probe" refers to a polynucleotide sequence that is capable of hybridizing or annealing to a target nucleic acid of interest and allowing for the specific detection of the target nucleic acid.

[0029] A "reporter moiety" or "reporter molecule" is a molecule that provides a detectable signal. The detectable phenotype can be, for example, colorimetric, fluorescent, or luminescent. Examples of fluorescent reporter moieties include, for example, fluorescein (FAM), hexachlorofluorescein (HEX), JA270 (Roche Molecular Systems), cyanine dyes (e.g., CY3.5, CY5, or CY5.5).

[0030] A "quencher moiety" or "quencher molecule" is a molecule capable of quenching a detectable signal from a reporter moiety. Examples of quencher moieties used with fluorescent reporters include, for example, so-called dark quenchers, such as Black Hole Quenchers (BHQ-1 or BHQ-2) (LGC BioSearch Technologies) or Iowa Black (Integrated DNA Technologies); and fluorescent moieties that use fluorescence resonance energy transfer (FRET), such as the cyanine dyes mentioned above.

[0031] A "mismatched nucleotide" or "mismatch" refers to a nucleotide that is not complementary to the target sequence at that position or positions. An oligonucleotide probe may have at least one mismatch, but may have 2, 3, 4, 5, 6, or 7 or more mismatched nucleotides.

[0032] The term "polymorphism" as used herein refers to an allelic variant. Polymorphisms can include single nucleotide polymorphisms (SNPs) as well as simple sequence length polymorphisms. Polymorphisms can result from one or more nucleotide substitutions at one allele compared to another, or can result from insertions or deletions, duplications, inversions, and other modifications known in the art.

[0033] The term "modification" as used herein refers to alterations of the oligonucleotide probe at the molecular level (e.g., base moiety, sugar moiety, or phosphate backbone). Nucleoside modifications include, but are not limited to, the introduction of cleavage blockers or cleavage inducers, the introduction of minor groove binders, isotopic enrichment, isotopic depletion, the introduction of deuterium, and halogen modifications. Nucleoside modifications can also include moieties that increase the stringency of hybridization or increase the melting temperature of the oligonucleotide probe. For example, nucleotide molecules can be modified with an extra bridge connecting the 2' and 4' carbons to result in locked nucleic acid (LNA) nucleotides that are resistant to cleavage by nucleases (as described in U.S. Patent No. 6,268,490 to Imanishi et al. and U.S. Patent No. 6,794,499 to Wengel et al.). The composition of the tag portion of the oligonucleotide probe and the quenching oligonucleotide molecule are limited only by their ability to form a stable duplex. Thus, these oligonucleotides may comprise DNA, L-DNA, RNA, L-RNA, LNA, L-LNA, PNA (peptide nucleic acid, as described in U.S. Pat. No. 5,539,082 to Nielsen et al.), BNA (bridged nucleic acid, e.g., 2',4'-BNA(NC) [2'-O,4'-C-aminomethylene bridged nucleic acid] as described in Rahman et al., J. Am. Chem. Soc. 2008;130(14):4886-96), L-BNA, etc. (where "L-XXX" refers to the L-enantiomer of the sugar unit of the nucleic acid), or any other known changes and modifications of the nucleotide base, sugar, or phosphodiester backbone.

[0034] Other examples of nucleoside modifications include various 2'-substitutions such as halo, alkoxy and allyloxy groups introduced into the sugar portion of oligonucleotides. Evidence has been presented that 2'-substituted-2'-deoxyadenosine polynucleotides resemble double-stranded RNA more than DNA. Ikehara et al. (Nucleic Acids Res., 1978, 5, 3315) show that 2'-fluoro substituents in duplexes with polyA, polyI or polyC and their complements are significantly more stable than ribonucleotide or deoxyribonucleotide polyduplexes as determined by standard melting assays. Inoue et al. (Nucleic Acids Res., 1987, 15, 6131) describe the synthesis of mixed oligonucleotide sequences containing 2'-OMe (O-methyl) substituents on all nucleic acid nucleotides. Mixed 2'-OMe-substituted oligonucleotides hybridized to their RNA complements as strongly as RNA-RNA duplexes, which were significantly stronger than RNA-DNA heteroduplexes of the same sequence. Examples of substitutions at the 2' position of the sugar include F, CN, CF3, OCF3, OMe, OCN, O-alkyl, S-alkyl, SMe, SO2Me, ONO2, NO2, NH3, NH2, NH-alkyl, OCH3=CH2, and OCCH.

[0035] The term "specific" or "specificity" in reference to the binding of one molecule to another, such as a probe for a target polynucleotide, refers to the recognition, contact, and formation of a stable complex between the two molecules, as well as the much weaker recognition, contact, or complex formation between the molecule and the other molecule. The term "annealing" as used herein refers to the formation of a stable complex between two molecules. In particular, "annealing" can refer to the formation of a stable double-stranded complex between complementary oligonucleotides.

[0036] A probe is "capable of annealing" to a nucleic acid sequence if at least one region of the probe shares substantial sequence identity with at least one region of the complement of the nucleic acid sequence. "Substantial sequence identity" is at least about 80%, preferably at least about 85%, more preferably at least about 90%, 95% or 99%, and most preferably 100% sequence identity. For purposes of determining sequence identity between DNA and RNA sequences, U and T are often considered to be the same nucleotide. For example, a probe containing the sequence ATCAGC can hybridize to a target RNA sequence containing the sequence GCUGAU.

[0037] The term "cleavage agent" as used herein refers to any means capable of cleaving an oligonucleotide probe to obtain fragments, including but not limited to enzymes. In the case of a method in which no amplification occurs, the cleavage agent may only serve to cleave, degrade or otherwise separate the second portion of the oligonucleotide probe or its fragments. The cleavage agent may be an enzyme. The cleavage agent may be natural or synthetic, modified or unmodified.

[0038] For methods in which amplification occurs, the cleavage agent is preferably an enzyme with synthetic (or polymerizing) activity and nuclease activity. Such enzymes are often nucleic acid amplification enzymes. An example of a nucleic acid amplification enzyme is a nucleic acid polymerase enzyme, such as Thermus aquaticus (Taq) DNA polymerase or E. coli DNA polymerase I. The enzyme may be naturally occurring, unmodified or modified.

[0039] "Nucleic acid polymerase" refers to an enzyme that catalyzes the incorporation of nucleotides into a nucleic acid. Exemplary nucleic acid polymerases include DNA polymerase, RNA polymerase, terminal transferase, reverse transcriptase, telomere extender, and the like.

[0040] "Thermostable DNA polymerase" refers to a DNA polymerase that is stable (i.e., resists degradation or denaturation) and retains sufficient catalytic activity when exposed to elevated temperatures for a selected period of time. For example, a thermostable DNA polymerase retains sufficient activity to cause a subsequent primer extension reaction when exposed to elevated temperatures for the time required to denature double-stranded nucleic acids. Heating conditions required for nucleic acid denaturation are well known in the art and are exemplified in U.S. Pat. Nos. 4,683,202 and 4,683,195. As used herein, a thermostable polymerase is typically suitable for use in a temperature cycling reaction, such as a polymerase chain reaction ("PCR"). Examples of thermostable nucleic acid polymerases include Thermus aquaticus (Taq) DNA polymerase, Thermus sp. Z05 polymerase, Thermus flavus polymerase, Thermotoga maritima polymerase, such as TMA-25 polymerase, TMA-30 polymerase, and Thermus thermophilus DNA polymerase.

[0041] A "modified" polymerase refers to a polymerase in which at least one monomer differs from the reference sequence, such as a native or wild type of polymerase, or another modified form of a polymerase. Such modified polymerases are described, for example, in US Patent Publication No. 20110294168 and US Patent Publication No. 20140178911. Exemplary modifications include monomer insertions, deletions, and substitutions. Modified polymerases also include chimeric polymerases that have identifiable component sequences (e.g., structural or functional domains) derived from two or more parents. Also included within the definition of modified polymerases are those that include chemical modifications of the reference sequence. Examples of modified polymerases include G46E E678G CS5 DNA polymerase, G46E L329A E678G CS5 DNA polymerase, G46E L329A D640G S671F CS5 DNA polymerase, G46E L329A D640G S671F E678G CS5 DNA polymerase, G46E E678G CS6 DNA polymerase, Z05 DNA polymerase, ΔZ05 polymerase, ΔZ05-Gold polymerase, ΔZ05R polymerase, E615G Taq DNA polymerase, E678G TMA-25 polymerase, and E678G TMA-30 polymerase.

[0042] The term "5' to 3' nuclease activity" or "5'-3' nuclease activity" refers to the activity of a nucleic acid polymerase typically associated with nucleic acid chain synthesis whereby nucleotides are removed from the 5' end of a nucleic acid chain, for example, E. coli DNA polymerase I has this activity, but the Klenow fragment does not. Among the enzymes that have 5' to 3' nuclease activity are 5' to 3' exonucleases. Examples of such 5' to 3' exonucleases include exonuclease from B. subtilis, phosphodiesterase from spleen, lambda exonuclease, exonuclease II from yeast, exonuclease V from yeast, and exonuclease from Neurospora crassa.

[0043] Various aspects of the invention are based on the special properties of nucleic acid polymerases, which can have several activities, among them a 5' to 3' nuclease activity, that allow them to cleave mononucleotides or small oligonucleotides from oligonucleotides annealed to a larger complementary polynucleotide. For cleavage to occur efficiently, the upstream oligonucleotide must also be annealed to the same larger polynucleotide.

[0044] Detection of target nucleic acid utilizing 5' to 3' nuclease activity can be performed by "TaqMan® assay" or "5'-nuclease assay" as described in U.S. Patent Nos. 5,210,015; 5,487,972; and 5,804,375; and Holland et al., 1988, Proc. Natl. Acad. Sci. USA 88:7276-7280. In the TaqMan® assay, there is a labeled detection probe that hybridizes within the amplified region during the amplification reaction. This probe is modified so that it does not act as a primer for DNA synthesis. The amplification is performed with a DNA polymerase that has 5' to 3' exonuclease activity. During each synthesis step of the amplification, any probe that hybridizes to the target nucleic acid downstream from the primer being extended is degraded by the 5' to 3' exonuclease activity of the DNA polymerase. Thus, synthesis of a new target strand also results in degradation of the probe, and the accumulation of degradation products is a measure of the synthesis of the target sequence.

