WO2018157844A1 - 一种利用磁珠提取核酸物质的方法以及试剂 - Google Patents

一种利用磁珠提取核酸物质的方法以及试剂 Download PDF

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WO2018157844A1
WO2018157844A1 PCT/CN2018/077797 CN2018077797W WO2018157844A1 WO 2018157844 A1 WO2018157844 A1 WO 2018157844A1 CN 2018077797 W CN2018077797 W CN 2018077797W WO 2018157844 A1 WO2018157844 A1 WO 2018157844A1
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magnetic beads
nucleic acid
sample
amplification
reagent
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French (fr)
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杨尚鑫
胡彬
刘杰
蔡媛媛
陶施芳
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绍兴迅敏康生物科技有限公司
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    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
    • C12Q1/689—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for bacteria
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    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/70—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving virus or bacteriophage
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    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/70—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving virus or bacteriophage
    • C12Q1/701—Specific hybridization probes
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • C12N15/1003—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor
    • C12N15/1006—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor by means of a solid support carrier, e.g. particles, polymers
    • C12N15/1013—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor by means of a solid support carrier, e.g. particles, polymers by using magnetic beads
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    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
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    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6844—Nucleic acid amplification reactions
    • C12Q1/6851—Quantitative amplification
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    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6844—Nucleic acid amplification reactions
    • C12Q1/686—Polymerase chain reaction [PCR]
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    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/16—Primer sets for multiplex assays

Definitions

  • the invention belongs to the technical field of molecular diagnostic biology, and particularly relates to a method for directly extracting biological samples by using magnetic beads for calculation, thereby directly contacting the amplified reagent nuclear magnetic beads, thereby completing amplification of the target target nucleic acid.
  • the real-time PCR technique was introduced in 1996 by Applied Biosystems, Inc., which added a fluorescent group to the PCR reaction system to monitor the entire PCR process in real time using fluorescence signal accumulation. Finally, the unknown template was quantitatively analyzed by a standard curve. .
  • pharyngeal isthmus culture In normal human pharyngeal isthmus culture, there should be normal oral flora, and no pathogenic bacteria grow.
  • the bacteria in the pharynx are from the outside world and do not cause disease under normal conditions. However, infection may occur due to decreased body or local resistance and other external factors. Therefore, bacterial culture of pharyngeal swabs can isolate pathogenic bacteria and contribute to the diagnosis of diphtheria, suppurative tonsillitis, acute pharyngitis and the like.
  • Throat swab detection is a medical test method that uses a medical cotton swab to take a small amount of secretions from the pharynx of the human body, inoculate it in a special culture dish, and then place it in a temperature-controlled device for cultivation.
  • Fluorescence PCR technology is often used to detect the patient's condition, oral mucosa and pharyngeal infection, but the nucleic acid nucleic acid needs to be extracted. The PCR takes a long time and the cost is high; the sample cannot be processed in large quantities, and the operation detects the risk of contamination.
  • the sample Whether it is the throat area or other samples, such as saliva, urine, and nasal swab samples, the sample contains viruses, bacteria, or fungi, or other biological cellular components, which require nucleic acid for nucleic acid detection.
  • the extraction process is to release the nucleic acid and remove the interfering substances in the tissue or cells, so that the subsequent effective amplification of the target nucleic acid can be performed.
  • magnetic beads to extract nucleic acids in a sample is a relatively reliable method, and can be automated, but the existing magnetic bead extraction method still appears to be more complicated, generally after magnetic beads are used to adsorb DNA or RNA.
  • the magnetic beads need to be cleaned, and then the nucleic acid material is eluted from the magnetic beads to amplify or remove impurities adsorbed on the magnetic beads, such as proteins, polysaccharides, other nucleic acids other than the target nucleic acid template, and Some other particulate components, although the magnetic beads are chemically modified, can adsorb nucleic acids, but the adsorption of magnetic beads is not selective, chemical and physical adsorption are also available.
  • the object of the present invention is to provide a method and a reagent for extracting nucleic acid substances by using magnetic beads, to overcome the deficiencies of the existing detection techniques, and to provide a rapid sample for throat swabs, saliva, urine, nasal swabs and serum.
  • Simple, efficient and practical detection method not requiring nucleic acid extraction process, simplifying the operation process, saving time and cost, and processing samples in large quantities, preventing contamination during operation detection.
