Amplification and detection of nucleic acids in biological samples

Abstract

Disclosed are methods for identifying the presence or absence of a target nucleic acid from one or more organisms in a biological sample, the methods comprising: (a) spinning the sample at a spin speed sufficient to precipitate cellular debris and fluorescence inhibitors present in the sample, reducing fluorescence interference or quenching in the sample; and (b) directly amplifying and detecting the target nucleic acid in the sample.

Description

[0001] This application is a divisional application of the invention patent application filed on November 24, 2015, with application number 201580074363.2 (international application number PCT / US2015 / 062376) entitled "Amplification and Detection of Nucleic Acids in Biological Samples".

[0002] Cross-reference to related applications

[0003] This application claims priority to U.S. Provisional Application No. 62 / 084,257, filed November 25, 2014, the contents of which are incorporated herein by reference in their entirety. Technical Field

[0004] This invention relates to methods for the diagnosis and detection of nucleic acids, which are used to detect organisms and genes in biological samples using direct amplification. Background Technology

[0005] The following discussion of the background of the present invention is provided only to help readers understand the present invention and should not be regarded as a description or constitute prior art of the present invention.

[0006] Nucleic acid detection in biological samples such as whole blood typically requires the extraction and purification of the nucleic acids from the sample prior to PCR. This is because components (e.g., hemoglobin in blood samples) and preservatives (e.g., anticoagulants) can interfere with PCR amplification (Wang, JT., et al., 1992, J. Clin. Microbiol. 30:750). Target nucleic acids are particularly difficult to detect because they are usually present at much lower levels than endogenous nucleic acids such as genomic DNA or its transcribed RNA. Extracting nucleic acids from biological samples is time-consuming and involves a high risk of contamination.

[0007] Given the high complexity involved in isolating and detecting nucleic acid molecules in biological samples, there is an increasing desire to directly detect nucleic acids in biological samples without any upstream nucleic acid extraction or extensive pretreatment steps. Several methods for direct PCR of pathogenic nucleic acids from blood samples have been reported, such as microwave irradiation (Ihhara, M., et al., 1994, BioTechniques 17(4):726), hydrogen peroxide treatment (Rudbeck, L. and Dissing, J., 1998, BioTechniques 25(4):588), and sodium hydroxide treatment (Queipo-Ortuna, M., et al., 1999, BioTechniques 27(2):248). However, in cases seeking rapid diagnostics, there is a need for rapid methods that involve only a few steps and minimal technical requirements, yet still achieve stable and successful amplification of nucleic acids in biological samples. Summary of the Invention

[0008] This invention is based on the discovery of a method that allows direct amplification of nucleic acids in whole blood samples without first extracting the nucleic acids from the sample. The method of this invention includes the following steps: rotating the whole blood sample at high speed to remove cell debris and substances that interfere with or quench fluorescence emission during PCR amplification; and directly amplifying and detecting the nucleic acid molecules.

[0009] Therefore, the present invention provides a method for identifying the presence or absence of the target nucleic acid, which is used for gene detection or detection of organisms in biological samples. The method comprises: (a) rotating a biological sample containing whole blood at a rotational speed sufficient to precipitate cell debris and fluorescence inhibitors present in the sample, thereby reducing fluorescence interference or quenching in the sample; and (b) directly amplifying and detecting the target nucleic acid in the sample. Preferably, the rotational speed is in the range of greater than 140 × g to 1500 × g, and the biological sample is placed in a centrifugal microfluidic disc.

[0010] In one aspect of the invention, the detection step includes detecting a visible signal emitted through an optical path in the precipitated sample. Preferably, the detectable signal is fluorescence emission. In another aspect of the invention, the amplification step includes real-time polymerase chain reaction analysis. In a preferred embodiment, the target nucleic acid is DNA. In another preferred embodiment, the target nucleic acid is RNA.

[0011] In a preferred aspect of the invention, the biological sample is whole blood, and the target nucleic acid is derived from one or more organisms found in the blood. In one aspect, the target nucleic acid is human nucleic acid. In another aspect, the target nucleic acid is derived from a microorganism. The microorganism can be a virus, such as Ebola virus, Marburg virus, influenza virus, respiratory syncytial virus, varicella-zoster virus, herpes simplex virus, enterovirus, dengue virus, or any combination thereof. In another aspect, the microorganism is a Gram-negative or Gram-positive bacterium. In a preferred embodiment, the bacteria are selected from one or more of Bacillus, Bordetella, Borrelia, Listeria, Escherichia, Salmonella, Campylobacter, Clostridium, Helicobacter, Mycobacterium, Staphylococcus, Camplobacter, Enterococcus, Neisseria, Shigella, Streptococcus, Vibrio, Yersinia, and Pseudomonas, or any combination thereof. Preferably, the bacteria are Bacillus anthracis. In yet another embodiment, the microorganism is a fungus. The cell debris may include lysed and intact red blood cells. The foregoing general and detailed descriptions are exemplary and illustrative, intended to provide further explanation of the claimed invention. For a detailed understanding of the invention, reference is made to the following detailed description of preferred embodiments in conjunction with the accompanying drawings. Other objects, advantages, and novel features will be apparent to those skilled in the art based on the following detailed description of the invention. Attached Figure Description

[0012] Figure 1The effect of high-speed rotation on the detection of artificial products is shown. The Bacillus anthracis chromosomal target was amplified from whole blood samples (10% of the reaction volume) collected in EDTA tubes, with 50 copies per reaction. The samples in the upper figure represent untreated samples, while the samples in the lower figure represent samples that were rotated off-board at approximately 900 × g for 10 minutes at 23°C as described in Example 1. Figure 1 The artificial product disappears during cycling between 5 and 30 cycles in a rotating sample.

[0013] Figure 2 This is a photograph of the centrifugal microfluidic disk described in Example 2. A top view of the example's individual well (top) and the disk (bottom) is shown. The disk on the left is operated using a measurement definition, which is a standardized method of rotating the sample at approximately 1500 rpm (approximately 140 × g) throughout the measurement, while the disk on the right is operated using a high-speed definition, which is rotating the sample at approximately 5000 rpm (1500 × g) throughout the measurement, except for an optical readout step where the sample is rotated at the standard speed of approximately 1500 rpm (approximately 140 × g). In the standard-speed disk, blood particles are observed throughout the well, while in the high-speed disk, the blood particles are deposited at the periphery of the well, leaving a clear optical path in the center of the well.