[0045] Any method suitable for detecting degradation products can be used for the 5'-nuclease assay. Often, the detection probe is labeled with two fluorescent dyes, one of which (the "quencher" or "quenching moiety") is capable of quenching the fluorescence of the other dye (the "reporter" or "reporter moiety"). The dyes are typically attached to the probe with the reporter or detection dye attached to the 5' end and the quenching dye attached to an internal site, such that quenching occurs when the probe is in an unhybridized state and cleavage of the probe by the 5' to 3' exonuclease activity of the DNA polymerase occurs between the two dyes. Amplification results in cleavage of the probe between the dyes, which is accompanied by the simultaneous elimination of quenching and an increase in observable fluorescence from the initially quenched dye. The accumulation of degradation products is monitored by measuring the increase in reaction fluorescence. US Pat. Nos. 5,491,063 and 5,571,673 describe alternative methods for detecting probe degradation that occurs concomitant with amplification.

[0046] The 5' nuclease assay for detection of target nucleic acid can use any polymerase with 5' to 3' exonuclease activity. In some embodiments, the polymerase with 5'-nuclease activity is a thermostable and thermoactive nucleic acid polymerase. Such thermostable polymerases include, but are not limited to, natural and recombinant forms of polymerases from various species of the eubacterial genera Thermus, Thermotoga, and Thermosipho, as well as chimeric forms thereof. For example, Thermus species polymerases that can be used in the methods of the invention include Thermus aquaticus (Taq) DNA polymerase, Thermus thermophilus (Tth) DNA polymerase, Thermus species Z05 (Z05) DNA polymerase, and Thermus species sps17 (sps17) DNA polymerase (described, e.g., in U.S. Pat. Nos. 5,405,774; 5,352,600; 5,079,352; 4,889,818; 5,466,591; 5,618,711; 5,674,738, and 5,795,762). Thermatoga polymerases that can be used in the methods of the invention include, for example, Thermatoga maritima DNA polymerase and Thermatoga neapolitana DNA polymerase, while an example of a Thermosipho polymerase that can be used is Thermosipho africanus DNA polymerase. Thermatoga maritima and Thermosipho africanus DNA polymerase sequences are published in International Patent Application No. PCT / US91 / 07035 and Publication No. WO92 / 06200. Thermatoga neapolitana sequence can be found in International Patent Publication No. WO97 / 09451.

[0047] In 5' nuclease assays, amplification detection is typically performed simultaneously with amplification (i.e., "real-time"). In some embodiments, amplification detection is quantitative and amplification detection is real-time. In some embodiments, amplification detection is qualitative (e.g., end-point detection of the presence or absence of a target nucleic acid). In some embodiments, amplification detection is post-amplification. In some embodiments, amplification detection is qualitative and amplification detection is post-amplification.

[0048] The term "tagged probe" or "tagged oligonucleotide probe" refers to an oligonucleotide probe based on a DNA probe architecture that allows discrimination of multiple targets in the same optical channel by measuring fluorescence at different temperatures, and is related to the "TAGS (Temperature assisted generation of signal)" technology disclosed in US Patent Application Publication No. 2018 / 0073064, which is incorporated herein by reference in its entirety. Multiplex PCR assays using only one reporter moiety (e.g., one fluorescent dye) are possible by designing tagged probes with tag moieties hybridized to respective quenching oligonucleotide molecules at different melting temperatures (Tm). A first tagged probe with a low Tm tag-quenching oligonucleotide duplex can be used to detect a first target nucleic acid by measuring a calculated fluorescence value at a first temperature equal to or greater than its Tm temperature. A second tagged probe with a high Tm tag-quenching oligonucleotide duplex can be used to detect a second target by measuring a calculated fluorescence value at a second temperature equal to or greater than its Tm temperature but higher than the first temperature. Thus, each optical channel of a given dye can be read in a different "thermal channel" representing the fluorescence measurement at a different temperature.

[0049] Stokes shift and large Stokes shift dyes The Stokes shift of a fluorescent dye is defined as the wavelength, frequency, or energy difference between the absorption peak maximum and the emission peak maximum for the same electronic transition. The majority of small molecule fluorophores exhibit Stokes shifts on the order of 10-25 nm, typically less than 80 nm (herein referred to as "small Stokes shift" (SSS) dyes). SSS dyes include traditional fluorescent dyes used in PCR assays, e.g., FAM, HEX, CFR610, and Quasar670. Fluorophores with significantly larger Stokes shifts are collectively referred to as "large Stokes shift" (LSS) dyes, "high Stokes shift" dyes, or "mega-Stokes" dyes. Although the term is not clearly defined, dyes with Stokes shifts greater than 80 nm are typically coined with the adjectives "large" or "high," although the term "mega" appears to be commonly used for dyes with Stokes shifts significantly greater than 100 nm. Two photophysical mechanisms have been discussed in the literature to explain the occurrence of the large Stokes shift. The molecular geometry type mechanism is based on the conformational relaxation of the excited state fluorophore and the resulting rearrangement of the solvent dipole. The Stokes shift grows as the difference between the (equilibrium) molecular geometry and dipole moment in the ground and excited states increases. Large Stokes shift fluorescence for the electronic type mechanism is due to intramolecular charge transfer (ICT) in the excited state.

[0050] A common problem with small Stokes shift fluorophores is internal quenching of fluorescence. Such self-quenching is caused by the spectral overlap of excitation and emission and is especially prevalent at high fluorophore concentrations. LSS dyes have better separated spectral bands, minimizing photon reabsorption. There is a non-zero probability that a fluorophore will be excited outside its main excitation peak. As a result, fluorescence from one dye will inevitably contribute to the total light detected in multiple emission channels. This spectral "crosstalk" or "bleed-through" can be compensated computationally to some extent by using predefined correction factors. Furthermore, scattering of the excitation light increases background fluorescence in adjacent channels. LSS dyes make it possible to reduce or even avoid crosstalk and scattering from other fluorophores. LSS dyes are particularly useful in experimental settings where many fluorophores generate strong background signals. A large spectral separation such as that of the LSS dyes allows for more effective filtering of the excitation light, thereby increasing the sensitivity of target detection. LSS dyes give access to fluorescence data from optical channels that were previously inaccessible. Facilitated by the wide spectral separation, LSS dyes, when used in combination with standard fluorophores, make it possible to increase the multiplexing capacity of fluorescent PCR devices. In this way, LSS labeling allows the implementation of additional channels into established 4- to 6-color instruments. In principle, 21 channels can be constructed from the combination of filters of a 6-color instrument (Figure 1). In practice, however, the number of channels is limited by the commercial availability of LSS dyes with a sufficiently large Stokes shift. Based on the 150 nm Stokes shift of currently commercially available LSS dyes, 9 additional channels can be implemented (Figure 1, white channels). The channels of standard dyes are highlighted in light gray, while dark gray indicates channels for which no suitable LSS dye is currently available. Alternatively, resonant electron transfer (RET) probes can also be used to generate large "virtual" Stokes shifts and access these channels.

[0051] Examples of commercially available LSS dyes include: ALEXA FLUOR 430, ATTO 430LS, ATTO 490LS, ATTO 390LS, CASCADE YELLOW, CF350, CHROMEO 494, CYTO 500 LSS, CYTO 510 LSS, CYTO 514 LSS, CYTO 520 LSS, DAPOXYL, DY 480XL, DY 481XL, DY 485XL, DY 510XL, DY 511XL, DY 520XL, Examples of suitable colorants include, but are not limited to, DY 521XL, DY 601XL, DY 350XL, DY 360XL, DY 370XL, DY 375XL, DY 380XL, DY 395XL, DY 396XL, DYLIGHT 515-LS, DYLIGHT 485-LS, DYLIGHT 510-LS, DYLIGHT 521-LS, FURA 2, INDO 1, KROME ORANGE, LUB 04, LUCIFER YELLOW, NBD X, NILE RED, PULSAR 650, PYMPO, STAR 440SXP, STAR 470SXP, STAR 520SXP, VIOGREEN, CF 350, SETAU 405, and PACIFIC ORANGE.

[0052] Despite the described advantages, the increase in the Stokes shift comes at a price. LSS dyes have a lower fluorescence quantum yield compared to standard fluorophores. Since the brightness of a fluorophore is defined as the multiplicative product of the molar extinction coefficient and the fluorescence quantum yield, LSS dyes are also less bright. Another aspect is that the 40-50 nm peak width of standard fluorophores can be doubled or tripled for LSS dyes. Nevertheless, for multiplexing purposes, the excellent spectral separation of LSS dyes is more important than the reduced brightness and larger peak width.

[0053] COBAS® LIAT, Multi-Segment Capillary PCR System The disclosure also describes multi-segment capillary PCR devices, consumables, and methods for processing samples using such devices and consumables. An example of such a system is the cobas® Liat® PCR System (Roche Molecular Systems, Pleasanton, Calif.).

[0054] The cobas® Liat® System is comprised of Liat® tubes and the Liat® analyzer (instrument). The assay utilizes single-use, disposable Liat® tubes that hold sample preparation and real-time PCR reagents and facilitate the sample preparation and real-time PCR process. Liat® tubes contain all necessary unit-dose reagents pre-filled in tube segments separated by frangible seals in the order of reagent use.

[0055] Liat® analyzers automate and integrate sample preparation, nucleic acid amplification, detection and quantification of target sequences in biological samples. Liat® analyzers perform all analytical steps from clinical samples and automatically report analytical results. During the testing process, the analyzer's multiple sample processing actuators compress Liat® tubes to selectively release reagents from tube segments, move samples from one segment to another, and control reaction volumes, temperatures and times for sample preparation, nucleic acid extraction, target enrichment, inhibitor removal, nucleic acid elution and real-time PCR. An embedded microprocessor controls and coordinates the operation of these actuators to perform all the necessary analytical processes in a closed Liat® tube. To run an assay, the user loads the sample into a Liat® tube and places the loaded Liat® tube into the Liat® analyzer. The analyzer performs sample preparation, real-time PCR, calculates and reports the results. All processes are controlled by an assay script.