  • a method and a reagent for extracting a nucleic acid substance by using magnetic beads comprising the following steps:
  • the cleavage reagent comprises a metal compound
  • the magnetic beads of the step (3) are directly contacted with the reagent for nucleic acid amplification, and a nucleic acid template is provided for nucleic acid amplification to perform amplification of the target nucleic acid.
  • the lysing reagent and the magnetic beads are a mixture, preferably a solution mixture.
  • the metal compound comprises one or both of NaCl and KCl.
  • the magnetic beads are monodisperse hydroxyl or carboxyl magnetic beads.
  • the lysing reagent is in the form of a lysis reagent and a nano magnetic bead solution, and the composition of the solution comprises: 0.1-2 M NaCl and 0-2 mM KCl, or a surface active agent of 0-0.3% by mass. , wherein the concentration of the magnetic beads is 0.00005 mg / ml.
  • the volume ratio of the sample to the lysate is 1:3 - 1:10.
  • the volume ratio of the magnetic beads to the lysate is 1:200, wherein the concentration of the magnetic beads is 0.1-100 mg/ML.
  • the sample comprises one or more of a throat swab, a nasal swab, a throat swab, serum, saliva, sputum, urine, serum.
  • the sample is a processed sample.
  • the carrier of the throat swab, nasal swab or throat swab sample is subjected to physiological saline, buffer eluted sample.
  • the lysis reagent and the magnetic beads are in a solution state and are located in a PCR tube, or the lysis reagent and the magnetic beads are in a dry state and are located in a PCR tube for PCR amplification.
  • the mixture is provided prior to contacting the sample with the mixture, the hydroxyl magnetic beads being included in the mixture.
  • the magnetic beads Preferably, wherein the magnetic beads have an average particle diameter of less than 1000 nm.
  • the sample or the magnetic beads and the lysate mixed with the sample are subjected to high temperature treatment at 60 ° C - 100 ° C, or the low temperature is room temperature or 20 Between °C and 30 °C.
  • the magnetic beads in the step (3) are simultaneously contacted with the reverse transcriptase and the nucleic acid amplification reagent for amplification.
  • the sample is simultaneously contacted with the lysis reagent and the magnetic beads.
  • the present invention provides an agent for extracting a nucleic acid, comprising a solution comprising 1 M NaCl, a solution having a mass ratio of 0.01% T surface activity and 0.001 MKCl, and 0.005 mg of magnetic beads.
  • the magnetic beads of the root are hydroxyl modified magnetic beads, or the magnetic beads have a dispersion coefficient of less than 5.
  • the invention provides an agent for extracting nucleic acid, comprising a cleavage component and a magnetic bead, the cleavage component comprising 1M NaCl, 0.01% Triton-X, 0.001MKCl and 0.005 mg of magnetic beads;
  • the average particle size is ⁇ 1000 nm.
  • the present invention provides an agent for extracting a nucleic acid, comprising a lysis solution comprising 0.00 M NaCl, a mass ratio of 0.01% T surface activity, and 0.001 MKCl, and a 0.005 mg magnetic bead.
  • the present invention provides the use of a nanomagnetic bead comprising a hydroxyl group as a nucleic acid extraction reagent, wherein the nanomagnetic bead has an average particle diameter of ⁇ 1000 nm.
  • the invention provides a method of nucleic acid extraction of a sample, the method comprising:
  • the sample is added to a PCR amplification tube containing a lysate, shaken and mixed, and then treated at a high temperature of 80-100 ° C for 10 min, and then allowed to stand at room temperature for 10 min;
  • step (3) adding magnetic beads to the PCR amplification tube in step (2) and reacting at 18-28 ° C for 5-10 min;
  • the PCR amplification tube is instantaneously centrifuged and placed in a magnetic frame. After the magnetic beads are adsorbed to one side of the magnetic frame, the supernatant is aspirated, and the magnetic beads are retained, and then the magnetic beads are not washed or washed. ;
  • the component of the lysate in the step (1) is 0-2M NaCl, 0-0.3% Triton-X and 0-0.2 mM KCl.
  • the high temperature in the step (2) is 100 °C.
  • the volume ratio of the sample to the lysate in the step (2) is 1:3 - 1:10.
  • the magnetic beads are commercially available nucleic acid extraction hydroxy magnetic beads, that is, a superparamagnetic core and an outer shell of inorganic silicon oxide, preferably monodisperse hydroxy magnetic beads having an average particle diameter of ⁇ 1000 nm.
  • reaction steps are carried out in the same tube.
  • the method of the invention can be manually operated as well as mechanically automated, and does not require a nucleic acid extraction process, which simplifies the operation process, saving time and cost.