[0014] Figure 3 Results of detecting bacterial chromosomal targets from Bacillus anthracis in whole blood during optimization of high-speed rotation assay parameters are shown. Samples were run using two different high-speed and duration settings. At the start of the run, samples were rotated at 2500 rpm (780 × g) for 6 minutes (A) or at 3500 rpm (1500 × g) for 2 minutes (B), followed by rotation at a standard speed of approximately 1500 rpm (approximately 140 × g) for the remainder of the run. Amplification curves and average Ct values ​​are shown by varying the initial rotation speed and duration.

[0015] Figure 4 This illustrates the amplification of RNA virus Ebola Reston from whole blood using VHF high-speed rotation assay parameters.

[0016] Figure 5 The effect of high-speed rotation on the area under the melting point curve is shown in genetic assays performed on MTHFR1298 heterozygous samples from whole blood.

[0017] Hybeacon, including primers and single nucleotide polymorphisms in the methylenetetrahydrofolate reductase (MTHFR) gene, was used. TM The probe reaction mixture was prepared in two identical universal discs. The reaction consisted of 8 μL of the reaction mixture plus 2 μL of whole blood or buffer as a sample, or 9 μL of the reaction mixture plus 1 μL of whole blood or buffer. The two discs were run with independent parameters: one performed the standard PCR followed by the melt analysis protocol (grey bars), while the other performed the standard protocol but added a high-speed rotation step (black bars) between the PCR cycle and the melt analysis step. In most blood samples, the high-speed rotation increased the area under the melting curve, resulting in a greater difference in fluorescence intensity between positive and negative samples.

[0018] Figure 6 The melting point curves for detecting MTHFR1298 heterozygous samples in whole blood are shown. Standard rotation speed (top) and high-speed rotation speed (bottom) parameters were used. Compared to the standard speed, high-speed rotation resulted in sharp peak differentiation in the heterozygous assay. Invention Details

[0019] This document discloses a method for identifying the presence or absence of target nucleic acids in biological samples without first isolating, extracting, and purifying the nucleic acids from the biological sample. The method disclosed herein is based on the unexpected discovery that applying high centrifugation force to whole blood samples causes the precipitation of cell debris and substances present in the sample that may interfere with the emission of PCR reaction chemistry or fluorescence, enabling successful real-time PCR amplification of the whole blood sample. The inventors have discovered that by adjusting the rotation speed of the sample, cell debris that may (i) inhibit the emission and / or detection of detectable signals from the sample, (ii) interfere with or quench the polymerase chain reaction chemistry itself, and / or (iii) block the light path through which detectable signals propagate during real-time PCR assays can precipitate within the sample, thereby allowing efficient amplification of the target nucleic acid and clearing the light path through the sample, enabling the detection of a visible amplification signal. As a result, the method disclosed herein allows for the detection of nucleic acids in whole blood samples without extracting the nucleic acids from the sample.

[0020] In a preferred embodiment, the present invention utilizes a 3M Integrated Cycler equipped with a consumable Direct Amplification Disc. The high rotational speed achieved by integrating the consumable disc into the 3M Integrated Cycler causes cell debris in the whole blood sample to move and settle around the periphery of the consumable disc, thereby enabling the separation of cell debris and successful real-time PCR amplification of the whole blood sample. The cell debris includes lysed and intact red blood cells and fluorescence inhibitors that typically cause quenching of the PCR reaction or interference with fluorescence emission. Preferably, the relative centrifugal force is from about 0 × g to about 1500 × g.

[0021] In other embodiments, high centrifugal force is applied to a multiplexed compact disc platform on which the whole blood sample is placed for testing, and the cell debris is separated by rotating at a high angular velocity, such as 1500 × g.

[0022] definition

[0023] As used in this article, the term "DNA" refers to a nucleic acid molecule that contains deoxyribose, which is the opposite of the ribose found in RNA.

[0024] As used herein, the term "RNA" refers to a nucleic acid molecule containing a ribose sugar opposite to the deoxyribose found in DNA. The term "RNA" as used herein refers to all types, including messenger RNA (mRNA), ribosomal RNA (rRNA), transfer RNA (tRNA), and small RNAs with regulatory functions. The term "small RNAs" has a specific meaning, referring to non-translated RNAs that perform housekeeping or regulatory functions in bacteria. "Small RNAs" are not rRNA or tRNA.

[0025] As used herein, the term "target nucleic acid" refers to any nucleic acid molecule or fragment that serves as a target for detection in a sample. In some embodiments, the target nucleic acid is of viral, bacterial, or fungal origin. In other embodiments, the target nucleic acid molecule is of human origin. The target nucleic acid can be a DNA or RNA molecule.

[0026] As used herein, the term “cycle” or “thermal cycling” refers to any technique that uses laboratory equipment to amplify fragments of nucleic acid sequences by employing primer extension reactions that utilize pre-programmed cycles of increasing and decreasing temperatures. Examples of thermal cycling include, but are not limited to, PCR, real-time PCR, and reverse transcription polymerase chain reaction (RT-PCR).

[0027] As used herein, the terms “reverse transcriptase-polymerase chain reaction” or “RT-PCR” refer to any technique used to synthesize and amplify DNA molecules using a sequence as a copy of an RNA sequence. RT-PCR is useful in detecting RNA species, such as in quantitative analysis of gene expression, and also in producing DNA copies of RNA for use in signal amplification in cloning, copy DNA library construction, probe synthesis, and in situ hybridization.

[0028] As used herein, the terms “reagent mixture” or “reaction mixture” or “reaction mixture” mean a composition having all the elements required for reverse transcription and / or reverse transcription polymerase chain reaction, or real-time polymerase chain reaction, including but not limited to polymerase and primers having specificity for sequences of diagnostic target RNA or DNA, respectively.

[0029] As used herein, the term "amplification mixture" refers to a mixture of reagents used in nucleic acid amplification reactions, but does not contain primers or samples. An amplification mixture includes buffer, dNTPs, and DNA polymerase. An amplification mixture may further contain at least one of MgCl2, KCl, and nonionic and ionic detergents.