[0056] A portion of the assay script that controls the thermal cycling profile is shown below in Table 1. In this embodiment, fluorescence readings from the FAM label were taken at 58° C. and at high temperature for each cycle starting with cycle #6. One of skill in the art will recognize that the parameters listed in Table 1 may be altered as needed, e.g., temperature, duration, and number of cycles may all be altered as needed. [Table 1]

[0057] In some embodiments, the segmented tubules provide a convenient container for receiving, storing, processing, and / or analyzing biological samples. In certain embodiments, the segmented tubules facilitate sample processing protocols that include multiple processing steps. In certain embodiments, a sample can be collected in a sample tubule and then the tubule can be placed into an analyzer. The analyzer can then manipulate the tubule and its contents to process the sample.

[0058] In one embodiment, the flexible capillary may be segmented into compartments by breakable seals. The individual segments may contain different reagents and buffers for processing the sample. Clamps and actuators within the analyzer may apply, hold, and / or release forces to the capillary in various combinations and at various times to direct fluid movement and rupture the breakable seals. This rupture of the breakable seals may create an inner capillary surface that is substantially free of obstacles to fluid flow. In some embodiments, the flow of the biological sample may be directed toward the distal end of the capillary as processing progresses, and the waste stream may be forced to move in the opposite direction toward the opening of the capillary where the sample was initially entered. This sample inlet may be sealed, optionally permanently, by a cap with a locking mechanism, and a waste chamber may be placed within the cap to receive the waste for storage. A major advantage of this approach is that the processed sample does not come into contact with surfaces touched by the unprocessed sample. As a result, trace amounts of reaction inhibitors present in the unprocessed sample that may coat the walls of the capillary are less likely to contaminate the processed sample.

[0059] In some embodiments, the capillary may be expandable such that it can receive an amount of fluid from each of multiple segments into one segment. This allows the sample and reagents to undergo a particular processing step in one segment and can result in a simpler mechanical structure for performing an assay. Another advantage of embodiments using capillaries that may be so expandable is that the same capillary structure can be used to package different volumes of reagents into segments, and the same capillary can be packaged in different ways depending on the assay being performed.

[0060] 7A-B and 8A-B, the transparent flexible capillary 10 can be configured into multiple segments, such as 16, 110, 120, 130, 140, 150, 160, 170, 180, and / or 190, and can be substantially flattened by compression. In one embodiment, the capillary can have at least two segments. The flexible capillary can provide operational functionality between about 2° C. and 105° C., compatibility with samples, targets and reagents, low gas permeability, minimal fluorescence properties, and / or elasticity during repeated compression and flexing cycles. The capillary can be made of a variety of materials, examples of which include, but are not limited to, polyolefins such as polypropylene or polyethylene, polyurethanes, polyolefin copolymers, and / or other materials that provide suitable properties. Capillary properties such as transparency, wetting properties, surface smoothness, surface charge, and thermoelasticity can affect the performance of the capillary. These properties can be improved by exemplary processes such as seeding, plasma treatment, addition of additives, and irradiation. In some embodiments, additive materials may be added to the plastic to improve selected properties. For example, slip additives such as erucamide and / or oleamide may be added. In some embodiments, so-called "antiblock" additives may be added. The additives may have concentrations in the plastic ranging from about 0.01% to about 5.0%.

[0061] The capillaries can be manufactured by a wide variety of suitable methods, such as extrusion, injection molding, and blow molding. In one embodiment, the capillaries are continuously extruded. Alternative techniques for manufacturing the capillaries include, for example, casting, extrusion, or blowing a film that can be formed into a suitable capillary by a secondary processing operation. The capillary wall material may include multiple layers by coextrusion or film lamination. For example, an inner layer may be selected for high biocompatibility, and an outer layer may be selected for low gas permeability. As a further example, the inner layer may be easily formed into a breakable seal 14 (FIGS. 8A-B), such as a peelable seal, and the outer layer may be elastic and highly impermeable. For example, the capillaries may have a wall thickness of about 0.03 mm to about 0.8 mm, preferably 0.03 mm to about 0.5 mm, and the capillaries may be substantially flattened with an applied external pressure of about 1 atmosphere.

[0062] Referring to FIG. 8A, in certain embodiments, segments of a sample capillary 10 are defined by breakable seals 14 to fluidically isolate adjacent segments. This sealing feature may be useful, for example, in separating dry reagents from liquid reagents until the two can be reconstituted to perform a particular assay, or to separate chemically reactive species until a reaction is desired. Breakable seals 14 may be formed in regions of the capillary 10 where opposing walls are substantially joined, but not joined strongly enough to prevent the walls from peeling apart later without significantly compromising the capillary or the previously sealed surfaces. Such seals are sometimes referred to as "peelable" seals. In some embodiments, the peelable seal region may be a band perpendicular to the axis of the capillary. It may span the length of the capillary ranging from about 0.5 mm to 5 mm, or from about 1 mm to about 3 mm, most preferably about 1 mm. The seal preferably spans the full width of the capillary to seal the segments. In some embodiments, the seal band may vary in height or shape and / or may be oriented at an angle across the axis of the capillary. Such variations can alter the release characteristics.

[0063] A breakable seal 14 in the form of a peelable seal can be formed between opposing walls of the capillary by applying a controlled amount of energy to the capillary at the location where the peelable seal is desired. For example, a temperature controlled sealing head can press the capillary at a specific pressure against a stationary anvil for a specific time interval. Various combinations of temperature, pressure and time can be selected to form a seal of the desired size and peel strength. The energy may be delivered by, for example, a temperature controlled sealing head maintained at a constant temperature of 105°C to 140°C to heat the polypropylene tubing material; an actuator capable of delivering a precise pressure of 3 to 100 atmospheres over the desired seal area; and a control system to drive the sequencing of the actuator to specific cycle times of 1 to 30 seconds. Using this method, good seals have been created in polypropylene capillaries that peel open when exposed to an internal pressure of about 1 atmosphere. Alternative techniques for delivering sealing energy to the capillary include RF welding and ultrasonic welding.

[0064] In other embodiments, alternative capillary materials and blends of materials can be used to optimize peelable seal performance. For example, two polypropylene polymers of different melting temperatures can be blended in ratios such that the composition and melting properties are optimized for peelable seal formation. Referring to FIG. 7B, in addition to or instead of the breakable seal 14, the flexible capillary can further have one or more pressure gates 194 that can be reversibly opened and closed during testing operations by applying a controlled force to a segment of the flexible capillary.

[0065] The filter can be embedded in the capillary segment. In a preferred embodiment, the filter can be formed by stacking multiple layers of flexible filter material. The top layer of the filter, which is in direct contact with the sample, may have a pore size selected for filtration; the bottom layer of the filter may comprise a material with a much larger pore size to provide a support structure for the top layer when pressure is applied during filtration. In this preferred embodiment, the filter can be folded to form a bag, the edges of whose open end are firmly attached to the capillary wall. The segment with the filter bag can be substantially flattened by compressing the exterior of the capillary.

[0066] In exemplary embodiments, one or more reagents may be stored in the capillary segments as dry substances and / or as liquid solutions. In embodiments where the reagents may be stored in a dry format, the liquid solutions may be stored in adjacent segments to facilitate reconstitution of the reagent solutions. Exemplary reagents include lysis reagents, elution buffers, wash buffers, DNase inhibitors, RNase inhibitors, proteinase inhibitors, chelating agents, neutralizing reagents, chaotropic salt solutions, detergents, surfactants, anticoagulants, germinant solutions, isopropanol, ethanol solutions, antibodies, nucleic acid probes, peptide nucleic acid probes, and phosphorothioate nucleic acid probes, and the like. In embodiments where one of the reagents is a chaotropic salt solution, the preferred components are guanidinium isocyanate or guanidinium hydrochloride, or combinations thereof. In some embodiments, the order in which the reagents may be stored in the capillary with respect to the opening into which the sample is introduced reflects the order in which the reagents may be used in a method utilizing the tube. In some embodiments, the reagent comprises a substance capable of specific binding to a preselected component of the sample. For example, a substance may specifically bind to nucleic acids, or a nucleic acid probe may specifically bind to nucleic acids having a particular base sequence.

[0067] In other embodiments, a solid phase substrate can be contained within the tubule segment and used to capture one or more selected components of the sample, such as target microorganisms or nucleic acids, if such components are present in the sample. Capture can serve to concentrate the target components and remove reaction inhibitors from the sample. The substrate can be a solid phase material that can capture target cells, virions, nucleic acids, or other selected components under defined chemical and temperature conditions and release the components under different chemical and temperature conditions.

[0068] In some embodiments, the reagents can be coated onto the substrate. Examples of reagents that can be coated are receptors, ligands, antibodies, antigens, nucleic acid probes, peptide nucleic acid probes, phosphorothioate nucleic acid probes, bacteriophages, silica, chaotropic salts, proteinases, DNase inhibitors, RNase inhibitors, DNase inhibitors, RNase inhibitors, and germination liquid. In some embodiments, the substrate can be stored in a dry segment of the capillary, and in other embodiments, immersed in a liquid. In some embodiments, the order in which the reagents can be stored in the capillary relative to the opening into which the substrate and sample are introduced reflects the order in which the reagents and substrates can be used in a method utilizing the device.

[0069] The substrate can be a bead, a pad, a filter, a sheet, and / or a part of a capillary wall or a collection tool. In embodiments where the substrate is a plurality of beads, the beads can be silica beads, magnetic beads, silica magnetic beads, glass beads, nitrocellulose colloid beads, and magnetized nitrocellulose colloid beads. In some embodiments where the beads can be paramagnetic, the beads can be captured by a magnetic field. Examples of reagents that can enable selective adsorption of nucleic acid molecules to surfaces coated with functional groups are described, for example, in U.S. Pat. Nos. 5,705,628; 5,898,071; and 6,534,262. Separation can be achieved by manipulating the ionic strength and polyalkylene glycol concentration of the solution to selectively precipitate and reversibly adsorb the nucleic acid to the solid phase surface.

[0070] If these solid surfaces are paramagnetic microparticles, the magnetic particles with the target nucleic acid molecules adsorbed thereto can be washed under conditions that retain the nucleic acid but not other molecules. The nucleic acid molecules isolated through this process are suitable for capillary electrophoresis, nucleotide sequencing, reverse transcription, cloning, transfection, transduction, microinjection of mammalian cells, gene therapy protocols, in vitro synthesis of RNA probes, cDNA library construction and polymerase chain reaction (PCR) amplification. Several companies offer magnetic-based purification systems, such as QIAGEN's MagAttract™, Cortex Biochem's MagZorb™, Roche Applied Science's MagNA Pure LC™, and Merck&Co's MagPrep® Silica. All of these products use negatively charged particles and manipulate buffer conditions to selectively bind various nucleic acids to the beads, wash the beads, and elute the beads in aqueous buffers. Many of the products used by these companies use chaotropic salts to aid in the precipitation of nucleic acids onto the magnetic beads. Examples are described in U.S. Patent Nos. 4,427,580; 4,483,920; and 5,234,809.