  • 1 is a diagram showing the results of real-time quantitative PCR detection of the method of the present invention applied to a clinical sample of B flow;
  • FIG. 2 is a graph showing the sensitivity results of the method of the present invention for detecting a positive B-pharynx swab sample
  • Figure 3 is a graph showing the reproducibility of a positive B-pharyngeal swab sample of the method of the present invention.
  • Figure 4 is a graph showing the results of detecting RSV by the method of the present invention of Example 7;
  • Fig. 5 is a graph showing the results of detecting RSV by the RSV nucleic acid detection kit (including nucleic acid extraction reagent) of Guangdong Huayin Pharmaceutical Technology Co., Ltd. of Example 7.
  • the sample comprises a biological sample, such as a sample obtained from a plant or animal individual.
  • a biological sample as used herein includes all clinical samples that can be used to detect nucleic acids in an individual, including but not limited to cells, tissues (eg, lung, liver, and kidney tissue), bone marrow aspirate, body fluids (eg, blood, blood) Derivatives and blood fractions (eg serum or yellow layer), urine, lymph, tears, prostatic fluid, cerebrospinal fluid, tracheal aspirate, sputum, pus, nasopharyngeal aspirate, oropharyngeal aspirate, saliva), eye swabs, Neck swabs, vaginal swabs, rectal swabs, stools and stool suspensions.
  • suitable samples include those obtained from middle ear fluid, bronchoalveolar lavage fluid, tracheal aspirate, sputum, nasopharyngeal aspirates, oropharyngeal aspirate or saliva.
  • the biological sample is obtained from an individual animal. Standard techniques for obtaining the sample are available. See, for example, Schluger et al., J. Exp. Med. 176: 1327-33 (1992); Bigby et al., American Respiratory Diseases Review (Am.
  • the sample includes an environmental sample, such as a surface sample (eg, obtained by wiping or vacuum processing), an air sample, or a water sample.
  • the sample includes isolated cells, such as animals, bacteria, fungi (eg, yeast), or plant cells and/or viruses. The isolated cells can be cultured using conventional methods and conditions suitable for the type of cell cultured.
  • the sample is typically sampled using a carrier, such as a water-absorbing carrier for carrying the sample.
  • a carrier such as a water-absorbing carrier for carrying the sample.
  • the carrying sample may be a straw, a storage device, a sampling device, and the components of the sampling device may be a filter paper, a cotton swab, or the like, to absorb and carry the carrier.
  • Contact with the lysate of the present invention may be a carrier or a sample mixture washed from the carrier.
  • the lysing liquid is contacted prior to the carrier carrying the sample to lyse and elute the sample, and then the lysing liquid is mixed with the magnetic beads, or the mixture of lysing liquid and magnetic beads is contacted with the carrier carrying the sample to complete the cleavage.
  • the lysate and magnetic bead solution can also be prepared in advance, and then, when used, the two solutions are mixed, and then directly contacted with the sample to contact the carrier carrying the sample, thereby completing the sample processing.
  • the cleavage component and the magnetic beads may be in a dry form, after contact with the liquid sample, the sample is then lysed or adsorbed by the magnetic beads to complete the release and adsorption of the nucleic acid.
  • the lysing liquid here may be a ionic solution, or a mixture of an ionic solution and a surface active agent, which may cause destruction of the cell wall or cell membrane to achieve release of nucleic acid (DNA or RNA).
  • physical heating may be employed, such as high temperature heating, for example, a problem of 60 degrees or more. The higher the temperature, the shorter the time, and the lower the temperature, the longer it takes.
  • the lysis reagent contains enough components to damage the cell wall or cell membrane, such as strong acid, strong base, or some enzyme reagents, these components can directly achieve the purpose of releasing the nucleic acid material, and can be contacted without physical heating or ultrasonication.
  • Physical methods are generally heating, grinding, sonication, and chemical methods are generally chemical reagent components.
  • the present invention employs a small amount of chemical reagents in conjunction with physical methods to achieve release of the nucleic acid material and adsorption of the magnetic beads. This prevents impurities from interfering with subsequent direct amplification.
  • the release of the nucleic acid is carried out using a chemical reagent, although the release of some non-nucleic material, such as proteins, polysaccharides, which may interfere with the amplification of the nucleic acid, is carried out using the magnetic beads of the present invention.
  • the adsorption of nucleic acids, even with impurities, can still perform normal nucleic acid amplification.
  • the nucleic acid is RNA.