[0030] The term “amplification master mixture” as used in this article includes the amplification mixture and the primers used to amplify the target nucleic acid, but does not include the sample to be amplified.

[0031] As used in this article, the term "reagent-sample mixture" refers to a mixture containing a reagent mixture plus a sample.

[0032] As used in this article, "primer" refers to a synthetic or naturally occurring oligonucleotide that, when placed under conditions where the synthesis of the complementary strand is catalyzed by a polymerase, functions as an initiation point for nucleic acid synthesis or replication along the template strand. In the case of reverse transcription, primers are composed of nucleic acids and guided by an RNA template. In the case of PCR, primers are composed of nucleic acids and guided by a DNA template.

[0033] As used herein, the term "DNA polymerase" refers to any enzyme that helps catalyze the polymerization of deoxyribonucleotides into DNA chains. DNA polymerases function by adding free nucleotides to the 3' end of the newly formed chain, causing the new chain to elongate in the 5'-3' direction.

[0034] This article uses This refers to a method for real-time PCR. In this method, the PCR reaction mixture includes components that hybridize with the amplified nucleic acid region. Probe. The... The probe comprises a donor and a quenching fluorophore, which are located at either end of the probe and sufficiently close to each other to allow the fluorescence of the donor to be absorbed by the quenching agent. However, when the probe hybridizes with the amplified fragment, the 5'-3' exonuclease activity of the Taq polymerase cleaves the probe, thereby allowing the donor fluorophore to emit detectable fluorescence.

[0035] As used in this article, "lysis" refers to any disturbance or alteration of the cell wall or viral particles that facilitates the acquisition or release of the cellular RNA or DNA. Complete destruction or rupture of the cell wall is not an essential requirement for lysis.

[0036] As used in this paper, the term "cycle threshold" or "Ct" refers to the cycle in which the increase in fluorescence due to product formation reaches a significant and detectable level above the background signal during thermal cycling.

[0037] As used herein, the term "direct amplification" refers to a nucleic acid amplification reaction that amplifies the target nucleic acid from the sample without prior purification, extraction, or concentration. The "cycle threshold" or "Ct" is a relative measure of the target concentration in a PCR reaction. Besides the target concentration, several factors influence the absolute value of Ct. However, artificial products from the reaction mixture or instrument that alter fluorescence measurements associated with Ct calculations will cause variations in the Ct value independent of the template.

[0038] As used herein, the term "extraction" refers to any operation that removes nucleic acids from other (non-nucleic acid) materials present in a sample. Such operations include, but are not limited to: mechanical or chemical lysis, the addition of detergents or proteases, or precipitation and removal of non-nucleic acid materials such as proteins.

[0039] As used in this article, the term "interfering substance" refers to any substance in a sample that is not the target nucleic acid. Such interfering substances include synthetic substances and biological substances. Such synthetic substances include chemicals and pharmaceuticals. Such biological substances include blood, urine, proteins, and other biomolecules.

[0040] As used in this article, the term "fluorescence inhibitor" refers to cellular components, cell debris, and substances that can substantially interfere with or quench fluorescence emission.

[0041] As used herein, the terms "rotational speed" or "rotational rate" refer to the number of complete rotations, turns, cycles, or rotations around a central point in each unit of time. Preferably, the relative centrifugal force is from about 140 × g to about 1500 × g.

[0042] As used herein, the term "amplification" or "performing an amplification" includes methods for replicating a target nucleic acid, thereby increasing the copy number of a selected nucleic acid sequence. Amplification can be exponential or linear. The target nucleic acid can be DNA or RNA. The sequence amplified in this manner forms an "amplifier." While exemplary methods involving amplification using polymerase chain reaction (PCR) are described below, numerous other methods for amplifying nucleic acids are known in the art (e.g., isothermal methods, rolling circle methods, etc.). Those skilled in the art will understand that these other methods can be used in place of or in conjunction with PCR methods. See, for example, Saiki, "Amplification of Genomic DNA" in PCR Protocols, Innis et al., Eds., Academic Press, San Diego, CA 1990, pp 13-20; Wharam et al., Nucleic Acids Res. 2001 Jun 1; 29(11): E54-E54; Hafner et al., Biotechniques 2001 Apr; 30(4): 852-860; Zhong, et al., Biotechniques 2001, 30(4): 852-6, 858, 860.

[0043] As used herein, in detecting signals from detectable markers to determine the presence of target nucleic acids in a sample, all terms “detection” do not require the method to provide 100% sensitivity and / or 100% specificity. It is well known that “sensitivity” is the probability of a positive test when a person has the target nucleic acid, while “specificity” is the probability of a negative test when a person does not have the target nucleic acid. A sensitivity of at least 50% is preferred, with at least 60%, at least 70%, at least 80%, at least 90%, and at least 99% being significantly more preferred. A specificity of at least 50% is preferred, with at least 60%, at least 70%, at least 80%, at least 90%, and at least 99% being significantly more preferred. Detection also covers assays with false positives and false negatives. False negative rates can be 1%, 5%, 10%, 15%, 20%, or even higher. False positive rates can be 1%, 5%, 10%, 15%, 20%, or even higher.

[0044] As used herein, the term "sample" or "test sample" may include clinical samples, isolated nucleic acids, or isolated microorganisms. In a preferred embodiment, the sample is obtained from a biological source (i.e., a "biological sample"), such as tissues, body fluids, or microorganisms collected from a subject. Sample sources include, but are not limited to, sputum (processed or unprocessed), bronchoalveolar lavage fluid (BAL), bronchoalveolar lavage fluid (BW), blood, whole blood, body fluids, cerebrospinal fluid (CSF), urine, plasma, serum, or tissue (e.g., biopsy material). Preferred sample sources include nasopharyngeal swabs, wound swabs, and nasal wash. As used herein, the term "patient sample" means a sample obtained from a person seeking diagnosis and / or treatment for a disease.