[0071] In some embodiments, the substrate may be a pad. In further embodiments, the substrate pad may comprise paper, alternating layers of paper with different hydrophobicity, glass fiber filters, or polycarbonate filters with a predetermined pore size. In some embodiments, the pad may be a filter or impermeable sheet for covering selected portions of the surface of the pad, the filter having a predetermined pore size. Such filtration devices can be used for separation of white and red blood cells (or other particles such as viruses or microorganisms) from whole blood and / or other samples. The pad can be attached to the capillary wall and / or the sample collection tool. In some embodiments, the pad can be immersed in a reagent solution, while in other embodiments, the pad can be coated with a dry reagent.

[0072] A preferred exemplary embodiment may include a linear arrangement of two or more capillary segments 110, 120, 130, 140, 150, 160, 170, 180, and / or 190 (FIG. 7B). The linear arrangement facilitates moving the sample and resulting waste and targets through the tube in a controlled manner. The raw biological sample can be loaded through the first opening 12 (FIG. 8B) of the first segment 110 (FIG. 7B) of the capillary. The waste from the processed sample can then be pushed back towards the first opening while the targets are pushed towards the opposite end, thereby minimizing contamination of the targets with reaction inhibitors that may have attached to the capillary walls and confining the targets to clean segments of the capillary that may contain appropriate reagents for further manipulation of the targets. Some embodiments may use a plurality of at least three segments, each containing at least one reagent. In some embodiments, these segments may contain reagents in the following order: the reagent in the second segment may be either a lysis reagent, a dilution or wash buffer, or a substrate. The reagent in the third segment can be a substrate, a lysis reagent, a wash buffer, or a neutralization reagent. The reagent in the fourth segment can be a wash buffer, a suspension buffer, an elution reagent, or a nucleic acid amplification and detection reagent. In some embodiments, the three segments can be arranged contiguously, while in other embodiments, the three segments can be separated by another segment or segments in between via a breakable seal.

[0073] In some embodiments, a pressure gate 194 (FIG. 7B) can be incorporated to selectively open and close a second opening located at the distal end of the capillary to collect products generated during testing from the capillary to the outside of the capillary for further processing. In some embodiments, this second opening can be located within a segment 198 defined by two pressure gates 194 and 196 to store products from the sample processing segment. In some embodiments, a combination of a breakable seal and pressure gate can be provided to transfer the contents of the capillary to the second opening.

[0074] In some embodiments, a tube closure device for closing the tube after sample loading can include a cap 20 (FIG. 7B) and / or a clamp 310. An interface or adapter 52 between the cap and the first opening of the flexible capillary can be used to ensure a reliable air-tight seal. In an exemplary embodiment, this interface can be threaded and can include a tapered feature 62 on the cap and / or a suitably rigid tube frame 50, such that when fastened together, threads 64 can engage and fit the tapered feature 62 between the tube frame and the cap to provide a suitable lock. In this exemplary embodiment, the cap may require ½ to 1 turn to fully remove or install from the tube holder. The combination of thread pitch and taper angle of the joint can be selected to be easy to manufacture while also providing a feedback resistance to inform the user that an effective seal has been formed. In other embodiments, the cap locking device can include a snap fit, press fit, and / or other type of "twist lock" mechanism between the cap and the tube holder, as well as similar configurations in which the cap is permanently attached to the tube, such as by hinging or tethering the cap.

[0075] Both the cap 20 and the tube frame 50 can be made of a suitable injection molded plastic, such as polypropylene. The tube frame 50 can then be secured to the flexible tube by a permanent airtight seal. The outer portion of the cap may be covered with ridges or finger grips to facilitate its handling. Additionally, the cap 20 may include an area for attaching a sample identification mark or label. As a further alternative, the cap may be attached directly to the first opening flexible tube via a press fit or collar that compresses the flexible tube opening against a protrusion in the cap to form an airtight seal. The lock between the tube cap and the tube holder may be keyed or guided so that the collection tool 36 or features incorporated in the cap may be clearly oriented relative to the tube to facilitate sample processing and flattening of the flexible tubule. Additionally, the cap may incorporate a mechanism, such as a ratchet or similar safety mechanism, to prevent the cap from being removed after it has been attached to the opening of the flexible tube.

[0076] In some embodiments, the cap 20 used to close the capillary may include a cavity 22 therein by making the cap body substantially hollow. In some embodiments, the hollow portion extends from the top of the cap body to an orifice at the base of the cap body. To form a chamber, the cavity top may be closed by fastening a cover to the cap body. The cover may be constructed of the same piece as the cap body. The cover may incorporate an air vent 26 or may further incorporate a fixed microbial barrier, a filter, or a material that expands to close the air vent when exposed to a liquid or a certain temperature. The bottom of the chamber may be kept open or closed by a breakable septum or valve. The hollow chamber may further incorporate a flexible membrane or septum 24. This flexible septum may be manufactured using dip molding, liquid injection silicone molding, blow molding, and / or other methods suitable for creating thin elastomeric structures. The flexible septum can be inserted into the cap body cavity 22 assembly to effectively isolate the interior of the tube from the external environment after the cap is in place on the tube. The flexible septum can be designed to ensure that its inherent stiffness is in a preferred known deformed state in the absence of externally applied pressure. As a further embodiment, the flexible septum may be replaced by a plunger. In an exemplary embodiment, the cap body, approximately 30 mm high by 14 mm in diameter, is injection molded with a suitable thermoplastic resin and may include an internal cavity with a usable volume of at least 500 uL. The chamber within the cap body may be adapted for useful purposes such as holding or dispensing reagents, acting as a reservoir to hold waste fluids, acting as a lead-in space for an integrated collection tool, or a combination thereof.

[0077] The cap 20 may have an integrated collection tool 30 (FIG. 8B), such as a swab, capillary tube, liquid dropper, inoculation loop, syringe, absorbent pad, forceps, scoop or stick, to facilitate collection of liquid and solid samples and their insertion into the tubule. The collection tool may be designed to collect and deposit a predetermined amount of material into the tube. Reagents may be stored in the collection tool itself. For example, the collection tool may include a swab impregnated with dry salts to suspend the salts from the swab into solution when the swab is hydrated. Additionally, the collection tool and cap may be designed such that the collection tool portion retracts into the cap body after depositing the sample into the tubule, leaving the tubule segment substantially undisturbed.

[0078] The chamber 22 in the cap 20 may be shaped to store a reagent. To accomplish this, for example, the base of the chamber may be closed by a breakable septum or valve (not shown) such that when the cap is squeezed, the septum breaks and the reagent is released. Such a feature may be useful, for example, when the cap is integrally formed with a collection tool such as a swab or stick. In this case, the reagent released from the cap chamber may be used to flush the sample from the collection tool into the tube segment or to lyse the sample contained in the collection tool. The reagent may also be released from the cap chamber by opening a breakable septum using pressure generated by compressing the flexible tube segment to force fluid up from the tube into the cap chamber. The chamber in the cap may be shaped to store waste liquid from processing in the capillary. In another embodiment, the base of the chamber may remain open such that when connected to the first opening of the flexible capillary, a fluid passageway is formed between the capillary and the chamber. As fluid moves into the cap chamber, a flexible septum 24 contained therein can move upward from an initial position to accommodate the new fluid flowing in. This septum movement can be facilitated by incorporating a vent hole 26 in the cap body cover.

[0079] Referring to FIG. 7B, after the fluid has been transferred into the cap chamber, a clamp 310 or actuator 312 in the analyzer can act to compress the tubing, effectively sealing the cap chamber volume from the tubing segment. As an alternative embodiment, the cap chamber can incorporate a pressure gate or check valve (not shown) to prohibit the flow of fluid from the cap chamber back into the tube segment. As a further alternative, the flexible septum can be omitted, with the cap chamber cover including a microbial barrier to allow free escape of contained gases, but retain all liquid volume and infectious agents in the tube. As a further alternative, the flexible septum can be replaced with a plunger that moves axially upward to accommodate the additional volume of fluid transferred from the tube segment to the cap chamber. Other methods of containing fluid waste in the cap chamber can be readily envisioned without departing from the scope of this disclosure.

[0080] A substantially rigid frame 50 (FIG. 7A) may be provided to hold the flexible capillary 10 in proper tension by restraining at least the proximal and distal ends of the capillary. In an exemplary embodiment, a first restraint may be provided to permanently attach and seal the capillary to the frame around the first opening of the tube. This seal may be formed by welding the flexible capillary to the frame using heat and / or ultrasonic sources. Alternatively, the seal may be created using a hot melt adhesive joint with ethylene vinyl acetate or by creating a joint using a UV cured epoxy or other adhesive. In a further embodiment, the capillary may be mechanically sealed or insert molded with the frame. A second restraint may be provided to attach and seal the capillary to the base of the frame. In an exemplary embodiment of this second restraint, this end of the capillary may be sealed flat and attached to the rigid frame by heat and / or ultrasonic welding techniques. Alternatively, this joint and seal may be formed using adhesive or mechanical techniques. In an alternative embodiment, the second seal may be similar to the first seal and is substantially open to allow access to the contents of the flexible capillary through the second opening. The capillary and frame materials may be optimized for joint fabrication. For example, the frame may be made of polypropylene, which has a lower melting point than the thinner capillary to ensure a more uniform melt across the weld zone or zones. To facilitate welding between the capillary and the frame, the joint area may be tapered or otherwise shaped so that energy directors or other commonly used features enhance the welding performance. In an exemplary embodiment, the rigid frame may be made of any suitable plastic by injection molding, whose dimensions are approximately 150 mm high x 25 mm wide.