  • amplification of RNA first requires a reverse transcription process followed by amplification of the reverse transcribed DNA.
  • the present inventors have found that when RNA is released, the method of the present invention is used to directly adsorb RNA by using magnetic beads, and then the lysate is removed, the magnetic beads are retained, and the magnetic beads are not washed or washed directly.
  • the reverse transcription of the C-NDA is contacted with the amplification reagent to complete the amplification.
  • the present invention utilizes a mixture of lysate and magnetic beads to cleave RNA, then removes the lysate and other materials, retains only the magnetic beads, and then contacts the magnetic beads with the reverse transcriptase and the amplification reagent. Enlarged. With such a method, amplification or the result of amplification can be efficiently performed.
  • the lysis reagent and the magnetic beads are contacted with the sample, subjected to a high temperature heat treatment, and then kept at a low temperature for a period of time.
  • the sample is subjected to a high temperature treatment, and then the lysis reagent is contacted with the magnetic beads and then kept at a low temperature for a while.
  • high temperature treatment is not necessary.
  • the lysis reagent contains a high concentration of ions, the cell membrane or the cell wall can be destroyed. In this case, high temperature treatment can be omitted.
  • target nucleic acids are to distinguish between organisms and to identify other biological samples.
  • a virus has a unique educational status, amplification and detection of too specific sequences, thereby confirming the particular virus without causing missed detection and false positives or false negatives.
  • the target nucleic acid is specific to the target organism, that is, the target nucleic acid is not found in other organisms or the target nucleic acid is not found in an organism similar to the target organism.
  • the target nucleic acid can be a nucleic acid that is present in an animal (eg, a human), a plant, a fungus (eg, a yeast), a protozoa, a bacterium, or a viral substance, or a nucleic acid material from the above sample.
  • the target nucleic acid can be present in the genome of the target organism (eg, on a chromosome) or on an extrachromosomal nucleic acid.
  • the target nucleic acid is an RNA, such as an mRNA.
  • the target nucleic acid can be present in a bacterium (eg, a Gram-positive or Gram-negative bacterium). Exemplary bacterial species include Acinetobacter sp.
  • strain ATCC 14396 Moraxella catarrhalis, Mycobacterium kansii ( Mycobacterium kansasii), Mycobacterium gordonae, Mycobacterium fortuitum, Mycoplasma pneumoniae, Mycoplasma hominis, Neisseria meningitis (eg, ATCC 6250), Neisseria gonorrhoeae O.
  • the target nucleic acid is present in a genus of bacteria selected from the group consisting of Acinetobacter, Aerococcus, Bacteroides, Bordetella, Campylobacter ( Campylobacter), Clostridium, Corynebacterium, Chlamydia, Citrobacter, Enterobacter, Enterococcus, Escherichia Genus (Escherichia), Helicobacter, Haemophilus, Klebsiella, Legionella, Listeria, Micrococcus Micrococcus), Mobilincus, Moraxella, Mycobacterium, Mycoplasma, Neisseria, Oligella, Pasteurella, Pu Prevotella, Porphyromonas, Pseudomonas, Propionibacterium, Proteus, Salmonella, Serratia Serratia, Portuguese Staphylococcus, Streptococcus, Treponema, Bacillus, Francisella or Yersinia.
  • Acinetobacter Aerococc
  • Exemplary Chlamydia target nucleic acids include sequences found on Chlamydia recessive plasmids.
  • Exemplary M. tuberculosis target nucleic acids are included in IS6110 (see US 5,731,150) and/or IS1081 (see Bahador et al., 2005, Journal of Agricultural Biosciences (Res. J. Agr. The sequence found in Biol. Sci.), 1:142-145).
  • Exemplary Neisseria gonorrhoeae target nucleic acids are included in NGO0469 (see Piekarowicz et al, 2007, BMC Microbiol. 7:66) and NGO0470.
  • Exemplary Group A Streptococcus target nucleic acids are included in Spy 1258 (see Liu et al., 2005, Res. Microbiol, 156: 564-567), Spy0193, lytA, psaA, and ply (see US 2010/0234245). The sequence found in ).
  • Exemplary Group B Streptococcus target nucleic acids are found in the cfb gene (see Podbielski et al., 1994, Med. Microbiol. Immunol., 183:239-256). the sequence of.
  • the target nucleic acid is a viral nucleic acid.
  • viral nucleic acids can be found in human immunodeficiency virus (HIV), influenza virus or dengue virus.
  • HIV target nucleic acids include sequences found in the Pol region.