[0045] As used herein, the term "primer-probe detection system" refers to a method for real-time PCR. This method utilizes bifunctional molecules (referred to herein as primer-probes) containing PCR primer elements covalently linked to probe elements via polymerase-blocking groups. Furthermore, each primer-probe molecule contains a fluorophore that interacts with a quencher to reduce background fluorescence. The primer-probes used herein may contain a 3' primer with a 5' extended probe tail (containing a hairpin structure) and a fluorophore / quencher pair. During PCR, polymerase extension to the probe tail is blocked by the inclusion of hexadecimal glycol (HEG). In the first round of amplification, the 3' target-specific primer anneals and extends with the target nucleic acid, thereby integrating the primer-probe into a newly synthesized strand with a newly synthesized target region against the 5' probe. In the next round of denaturation and annealing, the probe region of the primer-probe hairpin loop hybridizes with the target, thereby separating the fluorophore and quencher and generating a measurable signal. Such primer-probe combinations are described in Whitcombe et al., Nature Biotech 17:804-807 (1999). The probe is an example primer-probe.

[0046] biological samples

[0047] Biological samples from which the methods disclosed herein can be used to detect the target nucleic acids can be derived from sterile and / or non-sterile sites and include bodily fluids such as whole blood, plasma, serum, cell-free plasma, urine, cerebrospinal fluid (CSF), synovial fluid, pleural fluid, pericardial fluid, intraocular tamponade fluid, and fecal samples that may contain nucleic acids. In one embodiment, the biological sample is whole blood. As used herein, “cell-free plasma” means plasma containing less than 1% cells by volume.

[0048] Biological samples may be suspected of containing target nucleic acids. Target nucleic acids can be RNA and / or DNA. In some embodiments, the target nucleic acid is derived from microorganisms such as bacteria, fungi, or viruses. In other embodiments, the target nucleic acid is derived from a human. Furthermore, biological samples can be obtained from individuals suspected of being infected by microorganisms such as bacteria, fungi, or viruses. In some embodiments, the target nucleic acid can be an endogenous nucleic acid, such as a gene or transcript (RNA). In some embodiments, the target nucleic acid is a mutant form of a gene or transcript or a specific single nucleotide polymorphism (SNP).

[0049] The methods disclosed herein preferably use untreated biological samples (i.e., biological samples containing endogenous nucleic acids; and / or biological samples from which nucleic acids have not been extracted), resulting in a direct and simplified process from sample to answer. However, the detection methods disclosed herein are also effective if used on isolated nucleic acids (DNA and / or RNA) purified from biological samples according to any method known to those skilled in the art.

[0050] target nucleic acid

[0051] The target nucleic acid can be DNA (including genomic DNA) or RNA. Furthermore, the target nucleic acid can be any nucleic acid found in a microorganism or human host. DNA includes, for example, DNA derived from humans, bacterial species, fungi, and DNA viruses. Viral DNA suitable for evaluation includes DNA obtained directly from the viral capsid as well as DNA integrated into the host genome.

[0052] RNA types that can be used as target nucleic acid assays include rRNA, mRNA, transfer RNA (tRNA), or other RNA polynucleotides. rRNA types include 5S, 16S, and 23S polynucleotides, which may contain one or more sets of related bacteria-specific subsequences. The detection capability for characteristic sequences is variable and depends on the relevance level of the virus or bacteria to be detected using the assay. Other RNA polynucleotides can be used as target RNAs. Primers can be designed by those skilled in the art to guide the synthesis of copy DNA in a reverse transcription reaction using the target RNA as a template.

[0053] Those skilled in the art will also know how to design a pair of primers for amplifying the target DNA or target RNA in PCR using the copied DNA as a template. It is well known in the art that primers used simultaneously in PCR should have similar hybridization melting temperatures.

[0054] Reverse transcription and real-time PCR

[0055] Nucleic acid amplification

[0056] Target nucleic acids in biological samples can be amplified using various methods known to those skilled in the art. PCR is preferred for amplifying the target nucleic acid of interest. In this method, two or more oligonucleotide primers, joined or included in the opposite strand of the nucleic acid of interest and annealed thereto, are repeatedly annealed to their complementary sequences, extended by a DNA polymerase (e.g., AmpliTaqGold polymerase), and thermally denatured, resulting in exponential amplification of the target nucleic acid sequence. Cyclic parameters can be varied depending on the length of the nucleic acid to be extended. According to the present invention, those skilled in the art can design and prepare primers suitable for amplifying target sequences. The length of the amplification primers used in this invention depends on several factors, including nucleotide sequence identity and the temperature at which the nucleic acid hybridizes or is used during in vitro nucleic acid amplification. Considerations required to determine the preferred length of amplification primers for specific sequence identity are well known to those skilled in the art. For example, the length of a short nucleic acid or oligonucleotide can be related to its hybridization specificity or selectivity.

[0057] Methods that do not require a separation step before detecting the amplified nucleic acid products are often called real-time PCR or homogeneous detection. Most real-time methods detect the formation of amplified products by monitoring fluorescence changes during thermal cycling. These methods include, but are not limited to: dual labeled probes (Applied Biosystems, Foster City, Calif. 94404), Molecular Beacons (Tyagi S and Kramer FR (1996) Nat BiotechnoI14:303-308), Green dye (Molecular Probes, Inc. Eugene, Oreg. 97402-0469). Some of these same methods can also be used for endpoint detection of amplification products. An example of this type of method is... Greendye dissociation curve analysis. In dissociation curve analysis, the melting point can be detected by slowly increasing the temperature in the final stage combined with fluorescence monitoring, thereby detecting the presence of amplification products (Ririe et al., 1997, Anal. Biochem. 245:154-60).

[0058] In the method described herein, the presence of the target nucleic acid can be detected by reverse transcription (RT) and polymerase chain reaction (PCR). When reverse transcription and polymerase chain reaction are used together, they can be performed consecutively in two steps, or together in one step using all the reaction composition reagents added to the sample.