[0081] The rigid frame 50 can incorporate several features to facilitate the compression and flattening of the flexible capillary. For example, in an exemplary embodiment, the flexible capillary 10 may be constrained only at its two axial ends to allow maximum radial freedom and avoid impeding radial movement of the capillary when compressed. In another embodiment, compression can be facilitated by including a relief area in the frame near the first opening of the tube. This relief area can be used to facilitate the transition of the flexible capillary from a substantially compressed shape in the capillary segment to a substantially open shape at the first opening. Other useful features of the rigid frame that can facilitate compression of the flexible capillary can include an integrated capillary tensioning mechanism. In an exemplary embodiment, this tensioning mechanism can be manufactured by molding features such as a cantilever or leaf spring directly into the rigid frame to pull the capillary into tension at one of its attachment points with the frame.

[0082] The rigid frame 50 can facilitate tube identification, handling, sample loading, and interfacing with tube caps. For example, the frame can provide additional areas for identifying tubes through labels or writing 80 affixed thereto. The plastic material of the frame may be color coded with the cap material to aid in identifying the device and its function. The frame can incorporate special features such as thickness variations or keys to guide its orientation into the receiving instrument or during manufacturing. The frame can interface to a sleeve 90 or packaging that covers or protects the flexible tubing from damage from accidental handling, light exposure, and / or heat exposure. The body of the rigid frame can also provide a convenient structure for holding the tubes. The frame can have an integral collection tool 32, such as a deflector or scoop, to facilitate sample collection into the device. The sample receiving end of the frame may also incorporate a tapered or funnel-shaped inner surface to guide the collected sample into the flexible tube.

[0083] In some embodiments, a method of extracting nucleic acid from a biological sample by using the device described in the previous paragraph is contemplated. In certain embodiments, the sequence of events in such a test may include: 1) a biological sample may be collected using a collection tool; 2) the collected sample may be placed in a flexible capillary tube that may include multiple segments that may contain reagents required during the test through a first opening in the capillary tube; 3) at least one substrate may be set to a controlled temperature and / or other conditions to capture target organisms or nucleic acids during a set incubation period; 4) organisms or molecules in the unprocessed sample that cannot bind to the substrate may be removed by transferring the liquid to a waste reservoir; 5) the waste may be stored in the waste reservoir and separated from the target by a clamp and / or actuator compressed against the capillary tube; 6) a wash buffer released from another segment of the capillary tube may be added to remove reaction inhibitors; 7) an elution reagent from another segment may be added to release the target bound to the substrate after incubation at a controlled temperature; 8) the nucleic acid may be detected by techniques well known to those skilled in the art or collected through a second opening of the capillary tube. In an exemplary embodiment, the sample flow may flow from the first opening of the capillary toward the distal end as the test progresses, while the waste may flow toward the closed sample input opening of the capillary, and a waste chamber in the capillary's cap receives the waste for storage. As a result, undesirable contact between the processed sample and the surfaces of the reaction vessel touched by the unprocessed sample is avoided, thereby preventing reaction inhibition by trace amounts of reaction inhibitors that may be present in the unprocessed sample and coat the walls of the reaction vessel.

[0084] Some embodiments include a test tube 1 (FIGS. 7A-B) having a flexible capillary 10 divided into multiple segments, e.g., segments 16, 110, 120, 130, 140, 150, 160, 170, 180, and / or 190, that may traverse the longitudinal axis of the capillary and may contain reagents, e.g., reagents 210, 221, 222, 230, 240, 250, 260, 270, 280, and / or reagent 290; and actuators 312, 322, 332, 342. The use of an analyzer may include multiple actuators, such as 352, 362, 372, 382, ​​and / or 392, clamps, such as clamps 310, 320, 330, 340, 350, 360, 370, 380, and / or 390, and blocks, such as 314, 344, and / or 394 (others not numbered for simplicity), may be incorporated to process samples with opposing actuators and clamps. Various combinations of these actuators, clamps, and / or blocks may be used to effectively close and clamp the tubule, thereby isolating the fluids. In an exemplary embodiment, at least one of the actuators or blocks may include a thermal control element to control the temperature of the tubule segment for sample processing. The sample processing device may further include at least one magnetic field source 430 capable of applying a magnetic field to the segment. The sample processing device may further include a detection device 492, such as a photometer or CCD, to monitor reactions occurring or completed within the tubule.

[0085] The use of a tube in combination with an analyzer can enable many sample processing operations. Collection of a sample, such as blood, saliva, serum, soil, tissue biopsy, stool, or other solid or liquid sample, can be accomplished by using a sample collection tool 30, which may be incorporated into the cap 20, or a feature 32 on the tube frame 50. After an appropriate amount of sample has been collected, a cap can be placed on a first opening of the tube to close the tube and deposit the sample in the first segment. Following this step, the sample contained in the collection tool can be washed off or resuspended with a reagent contained in a separate chamber in the cap by compressing a portion of the cap. The tube can then be loaded into an analyzer for further processing. An identification feature, such as a barcode or RF tag, can be present on the tube to designate the identification of the sample in a format that can be read by the analyzer and / or user.

[0086] Opening the breakable seals of the capillary segments can be accomplished by applying pressure to the flexible capillary to irreversibly separate the bonded surfaces of the capillary walls. An actuator can be used to apply the necessary pressure to compress the capillary segment containing the fluid and open the breakable seals. In an embodiment where the segment is defined by two breakable seals A and B, the analyzer can preferentially break seal A by physically protecting the seal B area with an actuator or clamp to prevent seal B from breaking while pressure is applied to the segment to break seal A. Alternatively, seal A may be preferentially opened by applying pressure precisely to the segment adjacent to seal A; so that seal A is opened first by the pressure generated in the adjacent segment; after seal A is broken, the pressure between the two segments drops substantially due to the additional combined segment volume; the reduced pressure in the combined segment is insufficient to break seal B. This method can be used to open the breakable seals one at a time without the use of protective actuators and / or clamps. As a further alternative, the adhesion of seal A may be less than that of seal B such that seal A may break at a lower pressure than seal B.

[0087] The process of moving fluid from one segment to another may include, for example, releasing a clamp on one end of a first segment, compressing a clamp on the other end of the first segment, releasing an actuator on the second segment, and compressing the actuator on the first segment to move liquid from the first segment to the second segment. Alternatively, the clamp may be omitted or may be opened after releasing the actuator on the second segment.

[0088] The process of mixing two substances, at least one of which is a liquid, placed in adjacent segments can be accomplished by releasing a clamp between the two segments and allowing the liquid contained in the first segment to move through an open breakable seal to the second segment, or alternatively, compressing the second segment and the first segment to allow the liquid to flow between the segments.

[0089] Agitation can be achieved by alternately compressing and decompressing a capillary segment with an actuator while both clamps adjacent to the actuator compress the capillary. In another embodiment, agitation can be achieved by alternately moving liquid between at least two segments.

[0090] In embodiments where a tubule segment may contain a volume of liquid in excess of that required for the protocol, the process of adjusting the volume of liquid in the segment may be performed by: compressing the tubule segment to reduce the gap between the tubule walls, setting the volume of the segment to the desired level and allowing excess liquid to flow over a clamp at the end of the segment or an adjacent actuator into an adjacent segment; closing the tubule segment with a clamp or actuator, leaving an adjusted volume of liquid in the segment.

[0091] The process of removing air bubbles may include agitating the segment containing the bubbly liquid. Another process of removing air bubbles may include agitating a first segment containing liquid while closing a second segment; opening the second segment and transferring liquid from the first segment to the second segment; agitating the second segment and adjusting the position of the second actuator to move the liquid-air interface near or above the top end of the second segment, and then clamping the top end of the second segment to form a fully liquid-filled segment without air bubbles.

[0092] A liquid transfer process can be used to carry out the dilution process, with one of the segments containing the diluent and the other containing the substance to be diluted.

[0093] The process of reconstituting a reagent from dry and liquid components stored separately in different capillary segments or sub-segments may include compressing the capillary segment or sub-segment containing the liquid component to open a breakable seal connecting it to the dry reagent segment, transferring liquid to the dry reagent segment or sub-segment, and mixing the dry reagent and liquid components using a mixing process.

[0094] Incubation of the contents in a segment can be accomplished by setting the corresponding actuator and / or block temperatures and applying pressure to the segment to ensure sufficient surface contact between the capillary walls of the segment and the actuators and blocks, bringing the contents of the tubule segment to substantially the same temperature as the surrounding actuator and / or block temperatures. Incubation can be performed under all process conditions, as long as the temperatures of all involved segments are set as required.

[0095] Rapid temperature ramping for incubation can be accomplished by incubating a fluid in a first segment at a first temperature, setting a second temperature in a second segment adjacent to the first segment, and after incubation at the first temperature is completed, rapidly transferring the liquid from the first segment to the second segment and incubating at the second temperature.

[0096] Driving the flow through the flow channel process can be performed by compressing the capillary with a centrally located actuator, and its adjacent clamps, if present, to form a laminar flow channel with a gap of about 1 to about 500 μm, preferably about 5 to about 500 μm, through the segment. Adjacent actuators can be gently compressed on adjacent segments in liquid communication with the flow channel to generate offset internal pressures and ensure a substantially uniform gap in the laminar flow channel. Two adjacent actuators can then alternately compress and release the pressure of the capillary at their respective segments to generate flow at a controlled flow rate. Optional flow, pressure, and / or force sensors can be incorporated to allow for closed-loop control of the flow behavior. The flow channel process can be used for washing, enhancing substrate binding efficiency, and detection.

[0097] A magnetic bead immobilization and resuspension process can be used to separate the beads from the sample liquid. A magnetic field generated by a magnetic source 430 (FIG. 7B) can be applied to the segment 130 containing the magnetic bead suspension 230 to capture and fix the beads to the tube wall. An agitation process can be used during the capture process. In another embodiment, a flow path can be formed on the segment to which the magnetic field is applied, and the magnetic beads can be captured under flow to increase the capture efficiency. To resuspend the immobilized beads, the magnetic field can be turned off or removed, and an agitation or flow path process can be used for resuspension.

[0098] The washing process to remove residual debris and reaction inhibitors from the substrate can be performed using three basic steps: First, the actuator can compress a segment containing a substrate, such as immobilized beads or sheets, to substantially remove liquid from the segment. Second, a washing buffer can be moved to the segment using a process similar to that of reconstituting a reagent from dry and liquid components. For bead-based substrates, a bead resuspension process can be used, followed by recapturing the beads at the capillary wall. Third, after a mixing or agitation process, the actuator can compress the segment to remove the used washing solution from the segment. In another embodiment, a flow path can be formed in the segment containing the substrate, which can be either immobilized beads or sheets. A unidirectional flow wash with laminar flow characteristics is generated through the flow path with the substrate. Finally, all actuators and clamps (if present) can be closed to remove substantially all liquid from the segment. In a further embodiment, a combination of dilution-based and laminar flow-based washing can be used to further increase the washing efficiency.