  • the target nucleic acid is a protozoan nucleic acid.
  • it can be in the genus Plasmodium spp., Leishmania spp., Trypanosoma brucei gambiense, Trypanosoma brucei rhodesiense, Krebs Protozoan nucleic acids are found in Trypanosoma cruzi, Entamoeba spp., Toxoplasma spp., Trichomonas vaginalis, and Giardia duodenalis.
  • the target nucleic acid is a mammalian (eg, human) nucleic acid.
  • mammalian nucleic acids can be found in circulating tumor cells, epithelial cells, or fibroblasts.
  • the target nucleic acid is a fungal (eg, yeast) nucleic acid.
  • fungal nucleic acids can be found in Candida spp. (e.g., Candida albicans).
  • the target nucleic acid herein may be one end sequence on DNA, and may also be a sequence specific for one end of RAN reverse transcription.
  • the magnetic beads provided by the present invention may be any magnetic beads.
  • magnetic beads are classified according to different classifications.
  • Magnetic beads are a kind of nanoparticles, and chemical groups are generally attached to the surface of the particles, thus forming magnetic beads of the final product.
  • Such magnetic beads can be purchased commercially, for example from Xiamen Puruimai Biotech Co., Ltd. (http://www.purimagbead.com).
  • the magnetic beads are classified according to the diameter of the magnetic beads, and the other is classified according to the chemical groups encapsulated on the surface.
  • the size of the diameter it can be divided into average particle size less than 50 sodium, 60 sodium, 80 sodium, 90 sodium, 100 sodium, 150 sodium, 200 sodium, 300 sodium, 500 sodium, 600 sodium Rice, 700 sodium, 800 sodium, 900 sodium, 1000 sodium, 1500 sodium, 2000 sodium and other magnetic beads.
  • the chemical groups encapsulated by the magnetic beads may be classified into hydroxy magnetic beads, carboxyl groups or amino magnetic beads, or magnetic beads coated with mixed groups, depending on the nature of the chemical groups.
  • the magnetic beads or magnetic bead shells coated with a hydroxyl polymer are surface-modified with a large amount of silanol groups (hydroxyl groups).
  • the classification according to the shape environment of the magnetic beads may be a monodisperse magnetic bead or a non-monodisperse magnetic bead.
  • one or a combination of a hydroxyl group, a carboxyl group or an amino group is used to modify the surface of the magnetic beads to form a hydroxyl magnetic bead, a carboxyl magnetic bead or an amino magnetic bead, or a magnetic bead formed by modifying any two combinations.
  • the magnetic beads are hydroxy magnetic beads or carboxyl magnetic beads having a large amount of silanol groups (hydroxyl groups) modified on the surface of the magnetic bead shell.
  • the magnetic beads used have a uniform particle size or a polydispersity coefficient of ⁇ 0.2.
  • the monodisperse magnetic beads are selected such that they are superparamagnetic and have a magnetic response time of ⁇ 30 s.
  • the so-called "uniform" particle size does not necessarily mean that the particle size before each magnetic bead is the same, but in the same solution, at least 30% of the magnetic beads have a particle size close to or 35% of the magnetic beads.
  • Magnetic beads of different particle sizes are mixed, for example, magnetic beads of less than 10 nanometers and less than 50 nanometers, or magnetic beads of less than 50 nanometers and less than 100 nanometers, or less than 50 nanometers and less than 500 nanometers.
  • the magnetic beads are mixed, and the magnetic beads of any other particle size nanoparticles are mixed for nucleic acid adsorption.
  • Polydispersity is an important concept in polymer science, sometimes called molecular weight distribution coefficient, non-uniform index, dispersion and so on.
  • Mw/Mn weight average, number average molecular weight, respectively.
  • This definition measures the width of the molecular weight distribution. It is sometimes expressed as Lw/Ln (where Lw and Ln are weight average and number average contour lengths respectively), Dw/Dn (where Dw and Dn are weight average and number average particle diameter, respectively); the former represents the length of the polymer profile
  • Polydispersity indicates the polydispersity of the polymer diameter.
  • the dispersion coefficient referred to in the present invention refers to the latter, and the main index average particle diameter. It also indicates the uniformity of the ion diameter. Generally, the dispersion coefficient is ⁇ 0.2, or it may be less than 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, or 1, less than 1.5, and less than 2.