[0059] In the two-step method, the sample in the reverse transcription reaction composition is incubated to allow the synthesis of DNA copies from the target RNA. The reagent mixture contains primers that hybridize with the target RNA to initiate the synthesis of copy DNA. Additionally, the reagent mixture contains dNTPs, MgCl2, KCl, reverse transcriptase, and reverse transcriptase buffer. If DNA copies from more than one target RNA are required, the reagent mixture may contain more than one primer. The product of the reverse transcription reaction is then transferred to another assay tube in which PCR is performed according to a protocol well known in the art. The PCR composition typically includes a pair of primers that initiate the synthesis of the desired fragment of DNA from the reverse transcription template. Additionally, the PCR mixture typically contains dNTPs, MgCl2, KCl, a thermostable DNA polymerase such as Taq polymerase, and polymerase buffer. If the synthesis of multiple DNA fragments is desired, more than one pair of primers may be included. A new primer may also be added, which will act as a second primer in the pair with the original RT primers to amplify the DNA fragment. Other reverse transcriptases that can be used for viral samples include, but are not limited to: HIV reverse transcriptase (Ambion), Transcriptor reverse transcriptase (Roche), and Thermoscript reverse transcriptase (Invitrogen). Other DNA polymerases that can be used include, but are not limited to: Pfu, Vent, and Sequitherm DNA polymerase (EPICENTRE).

[0060] In one embodiment of the invention, a biological sample is combined with an RT-amplification mixture so that RT and PCR can be performed in a single assay.

[0061] Whether the RT-PCR is performed in two steps or one step, the RT step is run first and typically consists of a single-temperature incubation at a temperature between approximately 37°C and approximately 70°C. As is known to those skilled in the art, different temperatures are suitable for different RT enzymes and different primers. The subsequent PCR reaction typically consists of an initial incubation at approximately 94°C to approximately 97°C for approximately 2 to approximately 15 minutes. This step is used to denature the copy DNA and activate the heat-activated Taq polymerase. Multiple cycles of amplification of the copy DNA target are then performed. Three operations are performed during each cycle: target denaturation, primer annealing, and primer extension. Target denaturation typically occurs at temperatures above approximately 90°C. The primer annealing temperature is determined by the melting temperature of the specific primers used in the reaction, and primer extension is performed in a temperature range of approximately 50°C to approximately 72°C, depending on the thermostable polymerase used. When primer annealing and extension are performed at the same temperature, it is called two-temperature PCR; in contrast, in three-temperature PCR, each of the three steps occurs at a different temperature. After the amplification phase is completed, a final extension time is typically added to ensure the synthesis of all amplified products.

[0062] Without separate pre-sample preparation, the biological sample is directly loaded into the wells or chambers of a centrifugal microfluidic disc or a gene rotor disc chamber, followed by reverse transcription and real-time PCR amplification, as well as detection of the target nucleic acid (if present in the sample), all within the same disc. The sample may include an internal positive amplification control (IPC) using oligonucleotide primers and probes.

[0063] In some embodiments, the PCR is a multiplex PCR reaction. The integrated thermal cycler can heat at a rate of >5°C per second and cool at a rate of >4°C per second, and allows the cycling parameters to be varied according to the length of the amplification product to be extended.

[0064] Rotary platform technology

[0065] The method disclosed herein can be performed using any thermal cycler with a rotating platform capable of centrifuging or rotating the sample at high rotational speeds along a curved path around a central point. The rotational speed must be sufficient to move cell debris from the biological sample or the reagent-sample mixture to a portion of the sample chamber (preferably forming a precipitate), thereby isolating debris that could inhibit the emission and / or detection of detectable signals from the sample, interfere with or quench the polymerase chain reaction chemistry itself, and / or obstruct the optical path through which detectable signals propagate during real-time PCR assays. "High rotational speed" means a rotational speed greater than 140 g. Preferably, the relative centrifugal force is from about 140 × g to about 1500 × g.

[0066] In some implementations, the sample is contained in the chamber of a centrifugal microfluidic disk during amplification cycling. As used herein, a “centrifugal microfluidic disk” is a disk that rotates on its axis within a thermal cycler and includes a chamber where biological samples can be deposited. Exemplary centrifugal microfluidic disks are the Direct Amplification Disk (8 wells) from Focus Diagnostics and the conventional disk (96 wells), which are similar to those sold by 3M (St. Paul, MN, USA). TM Used together with the Integrated Cycler thermal cycler. The 3M... TMThe integrated cyclone can receive direct amplification discs and perform multiple assays on each disc. In some embodiments, the biological sample is deposited in the gene rotor disc. Preferably, the biological sample is a whole blood sample. As used herein, a "gene rotor disc" is a centrifuge rotor insert that maintains a tube or other chamber capable of containing the sample and / or sample amplification mixture at a uniform temperature. Examples of gene rotor discs are Qiagen Rotor Discs and / or Gene Discs used with the Qiagen Rotor-Gene Q thermal cycler.

[0067] In some embodiments, whole blood samples are deposited in a disc compartment (or well) separate from the disc compartment containing the reagent mixture. In this embodiment, the sample and reagent mixture can then be combined in the disc to allow amplification of target nucleic acids (if present). In some embodiments, the reagent mixture and sample are combined before deposition in a centrifugal microfluidic disc or gene rotor disc. In some embodiments, real-time PCR amplification and detection are performed in a direct amplification disc using SimplexaDirect assay and in a thermal cycler such as a 3M... TM This can be performed using an integrated cycler. Alternatively, PCR amplification and detection can be performed using Pall. Alternatively, the Gene POC diagnostic system can be used.

[0068] Rotational speeds achieved using centrifugal microfluidic discs and associated thermal cyclers, gene rotor discs and associated thermal cyclers, or any other thermal cycler with a rotating platform capable of producing such rotational speeds, can be used to precipitate cell debris from biological samples. The cell debris is isolated (preferably precipitated) to the periphery of the sample chamber. The cell debris from the biological sample includes, but is not limited to: lysed and intact red blood cells, fluorescence inhibitors, and reagents that can interfere with or quench the chemistry of the PCR reaction itself, or interfere with the optical pathways required to read fluorescence emission during real-time PCR assays.

[0069] To directly detect and amplify target nucleic acids in whole blood samples, it is necessary to increase the rotational speed or rotational rate of centrifugal microfluidic disks or gene rotor disks. The inventors have discovered that, under normal operating conditions, using 3M at the rotational speed set by the manufacturer... TM When using Integrated Cycler, successful real-time PCR amplification of whole blood patient samples is not permitted.