[0099] Lysis can be achieved by heating the sample at a set temperature or by using a combination of heat and chemical agents to disrupt open cell membranes, cell walls, or uncoated virus particles. In another embodiment, lysis can be achieved using chemical reagents such as proteinase K and chaotropic salt solutions. The chemical reagents can be stored in one or more tubule segments and combined with the sample using the processes disclosed above. In some embodiments, multiple processes such as chemical cell lysis, mechanical grinding, and heating can be combined to break down solid samples, such as tissue taken from a biopsy, to maximize performance.

[0100] The capture of the target microorganism can be achieved by using a substrate. In one embodiment, the surface of the substrate can be coated with at least one binding reagent, such as an antibody, ligand or receptor for an antigen, a receptor or ligand on the surface of the target organism (ASA), a nucleic acid (NA), a peptide nucleic acid (PNA) and a phosphorothioate (PT) nucleic acid probe, to capture a specific nucleic acid target sequence complementary to the probe or target organism. In another embodiment, the surface can be selected to have or be coated to form an electrostatically charged (EC) surface, such as a surface coated with silica or an ion exchange resin, to reversibly capture substantially only nucleic acids. In some embodiments, the substrate can be pre-loaded in a tubule segment or sub-segment in dry form, and a liquid binding buffer can be loaded into another segment. The substrate and buffer can be reconstituted by using the process described above.

[0101] In some embodiments, reagents from adjacent segments can be used to dilute the sample prior to incubation with the substrate. In some embodiments, target organisms can be captured on the substrate prior to lysing the microorganisms, while in other embodiments, a lysis step can be performed prior to the target capture step. In a preferred embodiment, incubation of the substrate under agitation can be performed at a desired temperature, e.g., at 4°C for viable capture or at room temperature for virus capture. Capture can be followed by a washing process to remove sample residues and undesired components from the tubule segments.

[0102] In some embodiments, magnetic beads can be used as a substrate for target capture, and a magnetic bead immobilization and resuspension process can be used to separate the beads from the sample liquid. In other embodiments, where the substrate can be a pad or sheet, the substrate can be incorporated into the collection tool and / or attached to the capillary walls within the segment.

[0103] Elution can be achieved by heating and / or incubating the substrate in solution in the tubule segments at an elevated temperature. Preferred temperatures for elution are 50° C.-95° C. In another embodiment, elution can be achieved by changing the pH of the solution in which the substrate is suspended or embedded. For example, in an exemplary embodiment, the pH of the wash solution can be 4-5.5 and the pH of the elution buffer can be 8-9.

[0104] The spore germination process can be carried out by mixing a sample containing bacterial spores with a germination solution and incubating the mixture under appropriate conditions. The germination solution may contain at least one of L-alanine, inosine, L-phenylalanine and / or L-proline, as well as some rich growth medium that allows partial growth of pre-vegetative cells released from the spores. The preferred incubation temperature for germination ranges from 20°C to 37°C. By coating the substrate with anti-spore antibodies, vegetative cells can be selectively enriched from a sample containing both live and / or dead spores. Live spores can release multiple vegetative cells from the substrate, which can be further processed to detect nucleic acid sequences characteristic of the bacterial species. In some embodiments, the germination solution can be absorbed into a pad.

[0105] In certain embodiments, the nucleic acids extracted from the biological sample may be further processed by amplifying the nucleic acids using at least one method from the group consisting of polymerase chain reaction (PCR), rolling circle amplification (RCA), ligase chain reaction (LCR), transcription-based amplification (TMA), nucleic acid sequence-based amplification (NASBA), and strand displacement amplification (SDA). In some embodiments, the nucleic acids extracted from the organism may be ribonucleic acid (RNA), and their processing may include coupled reverse transcription and polymerase chain reaction (RT-PCR) using a combination of enzymes such as Tth polymerase and Taq polymerase or reverse transcriptase and Taq polymerase. In some embodiments, the nicked circular nucleic acid probes may be circularized using T4 DNA ligase or Ampligase™ and a guide nucleic acid, such as a DNA or RNA target, and then the formation of the closed circularized probe may be detected after an in vitro selection process. Such detection may be by PCR, TMA, RCA, LCR, NASBA, or SDAR using enzymes known to those skilled in the art. In an exemplary embodiment, nucleic acid amplification can be detected in real time by using fluorescently labeled nucleic acid probes or DNA intercalating dyes and photometers or charge-coupled devices in molecular analyzers to detect the increase in fluorescence during nucleic acid amplification. These fluorescently labeled probes use detection schemes well known to those skilled in the art (i.e., TaqMan™, Molecular Beacons™, Fluorescence Resonance Energy Transfer (FRET) probes, Scorpon™ probes) and generally use fluorescence quenching and emission of quenching or fluorescence energy transfer from one reporter to another to detect the synthesis or presence of a specific nucleic acid.

[0106] Real-time detection of signals from the tubule segment can be achieved by using a sensor 492 (FIG. 7B), such as a photometer, spectrometer, CCD, etc., connected to a block such as block 490. In an exemplary embodiment, pressure can be applied to the tubule segment 190 by an actuator 392 to appropriately define the shape of the tubule segment. The format of the signal can be the intensity of light at a specific wavelength, such as fluorescence, spectrum, and / or image (such as an image of a cell or an artificial element such as a quantum dot). For fluorescence detection, light excitation from an optical system can be used to illuminate the reaction, and the emission can be detected by a photometer. To detect multiple signals with specific wavelengths, different wavelength signals can be detected in series or parallel by dedicated detection channels or spectrometers.

[0107] The disclosed devices and methods can be widely applied in medical, agricultural and environmental monitoring practices, as well as many other biological sample testing applications. Nucleic acid isolated from tissue biopsy samples surrounding tumors removed by surgeons can be used to detect precancerous tissue. In these applications, hotspot mutations in tumor suppressor genes and proto-oncogenes can be detected using genotyping techniques well known to those skilled in the art. Precancerous tissue often has somatic mutations that can be easily identified by comparing the results of genotyping tests using biopsy samples to the patient's genotype, using whole blood as the nucleic acid source. Genetic variants and germline mutations can be detected using nucleic acid isolated from white blood cells, using genotyping techniques well known to those skilled in the art. Examples of such mutations are the approximately 25 known variants of the CFTR gene that are recommended for prenatal testing by the American College of Medical Genetics and the American College of Obstetricians and Gynecologists. An example of a genetic variant is a high frequency allele of glucose-6-phosphate dehydrogenase that affects sensitivity to therapeutic agents such as the antimalarial drug primaquine.

[0108] Another example of a genetic variation with clinical relevance is an allele for increased risk of pathological conditions and increased risk of venous thrombosis, such as the factor V Leiden allele. Nucleic acid isolated from bacteria can be used to detect gene coding sequences to assess the pathogenicity of bacterial strains. Examples of such genes are the Lethal Factor, Protective Antigen A and Edema factor genes on the PXO1 plasmid of Bacillus anthracis and the Capsular Antigens A, B and C on the PXO2 plasmid of B. anthracis. The presence of these sequences allows researchers to distinguish B. anthracis from harmless soil bacteria. Nucleic acid isolated from RNA viruses can be used to detect gene coding sequences to detect the presence or absence of the virus or to quantify the virus to guide therapeutic treatment of infected individuals.

[0109] A particularly significant utility of such an assay is the detection of human immunodeficiency virus (HIV) to guide antiretroviral therapy. Nucleic acids isolated from DNA viruses can be used to detect gene coding sequences to detect the presence or absence of the virus in blood prior to use in the manufacture of blood-derived products. The detection of Hepatitis B virus in pools of blood samples is a well-known example of this utility to those skilled in the art. The presence of verotoxin E. coli in ground beef is a good example of a potential agricultural application of the device. The detection of Norwalk virus on surfaces is an example of a public health environmental monitoring application.

[0110] Some embodiments include a test tube 1 having a flexible device 10 divided into multiple segments, e.g., segments 16, 110, 120, 130, 140, 150, 160, 170, 180, and / or 190, that may traverse the longitudinal axis of the device and may contain reagents, e.g., reagents 210, 221, 222, 230, 240, 250, 260, 270, 280, and / or reagent 290; and actuators 312, 322, 332, 342. The use of an analyzer may include multiple compression members, such as 352, 362, 372, 382, ​​and / or 392, clamps, such as clamps 310, 320, 330, 340, 350, 360, 370, 380, and / or 390, and blocks, such as 314, 344, and / or 394 (others not numbered for simplicity), may be incorporated to process samples with opposing actuators and clamps. Various combinations of these actuators, clamps, and / or blocks may be used to effectively close and clamp the device, thereby isolating fluids. In an exemplary embodiment, at least one of the actuators or blocks may include a thermal control element to control the temperature of the device segments for sample processing. The sample processing device may further include at least one magnetic field source 430 capable of applying a magnetic field to the segments. The sample processing device may further include a detection device 492, such as a photometer or CCD, to monitor reactions occurring or completed within the device.

[0111] Fluid can be driven through the flow channel by compressing the device with a centrally located actuator and its adjacent clamps, if present, to form a flow channel with a gap of about 1 to about 500 μm, preferably about 5 to about 500 μm, through each segment. Adjacent actuators gently compress adjacent segments in liquid communication with the flow channel to generate offset internal pressures and ensure a substantially uniform gap in the flow channel. Two adjacent actuators can then alternately compress and release the pressure of the device at their respective segments to generate flow at a controlled rate. Optional flow, pressure, and / or force sensors can be incorporated to allow for closed-loop control of the flow behavior. The flow channel process can be used for washing, enhancing substrate binding efficiency, and detection.

[0112] A particle immobilization and resuspension process can be used to separate the particles from the sample liquid. A magnetic field generated by a magnetic source can be applied to a segment containing the magnetic particle suspension to capture and immobilize the particles to the tube wall. A stirring process can be used during the capture process. In another embodiment, a flow path can be formed in the segment to which the magnetic field is applied, and the magnetic particles can be captured in the flow to increase the capture efficiency. To resuspend the immobilized particles, the magnetic field can be turned off or removed, and a stirring or flow path process can be used for resuspension.