  • magnetic beads are extracted by magnetic beads, and any sample needs to be lysed by cells, tissues and organs containing nucleic acids in the sample, and the cleavage function is to release the nucleic acid substances, and then use magnetic beads for adsorption. After adsorption, it is generally divided into two paths. One is to elute the nucleic acid substance from the magnetic beads with a solution that dissolves the nucleic acid (for example, water or other reagents), and then add the solution containing the nucleic acid substance to the amplified nucleic acid. The necessary reagents are used for amplification.
  • a solution that dissolves the nucleic acid for example, water or other reagents
  • the other is to separate the magnetic beads from the lysis solution, then wash the magnetic beads, remove the washing solution, and finally add the magnetic beads to the nucleic acid amplification reagent for amplification (for example, China).
  • the method disclosed in the invention patent application 200610022581 but the patent is only for serum samples, and the magnetic beads are required to be cleaned before amplification).
  • the prior art is transferred to China.
  • the washing process may be to dissolve the nucleic acid on the magnetic beads, or to remove the non-nucleic acid substances adsorbed by the magnetic beads, thereby reducing The effect of impurities on amplification.
  • the present invention has been experimentally found that it is possible to directly add the magnetic beads separated from the lysate to the nucleic acid amplification reagent or reverse transcriptase for reverse transcription without eluting or washing the nucleic acid in any step.
  • the amplification of the nucleic acid can occur, thereby simplifying the steps of nucleic acid extraction, and realizing the extraction and detection of fully automated nucleic acids.
  • the term "washing or washing” as used herein means the same meaning, that is, after the magnetic beads adsorb the nucleic acid, the magnetic beads are subjected to secondary treatment to remove impurities adsorbed on the surface or to dissolve the nucleic acid substance adsorbed on the surface of the magnetic beads.
  • Magnetic beads adsorption is generally not selective for nucleic acids. As long as it is a nucleic acid, it will adsorb to the surface of the magnetic beads. After adsorption, the prior art elution or washing is to remove. Proteins, polysaccharides, phenolics, or templates that remove other non-target nucleic acids. For example, in order to detect influenza virus, but the sample may contain other viruses or bacteria, such as StrepA, RSV, and the like. The DNA or RNA of these viruses or bacteria will also be adsorbed on the magnetic beads.
  • the specificity and sensitivity of the primers need to be considered when designing the amplification primers, if the target template is low in content or insufficient in purity, it will affect the later expansion of the nucleic acid. Increases, especially in the presence of other non-target nucleic acid templates, can also affect later amplification.
  • RNA nucleic acid the traditional nucleic acid needs to be purified and extracted in multiple steps. Because RNA is relatively easy to degrade, it is necessary to increase the protective reagent for RNA protection and prevent degradation during reverse transcription. The extraction process is cumbersome, the steps are consumable, and the effect is not good, because the longer the extraction cycle, the need to protect the RNA for RNA protection, and the final effect of the extraction is not good.
  • the present invention directly uses magnetic beads for adsorption, and unexpectedly discovers that after directly contacting the magnetic beads and the reverse transcriptase, the reverse transcription reagent is removed, and then the magnetic beads are directly contacted with the amplification reagent, which can be effective or effective.
  • the panel of the present invention unexpectedly discovered that after the magnetic beads are adsorbed by the magnetic beads, the magnetic beads are not washed or washed, and the magnetic beads and the amplification reagent are directly contacted to achieve specificity and sensitivity amplification, regardless of the blood sample. , or a swab sample, or saliva, urine samples are all possible.
  • the present invention provides a nucleic acid extraction lysate comprising a compound of the latter several metal ions, preferably an valence metal ion such as a potassium ion, a sodium ion, a calcium ion.
  • a valence metal ion such as a potassium ion, a sodium ion, a calcium ion.
  • one or more surface active agents may also be included.
  • the monovalent metal ion compound comprises one or more of NaCl, KCl, and LiCl
  • the surface active agent includes a surface active material such as Tween and Triton-X.
  • the concentration of the monovalent metal ion compound is 0.000001-3 M, and the concentration of the surface active agent is 0.1-0.5% by mass.
  • the following components may be included: 0-2M NaCl, 0-0.3% Triton-X (mass percent), and 0-0.2 mM KCl; or only one of NaCl, KCl or Both contain no surface active agents. Generally, the NaCl content is higher and the cell membrane can be destroyed. The regulation of KCl allows the magnetic beads to have the maximum adsorption capacity and the adjustment of the components in the solution.
  • the lysate comprises magnetic bead particles and a lysis reagent component.
  • the magnetic beads may be other volumes, such as 0.2-5 ul, or 0.2-3 ul, or 0.1-2 ul (e.g., a magnetic bead concentration of 10 milligrams per milliliter).