[0070] This invention relates to modifications of system parameters that allow the precipitation of said fragments to be used for successful nucleic acid amplification without extracting said nucleic acid from said whole blood sample. The invention also relates to rotation speeds during certain steps of said real-time PCR assays, optimized and modified to allow clearing of the optical path before determining real-time PCR fluorescence emission in each cycle. The rotation speeds used for each step in the real-time PCR process are shown below. Rotation speeds can be introduced at any stage of the PCR cycle parameters (Table 1). For rapid rotation, it can be performed throughout the run of each cycle, except before fluorescence reading (Table 1), or at any stage of the PCR cycle before fluorescence reading during said annealing / extension steps (Tables 2-4).

[0071] Table 1: Valving and Spinning Parameters

[0072]

[0073]

[0074] * Select the valve for the reaction mixture or sample before mixing. Prioritizing the valve can improve stable results, especially near the detection limit.

[0075] **The rotation speed setting can be introduced at various stages of the PCR cycle parameters.

[0076] Table 2: Final Definitions for the Assay of Bacillus anthracis

[0077]

[0078] Table 3: Definition of final viral hemorrhagic fever

[0079]

[0080] Table 4: Definitions of the final determination used for coagulation melting assay

[0081]

[0082] This invention is the first report of a real-time PCR system for direct PCR reactions capable of removing cell debris and inhibitors using high rotational speeds. The methods disclosed herein demonstrate, as shown in Example 3, improved detection and consistency using melt curve analysis with human DNA directly amplified from whole blood.

[0083] The high-speed rotation assay is performed by increasing the relative centrifugal force of a centrifugal microfluidic disk, such as a direct amplification disk, from approximately 140 × g to approximately 1500 × g. For centrifugal microfluidic disks or gene rotor disks with different radii, the rotation speed required to adequately precipitate / isolate cell debris may vary. Once the process is found to be advantageous, the rotation speed parameter can be further optimized on a per-assay basis. The method disclosed in this invention can directly amplify nucleic acids from any biological sample without additional extraction and purification steps. The software controls the rotation speed, allowing nucleic acids from different organisms to be separated and detected in whole blood samples. The duration of rotation varies with the rotation speed. Higher rotation speeds require shorter rotation cycles. Thus, rotation at 1500 × g results in complete separation of debris from the sample within two minutes. The rotation speed parameter can be further optimized on a per-assay basis. The inventors have successfully applied this method to directly amplify nucleic acids from whole blood for the detection of anthrax bacilli, viral hemorrhagic fever panels, dengue fever serological typing, and detection of single nucleotide polymorphisms (SNPs).

[0084] In some implementations, the 3M TM Integrated Cycler or equivalent instruments, used in conjunction with direct amplification disc consumables or equivalent consumables and direct chemistry, generate increased rotational speeds that allow for the direct detection of DNA and RNA from biological samples, such as whole blood samples. Examples of successful detection include, but are not limited to, DNA targets in the bacterial pathogen Bacillus anthracis, and RNA viruses Ebola and Marburg.

[0085] Detectable signal

[0086] In the method disclosed herein, the presence or absence of a target nucleic acid in a whole blood sample is determined by detecting the generation of a signal that amplifies the target nucleic acid (if present in the sample). The emission of a detectable signal during nucleic acid amplification is a marker of a real-time polymerase chain reaction. Therefore, in one embodiment, a real-time PCR reaction is performed on the sample, and if the target nucleic acid is present in the sample, its presence is detected by detecting a detectable tag.

[0087] A common method for real-time PCR is to use fluorescent probes, such as... Probes, molecular beacons, and scorpions. In some embodiments, the real-time PCR reaction includes the use of a quencher / donor probe detection system, such as... PCR detection system. As used herein, "quencher / donor probe detection system" refers to a method for real-time PCR, wherein the reagent master mixture contains a quencher / donor probe that hybridizes with the target nucleic acid to be amplified. Quencher / donor probe (e.g. The probe comprises a donor and a quencher fluorophore at either end of the probe, and are sufficiently close to each other such that the fluorescence of the donor is absorbed by the quencher. However, when the probe hybridizes with the amplified fragment, the 5'-3' exonuclease activity of the Taq polymerase cleaves the probe, thereby allowing the donor fluorophore to emit detectable fluorescence.

[0088] Compared to other forms of quantitative reverse transcriptase PCR that detect the amount of final amplified product, real-time PCR quantifies to the initial amount of the template with greater specificity, sensitivity, and reproducibility. Real-time RT-PCR cannot detect the size of the amplicons. The probe and molecular beacon technology used is based on the principle of fluorescence quenching, including a donor fluorophore and a quenching component.

[0089] In some embodiments, the detectable tag is a fluorophore. As used herein, the term "fluorophore" refers to a molecule that absorbs light of a specific wavelength (excitation frequency) and subsequently emits light of a longer wavelength (emission frequency). As used herein, the term "donor fluorophore" refers to a fluorophore that contributes or transfers emission energy to the quencher when it is very close to the quencher portion. As a result of contributing energy to the quencher portion, the donor fluorophore itself will emit light at a specific emission frequency, less than it would have if the nearby quencher portion were not present.

[0090] Primers and probes such as Scorpions, or Taqman and Hybeacon probes, can be used to perform the method according to the present invention. Other real-time PCR fluorescence amplification techniques can also be applied by those skilled in the art.

[0091] The probe can be detectably labeled using methods known in the art. Other useful labels include, for example, those that fluoresce in the red region of the visible spectrum and can be quenched by agents such as Black Hole Quencher. TM (BHQ TM Fluorescent dyes that are effectively quenched by BHQ-1, BHQ-2, and BHQ-3 include Cy5α, Cy3α, FITC, rhodamine, lanthanum phosphors, Texas Red, carboxyfluorescein fluorophores such as hydroxyfluorescein (FAM), JOE, and oxanthine dyes such as Cal Fluor Red. (“CFR610”) and Quasar The tag can be attached to an oligonucleotide probe that hybridizes with or binds to the target nucleic acid to be detected.