[0113] Embodiments of the present invention are further described in the following examples, which do not limit the scope of the invention described in the claims. EXAMPLES

[0114] Example 1: Large Stokes shift fluorescent dyes The concept of multiplexing PCR with LSS dyes was demonstrated by developing a Sexually Transmitted Infection (STI) panel for the cobas® Liat® system. Two fluorogenic PCR probes (TaqMan probes) were prepared from commercially available LSS dyes; Dy396XL "Mega-Stokes" dye (Dyomics, GmbH, Jena, Germany) and Chromeo 494 dye (Active Motif, Carlsbad, CA, USA). Figure 2 summarizes the excitation and emission spectra of four standard fluorescent dyes and Dy396XL (392 nm / 572 nm), excited in the UV channel and readout in the green channel. Similarly, Figure 3 summarizes a similar setup with Chromeo 494 dye (494 nm / 628 nm), excited with light from the blue channel and readout in the amber and / or red channels.

[0115] Example 2: Multiplex PCR with LSS dyes on the Cobas® Liat system With the introduction of the LSS dyes (Dy395XL and Chromeo494), the multiplexing level of the cobas® Liat® System increased from four detection channels (bb, gg, aa, rr) to six detection channels (ug and br added). Table 2 provides an example of a six-channel cobas® Liat® CT / NG / TV / MG assay. The LSS dyes Dy395XL and Chromeo494 were used to label the MG and NG probes, respectively. Figure 4 summarizes the real-time PCR growth curves of the six TaqMan probes in the cobas® Liat® CT / NG / TV / MG assay. [Table 2]

[0116] The 6-channel cobas® Liat® CT / NG / TV / MG study demonstrated the feasibility of combining four conventional channels (FAM, HEX, CFR610, Quasar670) with ug and br channels (Dy395XL and Chromeo494, respectively) (Table 3). After crosstalk correction, no signal was observed without the corresponding target. [Table 3]

[0117] Example 3: Comparison of Dy395XL and Dy396XL Further functionality and manufacturability evaluation of the LSS dyes revealed that the fluorescence intensity of Dy395XL was rather weak (ε = 20,600 M -1 cm -1 ), and coupling with DNA was shown to be unscalable due to exceptionally low solubility in water-miscible organic solvents and problematic purification. -1 cm -1 Dy396XL, a Dy395XL analog with Dy395XL, showed feasible manufacturability of coupling with DNA. Figures 5-6 show the performance comparison of two MG probes, each labeled with either Dy395XL or Dy396XL, respectively. The baseline of the MG-Dy396XL probe was 1.5-2.5 times higher than that of the MG-Dy395XL probe. Statistical analysis showed that the performance of the MG-Dy396XL probe was superior in terms of Ct and amplitude (faster Ct and higher amplitude) than the MG-Dy395XL probe. The higher Kexp value (slope of the exponential phase) of the MG-Dy396XL probe also suggested a more robust growth curve for the MG-Dy396XL probe.

[0118] Example 4: Experimental setup for multiplex PCR using LSS dyes and TAGS technology on a LightCycler® 480 analyzer The following examples show that the concept of extended optical multiplexing with LSS dyes can be combined with other multiplexing methods. PCR using temperature assisted generation of signal (TAGS) technology is based on a DNA probe architecture that allows discrimination of multiple targets in the same optical channel by measuring fluorescence at different temperatures (disclosed in US Patent Application Publication No. 2018 / 0073064, which is incorporated herein by reference in its entirety). TAGS-based thermal multiplexing can be combined with optical multiplexing, provided that the fluorescence signal intensity of the LSS dyes remains stable at temperatures up to 100°C.

[0119] Multiplex PCR using a TAGS technology model system with three thermal channels (TCs) has been constructed to demonstrate higher order multiplexing. For TC1, a standard TaqMan probe containing a 5'-fluorophore and an internal BHQ-2 quencher was used, whereas TC2 and TC3 used tagged TAGS probes. The tagged probes consisted of a target-specific DNA sequence with a 5'-BHQ-2 fluorescent quencher and a covalently attached "R-tag" sequence specific for the respective thermal and light channel. The R-tag sequence was made of a non-natural L-DNA carrying a fluorescent dye and with a defined melting temperature relative to another complementary L-DNA strand carrying a second 3'-BHQ-2 fluorescent quencher (quenching oligonucleotide, "Q-tag"). The tagged probes for TC2 and TC3 differ only in the length of the L-DNA fragment.

[0120] Regular TaqMan probes and tagged TAGS probes containing LSS dyes were prepared by introducing amino modifications during solid-phase DNA synthesis and post-synthetic labeling with in situ activated carboxylic acids of the dyes (as disclosed in US Patent Publication No. 2020 / 0017895, which is incorporated herein by reference in its entirety). Oligonucleotides were purified by reverse-phase chromatography using triethylammonium acetate buffer and acetonitrile. The final tagged probes were purified by polyacrylamide gel electrophoresis.

[0121] To demonstrate compatibility with TAGS technology, two LSS dyes with thermostable fluorescence have been selected; the commercially available ATTO 490LS dye (ATTO-TEC GmbH, Siegen, Germany) and another proprietary dye called RLS. On the LightCycler® and cobas® x800 systems, the ATTO 490LS (496 nm / 661 nm) can be excited with the FAM excitation channel (495 nm) and readout is performed in the LCR emission channel (645 nm). The RLS dye (468 nm / 553 nm) can be excited with light from the COU excitation channel (435 nm) and readout is performed in the HEX emission channel (580 nm). The light channel assignment matrix of the five standard dyes (COU, FAM, HEX, LCR, Cy5.5) and the two LSS dye channels (ATTO 490LS and RLS) on the five-color LightCycler® 480 and cobas® x800 analyzers is shown in Figure 9. The excitation and emission spectra of five standard fluorescent dyes in combination with the ATTO 490LS and RLS dyes are outlined in Figures 10 and 11, respectively.

[0122] Example 5: Multiplex PCR using Atto490LS dye and TAGS technology In this example, a PCR reaction with thermal multiplexing and detection in the LSS light channel was performed. The branched probe was incubated with the quenching oligonucleotide at a molar ratio of 1:20. The mixture was typically cycled in a 50 μL reaction containing 60 mM Tricine, 120 mM potassium acetate, 5.4% DMSO, 0.027% sodium azide, 3% glycerol, 0.02% Tween® 20, 43.9 μM EDTA, 0.2 U / μL UNG, 400 μL dATP, 400 μM CTP, 400 μM dGTP, 800 μM dUTP, 3.3 mM manganese acetate, 0.9 U Z05 enzyme, 800 nM Q-tag, 400 nM of each primer, and 40 nM branched probe. Cycling conditions similar to a typical PCR amplification reaction are shown in Table 4 below. [Table 4]

[0123] Figure 12 shows real-time PCR growth curves in the ATTO 490LS channel across the three thermal channels. Data were generated on a LightCycler® 480 analyzer. TC1 is based on the ATTO 490LS dye and a standard TaqMan probe with a fluorescent readout at 58°C. TC2 and TC3 are based on the tagged TAGS probe design with the ATTO 490LS dye that produces a fluorescent readout at 80°C and 91°C, respectively. All possible target combinations across the three thermal channels were tested in eight individual PCR reactions (samples A-H). The growth curves for each thermal channel where a positive PCR signal is expected are marked with an asterisk. As expected, a signal was only obtained when the target was present. No signal was observed without the corresponding target. In the higher thermal channels, the slight positive or negative slope in the absence of target was caused by non-optimized thermal crosstalk compensation of the fluorescent dyes. The concentration of the target was 1000 cp / reaction.

[0124] Example 6: Multiplex PCR using RLS dyes and TAGS technology A similar experiment to that described in Example 5 was performed, except that the fluorescence detection was performed in a different LSS channel using a proprietary LSS dye called RLS. As before, a signal was obtained only if the target was present. Notably, the ATTO 490LS and RLS occupy two separate LSS channels. Crosstalk between the standard light channel and the LSS dye channel is negligible, indicating that the ATTO 490LS and RLS can be used simultaneously. Figure 13 shows the corresponding real-time PCR growth curves.

[0125] Conclusion: The above examples demonstrate that the introduction of LSS dyes can increase the multiplexing level on the LightCyler® and cobas® x800 systems from five conventional detection channels (COU, FAM, HEX, LCR, Cy5.5) to at least seven detection channels (ATTO 490LS and RLS added). As demonstrated herein, multiplexing with LSS dyes combined with TAGS multiplexing with three temperature channels allows the detection of 21 individual targets.

[0126] In principle, higher order optical multiplexing is compatible with any fluorescence-based PCR platform if the fluorescent properties of the dyes match the optical filters of the instrument. In this way, no changes in the instrument hardware are required. The platform independence of the optical multiplexing technology was also demonstrated by successfully applying the concept to two different PCR systems, the cobas® Liat and the LightCycler® 480, which is directly transferable to the cobas® x800 system.