  • the lysing liquid comprises magnetic beads in addition to the above metal ion salt, and the mass percentage of the magnetic beads may be between 0.0000001 and 0.1% mg/ML, and the lysate herein may not contain any The pure water of the salt is mixed with the magnetic bead solution. The content of the magnetic beads and its trace amount, but rely on the nanoparticles of the magnetic beads to adsorb and adsorb the nucleic acid material as a template for amplification.
  • the lysing reagent is provided in a dry powder state in a test tube, and then the desired lysed sample is directly added to a vessel such as a test tube for lysis, and then magnetic beads are added.
  • the lysis reagent and the magnetic beads are both in dry form, and then the sample is directly contacted with the dry mixture to form a new mixture, and then the magnetic beads are separated from the mixture.
  • the liquid taken out here can also be automatically taken out by a straw, and the magnetic beads and other liquid components are separated, so that only the magnetic beads are retained in the PCR tube.
  • a protective sleeve of iron so that the magnetic beads are adsorbed on the outside of the protective sleeve, and then the protective sleeve is placed in the PCR tube, the iron is removed, and the magnetic beads are released in the PCR tube.
  • the lysis reagents and/or magnetic beads are present in a PCR amplified tube. After lysing the cells with the lysing liquid, remove the other substances, leaving only the magnetic beads in the test tube, and then directly adding the magnetic beads to the PCR tube or directly adding the amplification reagent for nucleic acid amplification, such as isothermal amplification, PCR amplification .
  • the magnetic beads may be adsorbed by a magnetic adsorption magnetic bead or the magnetic beads may be adsorbed outside the PCR tube by suction, and then the lysing liquid may be removed, and then the necessary reagent for nucleic acid amplification may be directly added to the PCR tube to perform PCR. Amplification.
  • a test tube containing an adsorption magnet for example, a magnet contained in a test tube
  • the necessary reagent for nucleic acid amplification is added to the bead PCR tube for subsequent amplification.
  • the so-called cleavage mode of the present invention may be a high temperature heat cracking.
  • the magnetic bead suspension and lysate are mixed together, then added to a PCR tube, and then the desired lysed sample is added to the PCR tube, such as a throat swab, nasal swab, sputum, cerebrospinal fluid. , room water, etc., then heat the PCR tube for a few minutes, then stand at a certain temperature (room temperature or 15-30 degrees Celsius) for a few minutes, then adsorb the magnetic beads from the magnet, remove the liquid in the PCR tube The magnetic beads are retained, and then reagents necessary for nucleic acid amplification, such as a polymerase, a primer, a probe or other reagents, are added to the PCR tube. In this process, it is not necessary to elute the magnetic beads, and the nucleic acid adsorbed on the magnetic beads is directly used as a template for amplification.
  • reagents necessary for nucleic acid amplification such as a polymerase, a primer, a
  • the mixture of the lysing liquid and the magnetic bead suspension is preselected in a mixed form in a PCR tube, and then the sample is mixed as in the above step, followed by heating and cleavage, and the magnetic beads are separated from the mitigating liquid. The magnetic beads are retained and no processing is performed on the magnetic beads.
  • any lytic enzyme, or other chemical reagents such as a strong acid or a strong base reagent may be added.
  • the present invention may also carry out the release of nucleic acid (rather than high temperature heating) in a chemical manner, for example, a mixture of some Tris hydrochloric acid, EDTA, a surface active agent, a protease, etc. as a lysing liquid, and then these lysing liquids and
  • the magnetic bead suspension is mixed in the PCR tube, and then the sample (for example, throat swab, nasal swab, sputum, cerebrospinal fluid, aqueous humor, etc.) is added, mixed, and allowed to stand; then the liquid in the test tube is removed, and the magnetic beads are retained.
  • the necessary reagent for nucleic acid amplification is directly added to the tube in which the magnetic beads are retained to perform amplification of the nucleic acid, for example, PCR amplification.
  • the above samples are selected from throat swabs, nasal swabs, sputum, cerebrospinal fluid, aqueous humor, and the like.
  • the throat swabs, nasal swab samples are stored on a cotton swab or in a solution eluted from the cotton swab.
  • the eluted solution is typically a solution taken from a commercially available sample, such as a viral transport solution in a Nikon virus sampling tube, or eluted using physiological saline.