[0092] In some embodiments, a detectable signal from the sample is read while the sample is rotated in a thermal cycler. The detectable signal is read via a cleared optical path after the cell debris has been deposited or isolated, thereby reducing background fluorescence and undesirable amplification curve variations that could lead to misreading and / or masking of the amplification curve. See U.S. Patent Application Publications 2011 / 0117656 and 2012 / 0171677, the entire contents of which are incorporated herein by reference. In some embodiments, the detectable signal from the deposited sample is read shortly after the sample rotation has decreased. Preferably, high-speed rotation is performed only before thermal cycling, and the sample is rotated at a low speed (approximately 140 × g) during optimal reading to allow optical reading before high-speed rotation resumes. In an alternative embodiment, the sample is rotated at a high speed (1500 × g) throughout the assay.

[0093] Determination of sensitivity

[0094] The sensitivity of several amplification assays using untreated samples can be increased by adding one or more sensitivity-enhancing components to the buffer solution used in the assay. These components include, but are not limited to, KCl, surfactants, and albumin. In some embodiments, the albumin is bovine serum albumin. In some embodiments, the surfactant is a cationic surfactant. The sensitivity of the direct amplification assay can also be increased by providing additional heating, such as preheating the sample before adding the reagents. In some embodiments, the sensitivity can be increased by combining sensitivity-enhancing components with additional heating. Example

[0095] The methods described herein will be more readily understood by referring to the following examples, which are provided for illustrative purposes only and are not intended to limit the methods and kits of the present invention.

[0096] Example 1: Off-Board rotation test in whole blood

[0097] Off-board centrifugation experiments were performed using whole blood samples. The samples were centrifuged at 900 × g for 10 minutes at 23°C. Results showed that centrifugation using pre-spinned blood samples reduced background fluorescence and undesirable changes in the amplification curve, while no reduction in background fluorescence was observed in blood samples without pre-spinning. Figure 1 ).

[0098] Example 2: The effect of high-speed rotation of a centrifugal microfluidic disk on the detection of viral RNA or bacterial DNA in whole blood.

[0099] Increasing the rotational speed or rotation rate of the centrifugal microfluidic disk on the 3M Integrated Cycler system yielded unexpected results. Using specially designed software, the rotational speed or rotation rate was controlled within the range of approximately 140 × g to approximately 1500 × g. As shown below, the rotational speed parameter was further optimized on a per-assay basis. As shown below, this method has been successfully applied to the detection of Bacillus anthracis, viral hemorrhagic fever virus panels, and dengue serotyping assays by directly amplifying nucleic acids from whole blood.

[0100] A. Blood contaminated with dengue virus

[0101] Inactivated dengue virus was diluted in whole blood or PBS buffer. For the same sample, one assay run was a standardized method of rotating the sample at approximately 1500 rpm (approximately 140 × g) throughout the assay, and another assay run was a method of rotating the sample at approximately 5000 rpm (1500 × g) throughout the assay, except for an optical readout step in which the sample was rotated at the standard speed of approximately 1500 rpm (approximately 140 × g).

[0102] Table 5: Effect of rotation speed on the Ct value of the dengue virus target

[0103]

[0104] B. Anthrax

[0105] Anthrax bacteria were diluted in whole blood. For the same sample, one assay run involved rotating the sample at 780 × g for 6 minutes (slower rotation) at the start of the run, while another assay run involved rotating the sample at approximately 5000 rpm (1500 × g) for 2 minutes (higher rotation) at the start of the run. In both cases, the sample was rotated at a standard speed of approximately 1500 rpm (approximately 140 × g) for the remainder of the run. Figure 3 The effect of initial rotation speed on the optimization of fluorescence intensity and Ct value for anthrax detection is shown.

[0106] C. Viral hemorrhagic fever Panel

[0107] Dilute inactivated Ebola Reston virus in whole blood or PBS buffer. Run the same sample using reagents capable of detecting Ebola and Marburg viruses and distinguishing between Ebola Reston and Ebola Zaire viruses. Compare the results to runs defined as follows: one run is defined as a standardized method of rotating the sample at approximately 1500 rpm (approximately 140 × g) throughout the assay; the other run is defined as rotating the sample at approximately 5000 rpm (1500 × g) throughout the assay, except for an optical readout step where the sample is rotated at the standard speed of approximately 1500 rpm (approximately 140 × g). Figure 4 The amplification curve for detecting viral hemorrhagic fever from the Ebola Reston virus is shown using a high rotation speed of 5000 rpm.

[0108] The above results demonstrate that nucleic acids can be successfully amplified and detected directly from whole blood samples of anthrax bacilli, viral hemorrhagic fever panels, and dengue fever serotypes.

[0109] Example 3: Effects of high-speed disk rotation on amplification and melt analysis of human DNA in whole blood.

[0110] Molecular testing was performed on mutations in genes responsible for coagulation associated with thrombosis (such as factor V, factor II, and MTHFR genes) to determine the effect of high-speed rotation on the detection and amplification of human DNA in whole blood.

[0111] Hybeacon, which includes primers and single nucleotide polymorphisms for the methylenetetrahydrofolate reductase (MTHFR) gene, was used. TM The probe reaction mixture was prepared in two identical standard pans. The reaction consisted of 8 μL of the reaction mixture plus 2 μL of whole blood or buffer as a sample, or 9 μL of the reaction mixture plus 1 μL of whole blood or buffer. The two pans were run with independent parameters: one performed the standard PCR followed by a melt analysis procedure, while the other performed the standard procedure but with a high-speed rotation step added between the PCR cycle and the melt analysis step. Figure 5 As shown, the results indicate that the high-speed rotation increased the area under the melting point curve values ​​in most blood samples, thereby increasing the difference between positive and template-free samples. In a similar experiment observing the detection of MTHFR1298 heterozygotes from whole blood patient samples, the introduction of high rotation led to a sharper separation of the heterozygote assay compared to the standard speed. Figure 6 ).

[0112] These results demonstrate improved detection and amplification of nucleic acids from any microorganism (including, but not limited to, bacteria, viruses, and fungi) and human nucleic acids in whole blood. These results also show that high-speed centrifugation of whole blood samples reduces background fluorescence, eliminates cell debris such as lysed and intact red blood cells, reduces fluorescence interference and quenching, and prevents undesirable amplification curve changes that lead to misreading and / or masking of amplification curves.