Claims

1. 1. A method for detecting at least two target nucleic acid sequences in a sample, comprising: (a) subjecting said sample suspected of containing said at least two target nucleic acid sequences in a single reaction vessel to the following: i. a first pair of oligonucleotide primers having nucleotide sequences complementary to each strand of a first target nucleic acid sequence, and a second pair of oligonucleotide primers having nucleotide sequences complementary to each strand of a second target nucleic acid sequence; ii. a first oligonucleotide probe comprising a nucleotide sequence at least partially complementary to the first target nucleic acid sequence and annealing within the first target nucleic acid sequence bounded by the first pair of oligonucleotide primers, the first oligonucleotide probe being labeled with a large Stokes shift (LSS) fluorescent dye capable of generating a detectable signal and a first quencher moiety capable of quenching the detectable signal generated by the LSS fluorescent dye, the LSS fluorescent dye being separated from the first quencher moiety by a nuclease-sensitive cleavage site; iii. a second oligonucleotide probe comprising a nucleotide sequence at least partially complementary to the second target nucleic acid sequence and annealing within the second target nucleic acid sequence bounded by the second pair of oligonucleotide primers, the second oligonucleotide probe being labeled with a small Stokes shift (SSS) fluorescent dye capable of generating a detectable signal and a second quencher moiety capable of quenching the detectable signal generated by the SSS fluorescent dye, the SSS fluorescent dye being separated from the second quencher moiety by a nuclease-sensitive cleavage site, the SSS fluorescent dye having an absorption peak maximum significantly different from an absorption peak maximum of the LSS fluorescent dye on the first oligonucleotide probe and an emission peak maximum similar to an emission peak maximum of the LSS fluorescent dye on the first oligonucleotide probe, the significant difference being at least 80 nanometers in wavelength. contacting with (b) amplifying the first and second target nucleic acid sequences by polymerase chain reaction (PCR) using a nucleic acid polymerase having 5' to 3' nuclease activity, whereby during an extension step of each PCR cycle, the 5' to 3' nuclease activity of the nucleic acid polymerase enables cleavage and separation of the LSS fluorescent dye from the first quenching moiety on the first oligonucleotide probe and cleavage and separation of the SSS fluorescent dye from the second quenching moiety on the second oligonucleotide probe; (c) measuring the detectable signal from the LSS fluorescent dye by excitation at or near the wavelength of the absorption peak maximum of the LSS fluorescent dye, and measuring the detectable signal from the SSS fluorescent dye by excitation at or near the wavelength of the absorption peak of the SSS fluorescent dye; (d) repeating steps (b) and (c) for multiple PCR cycles to produce a desired amount of amplification product from said first and second target nucleic acid sequences; (e) detecting the presence of the first target nucleic acid sequence from the signal detected from the LSS fluorescent dye, and detecting the presence of the second target nucleic acid sequence from the signal detected from the SSS fluorescent dye. A method comprising:

2. 2. The method of claim 1, wherein the difference between the absorption peak maximum of the LSS fluorescent dye and the absorption peak maximum of the SSS fluorescent dye is greater than 100 nanometers in wavelength.

3. The LSS fluorescent dye is ALEXA FLUOR 430, ATTO 430LS, ATTO 490LS, ATTO 390LS, CASCADE YELLOW, CF350, CHROMEO 494, CYTO 500 LSS, CYTO 510 LSS, CYTO 514 LSS, CYTO 520 LSS, DAPOXYL, DY 480XL, DY 481XL, DY 485XL, DY 510XL, DY 511XL, DY 520XL, DY 521XL, DY 601XL, DY 350XL, DY 360XL, DY 370XL, DY 375XL, DY 380XL, DY 395XL, DY 396XL, DYLIGHT 515-LS, DYLIGHT 485-LS, DYLIGHT 510-LS, DYLIGHT 521-LS, FURA 2, INDO 1, KROME ORANGE, LUB 04, LUCIFER YELLOW, NBD X, NILE RED, PULSAR 650, PYMPO, STAR 440SXP, STAR 470SXP, STAR 520SXP, VIOGREEN, CF 350, SETAU 405, and PACIFIC 3. The method of claim 1 or 2, wherein the soluble soluble soluble protein is selected from the group consisting of: ORANGE.

4. 4. The method of claim 3, wherein the LSS fluorescent dye is selected from DY 396XL or CHROMEO 494.

5. The method of any one of claims 1 to 3, wherein the LSS fluorescent dye has a fluorescent signal intensity that remains stable at temperatures up to 100°C.

6. 6. The method of claim 5, wherein the LSS fluorescent dye is ATTO 490LS.

7. The reaction vessel is a capillary tube, (i) a proximal end having an opening through which a sample can be introduced; (ii) a distal end; and (iii) at least a first segment containing at least one nucleic acid extraction reagent, a second segment distal to the first segment containing a wash reagent, and a third segment distal to the second segment containing one or more amplification reagents, each of the segments comprising: (A) defined by the tubule; (B) being at least partially fluidly isolated by a fluid-tight seal formed by bonding opposing wall portions of the capillary to one another, such that (1) the seal is broken by applying fluid pressure to a segment that is partially fluidly isolated by the seal; and (2) the seal is capable of being clamped where the opposing wall portions of the capillary are joined without destroying the seal to prevent the seal from being destroyed by application of fluid pressure to a segment partially fluidly isolated by the seal; (C) at least a first segment, a second segment, and a third segment, each of which is expandable to receive a volume of fluid ejected from another segment and compressible to be substantially free of fluid when compressed; (iv) a cap for closing said opening, the cap including a chamber in fluid communication with said capillary, allowing free flow of gas but retaining all liquid volume and infectious agents within the tube; (v) a rigid frame in which the proximal and distal ends of the capillary are held; and (vi) an integral capillary tensioning mechanism or attachment of the capillary to the frame that pulls the capillary under sufficient tension to facilitate compression and flattening of the capillary; The method according to any one of claims 1 to 6, comprising:

8. 1. A method for detecting at least two target nucleic acid sequences in a sample, comprising: (a) subjecting said sample suspected of containing said at least two target nucleic acid sequences in a single reaction vessel to the following: i. a first pair of oligonucleotide primers having nucleotide sequences complementary to each strand of a first target nucleic acid sequence, and a second pair of oligonucleotide primers having nucleotide sequences complementary to each strand of a second target nucleic acid sequence; ii. a first oligonucleotide probe comprising a nucleotide sequence at least partially complementary to the first target nucleic acid sequence and annealing within the first target nucleic acid sequence bounded by the first pair of oligonucleotide primers, the first oligonucleotide probe being labeled with a large Stokes shift (LSS) fluorescent dye capable of generating a detectable signal and a first quencher moiety capable of quenching the detectable signal generated by the LSS fluorescent dye, the LSS fluorescent dye being separated from the first quencher moiety by a nuclease-sensitive cleavage site; iii. a second oligonucleotide probe comprising a nucleotide sequence at least partially complementary to the second target nucleic acid sequence and annealing within the second target nucleic acid sequence bounded by the second pair of oligonucleotide primers, the second oligonucleotide probe being labeled with a small Stokes shift (SSS) fluorescent dye capable of generating a detectable signal and a second quencher moiety capable of quenching the detectable signal generated by the SSS fluorescent dye, the SSS fluorescent dye being separated from the second quencher moiety by a nuclease-sensitive cleavage site, the SSS fluorescent dye having an absorption peak maximum significantly different from an absorption peak maximum of the LSS fluorescent dye on the first oligonucleotide probe and an emission peak maximum similar to an emission peak maximum of the LSS fluorescent dye on the first oligonucleotide probe, the significant difference being at least 80 nanometers in wavelength. contacting with; (b) amplifying the first and second target nucleic acid sequences by polymerase chain reaction (PCR) using a nucleic acid polymerase having 5' to 3' nuclease activity, whereby during an extension step of each PCR cycle, the 5' to 3' nuclease activity of the nucleic acid polymerase enables cleavage and separation of the LSS fluorescent dye from the first quenching moiety on the first oligonucleotide probe and cleavage and separation of the SSS fluorescent dye from the second quenching moiety on the second oligonucleotide probe; (c) measuring the detectable signal from the LSS fluorescent dye by excitation at or near the wavelength of the absorption peak maximum of the LSS fluorescent dye, and measuring the detectable signal from the SSS fluorescent dye by excitation at or near the wavelength of the absorption peak of the SSS fluorescent dye; (d) repeating steps (b) and (c) for multiple PCR cycles to produce a desired amount of amplification product from said first and second target nucleic acid sequences; (e) detecting the presence of the first target nucleic acid sequence from the signal detected from the LSS fluorescent dye, and detecting the presence of the second target nucleic acid sequence from the signal detected from the SSS fluorescent dye. A method comprising:

9. 9. The method of claim 8, wherein the difference between the emission peak maximum of the LSS fluorescent dye and the emission peak maximum of the SSS fluorescent dye is greater than 100 nanometers in wavelength.

10. The LSS fluorescent dye is ALEXA FLUOR 430, ATTO 430LS, ATTO 490LS, ATTO 390LS, CASCADE YELLOW, CF350, CHROMEO 494, CYTO 500 LSS, CYTO 510 LSS, CYTO 514 LSS, CYTO 520 LSS, DAPOXYL, DY 480XL, DY 481XL, DY 485XL, DY 510XL, DY 511XL, DY 520XL, DY 521XL, DY 601XL, DY 350XL, DY 360XL, DY 370XL, DY 375XL, DY 380XL, DY 395XL, DY 396XL, DYLIGHT 515-LS, DYLIGHT 485-LS, DYLIGHT 510-LS, DYLIGHT 521-LS, FURA 2, INDO 1, KROME ORANGE, LUB 04, LUCIFER YELLOW, NBD X, NILE RED, PULSAR 650, PYMPO, STAR 440SXP, STAR 470SXP, STAR 520SXP, VIOGREEN, CF 350, SETAU 405, and PACIFIC 10. The method of claim 8 or 9, wherein the soluble soluble soluble agent is selected from the group consisting of: ORANGE.

11. 11. The method of claim 10, wherein the LSS fluorescent dye is DY 396XL or CHROMEO 494.

12. The method of any one of claims 8 to 10, wherein the LSS fluorescent dye has a fluorescent signal intensity that remains stable at temperatures up to 100°C.

13. 13. The method of claim 12, wherein the LSS fluorescent dye is ATTO 490LS.

14. The reaction vessel is a capillary tube, (i) a proximal end having an opening through which a sample can be introduced; (ii) a distal end; and (iii) at least a first segment containing at least one nucleic acid extraction reagent, a second segment distal to the first segment containing a wash reagent, and a third segment distal to the second segment containing one or more amplification reagents, each of the segments comprising: (A) defined by the tubule; (B) being at least partially fluidly isolated by a fluid-tight seal formed by bonding opposing wall portions of the capillary to one another, such that (1) the seal is broken by applying fluid pressure to a segment that is partially fluidly isolated by the seal; and (2) the seal is capable of being clamped where the opposing wall portions of the capillary are joined without destroying the seal to prevent the seal from being destroyed by application of fluid pressure to a segment partially fluidly isolated by the seal; (C) at least a first segment, a second segment, and a third segment, each of which is expandable to receive a volume of fluid ejected from another segment and compressible to be substantially free of fluid when compressed; (iv) a cap for closing said opening, the cap including a chamber in fluid communication with said capillary, allowing free flow of gas but retaining all liquid volume and infectious agents within the tube; (v) a rigid frame in which the proximal and distal ends of the capillary are held; and (vi) an integral capillary tensioning mechanism or attachment of the capillary to the frame that pulls the capillary under sufficient tension to facilitate compression and flattening of the capillary; The method according to any one of claims 8 to 13, comprising:

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