[0113] All articles, patents, patent applications, and all other documents and electronic information mentioned or cited herein are incorporated herein by reference in their entirety as if each individual publication were incorporated separately by reference. The applicant reserves the right to physically incorporate any and all materials and information from any such articles, patents, patent applications, or other physical and electronic documents into this application.

[0114] The invention described illustratively herein can be suitably practiced in the absence of any one or more elements, any one or more limitations, the omission of which is not specifically disclosed herein. Furthermore, the terminology and expressions used herein are descriptive rather than limiting, and their use is not intended to exclude any equivalents of the features and portions thereof shown and described, but rather to acknowledge that various modifications are possible within the scope of the invention. Therefore, it should be understood that while the invention has been specifically disclosed through preferred embodiments and optimal features, modifications, alterations, and variations of the invention disclosed herein embodied are possible for those skilled in the art and are considered to be within the scope of the invention.

[0115] This document describes the invention broadly and generally. Each narrower group of categories and subcategories falling within the general disclosure also forms part of the invention. This includes the general description of the invention, as well as any accompanying conditions or negative limitations removing any subject matter from that category, regardless of whether the excised material is specifically described herein. Other embodiments are shown in the appended claims. Furthermore, when features or aspects of the invention are described in the Markush group, those skilled in the art will recognize that the invention can also be described in any single member or subgroup of the Markush group.

[0116] This invention also includes the following:

[0117] Item 1. A method for identifying the presence or absence of a target nucleic acid in a biological sample, the method comprising:

[0118] (a) Rotate biological samples, including whole blood, at a rotational speed sufficient to precipitate cell debris and fluorescence inhibitors present in the sample, in order to reduce fluorescence interference or quenching in the sample; and

[0119] (b) Direct amplification and detection of target nucleic acids in samples.

[0120] Item 2. The method according to Item 1, wherein the rotational speed is in the range of greater than 140×g to 1500×g.

[0121] Item 3. The method according to Item 1, wherein the biological sample is placed in a microfluidic disk capable of withstanding centrifugal force.

[0122] Item 4. The method according to Item 1, wherein the detection step includes detecting a visible signal emitted through an optical path in the precipitated sample.

[0123] Item 5. The method according to Item 1, wherein the detectable signal is fluorescence emission.

[0124] Item 6. The method according to Item 1, wherein the amplification step includes real-time polymerase chain reaction analysis.

[0125] Item 7. The method according to Item 1, wherein the target nucleic acid is DNA.

[0126] Item 8. The method according to Item 1, wherein the target nucleic acid is RNA.

[0127] Item 9. The method according to Item 1, wherein the biological sample is whole blood.

[0128] Item 10. The method according to Item 1, wherein the target nucleic acid is derived from one or more organisms found in blood.

[0129] Item 11. The method according to Item 10, wherein the target nucleic acid is human nucleic acid.

[0130] Item 12. The method according to Item 10, wherein the target nucleic acid is derived from microorganisms.

[0131] Item 13. The method according to Item 12, wherein the microorganism is a virus.

[0132] Item 14. The method according to Item 13, wherein the virus is one or more selected from Ebola virus, Marburg virus, influenza virus, respiratory syncytial virus, varicella-zoster virus, herpes simplex virus, enterovirus, dengue virus, or any combination thereof.

[0133] Item 15. The method according to item 12, wherein the microorganism is bacteria.

[0134] Item 16. The method according to Item 15, wherein the bacteria are Gram-negative or Gram-positive bacteria.

[0135] Item 17. The method according to Item 16, wherein the bacteria is selected from one or more of Bacillus, Bordetella, Leptospira, Listeria, Escherichia coli, Salmonella, Campylobacter, Clostridium, Helicobacter pylori, Mycobacterium, Staphylococcus, Campylobacter, Enterococcus, Neisseria, Shigella, Streptococcus, Vibrio, Yersinia, and Pseudomonas, or any combination thereof.

[0136] Item 18. The method according to Item 17, wherein the bacteria is Bacillus anthracis.

[0137] Item 19. The method according to Item 12, wherein the microorganism is a fungus.

[0138] Item 20. The method of Item 1, wherein the cell debris comprises lysed and intact red blood cells, as well as denatured proteins, which can physically block or quench fluorescence.

Claims

1. An in vitro method of amplifying a target nucleic acid in a biological sample for non-diagnostic purposes, the method comprising: (a) directly amplifying a target nucleic acid in a sample using a real-time polymerase chain reaction (PCR) assay, wherein the target nucleic acid is not extracted from the sample prior to amplification; and (b) rotating the sample during one or more steps of the real-time PCR assay at a rotational speed in the range of greater than 140 x g and less than or equal to 1500 x g, wherein the rotation is performed in a thermal cycler, wherein the sample is placed in a microfluidic disc capable of withstanding centrifugal forces and generating a rotational speed, the microfluidic disc is rotated on a rotational platform in the thermal cycler, wherein the rotational speed is sufficient to precipitate cellular debris and fluorescence inhibitors present in the sample to reduce fluorescence interference or quenching in the sample, wherein the biological sample is whole blood.

2. The method of claim 1, wherein the step of amplifying comprises a real-time polymerase chain reaction analysis.

3. The method of claim 1, wherein the target nucleic acid is DNA.

4. The method of claim 1, wherein the target nucleic acid is RNA.

5. The method of claim 1, wherein the target nucleic acid is from one or more organisms found in blood.

6. The method of claim 5, wherein the target nucleic acid is a human nucleic acid.

7. The method of claim 5, wherein the target nucleic acid is from a microorganism.

8. The method of claim 7, wherein the microorganism is a virus.

9. The method of claim 7, wherein the microorganism is a bacterium.

10. The method of claim 9, wherein the bacterium is a gram-negative bacterium or a gram-positive bacterium.

11. The method of claim 7, wherein the microorganism is a fungus.

12. The method of claim 1, wherein cellular debris includes lysed red blood cells and intact red blood cells and denatured proteins that can physically block or quench fluorescence.

13. The method of claim 1, wherein the biological sample is rotated during one or more steps of the real-time PCR assay at a rotational speed in the range of greater than 780 x g and less than or equal to 1500 x g.

14. The method of claim 1, wherein the biological sample is rotated during one or more steps of the real-time PCR assay at a rotational speed in the range of greater than 900 x g and less than or equal to 1500 x g. ​

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