  • the nucleic acid material proposed by the method of the present invention can be used in a method of amplifying a plurality of nucleic acids. These methods use magnetic beads directly mixed with the reagents necessary for nucleic acid amplification, and then amplified, for example, using isothermal amplification and PCR amplification. Isothermal amplification is the reagent necessary for the addition of isothermal amplification, and the nucleic acid adsorbed on the magnetic beads is used as a template. If PCR amplification is used, the necessary reagents for PCR amplification are added, and the nucleic acid adsorbed on the magnetic beads is used as a template for amplification.
  • allelic nucleic acid amplification techniques including, for example, recombinase polymerase amplification (RPA), transcription-mediated amplification, nucleic acid sequence-based amplification, signal-mediated RNA amplification, strand displacement amplification. , rolling circle amplification, loop-mediated isothermal amplification of DNA, isothermal multiple displacement amplification, melting enzyme-dependent amplification, single-primer isothermal amplification, cyclase-dependent amplification, nicking and extended amplification (See US 2009/0017453).
  • RPA recombinase polymerase amplification
  • transcription-mediated amplification transcription-mediated amplification
  • nucleic acid sequence-based amplification e.g., RNA sequence-based amplification
  • signal-mediated RNA amplification strand displacement amplification
  • rolling circle amplification loop-mediated isothermal amplification of DNA
  • isothermal multiple displacement amplification melting enzyme-dependent amplification
  • RPA is an exemplary isothermal nucleic acid amplification method.
  • RPA employs an enzyme called a recombinase that is capable of pairing oligonucleotide primers with homologous sequences in double helix DNA. In this way, DNA synthesis involves defined points in the sample DNA. If a target sequence is present, an exponential amplification reaction is initiated using two gene-specific primers. The reaction progresses rapidly and specifically amplifies from several target copies to detectable levels within 20 to 40 minutes.
  • the RPA method is disclosed in, for example, US 7, 270, 981, US 7, 399, 590, US 7, 777, 958, US 7, 435, 561, US 2009/0029421, and PCT/US2010/037611, all of which are incorporated herein by reference.
  • the RPA reaction contains a blend of proteins and other factors required to support the activity of the recombinant elements of the system, as well as a blend of factors that support DNA synthesis from the 3' end of the oligonucleotide paired with the complementary substrate.
  • the key protein component of the recombinant system is the recombinase itself, which may be derived from a prokaryotic, viral or eukaryotic source.
  • single-stranded DNA binding proteins are required to stabilize nucleic acids during various exchange transactions that are performed in the reaction. Since many substrates are still characterized by partial duplexes, polymerases with strand displacement characteristics are particularly desirable.
  • in vitro conditions including the use of crowded reagents (eg, polyethylene glycol) and loaded proteins can be used.
  • crowded reagents eg, polyethylene glycol
  • An exemplary system comprising a phage T4UvsX recombinase, a phage T4UvsY loading reagent, a phage T4gp32, and a large fragment of Bacillus subtilis polymerase I has been reported.
  • the components of the isothermal amplification reaction can be provided in solution and/or in a dry (e.g., lyophilized) form. Resuspension or reconstitution buffers may also be employed when one or more components are provided in dry form.
  • the reaction mixture may contain buffers, salts, nucleotides, and other components necessary to carry out the reaction.
  • the reaction mixture can be incubated at a specific temperature suitable for the reaction. In some embodiments, the dimensions are maintained at 80 ° C or below, for example, 70 ° C or less, 60 ° C or less, 50 ° C or less, 40 ° C or less, 37 ° C or less, or 30 ° C or less. In some embodiments, the reaction mixture is maintained at room temperature. In some embodiments, the Celsius temperature of the mixture is changed by less than 25% (eg, less than 20%, less than 15%, less than 10%, or less than 5%) throughout the reaction time and/or the mixture is allowed throughout the reaction time. The temperature change is less than 15 ° C (eg, less than 10 ° C, less than 5 ° C, less than 2 ° C or less than 1 ° C).
  • the amplification of the nucleic acid can also be any other PCR method.
  • real-time fluorescent quantitative PCR technology was introduced in 1996 by Applied Biosystems, Inc., which refers to the addition of a fluorescent group in the PCR reaction system, and real-time monitoring using fluorescence signal accumulation. Throughout the PCR process, the unknown template was finally quantified by a standard curve.
  • Detection of amplification products typically involves the use of labeled probes that are sufficiently complementary and hybridize to amplification products corresponding to the target nucleic acid.
  • the presence, amount, and/or identity of an amplification product can be detected by hybridizing a labeled probe (eg, a fluorescently labeled probe) that is complementary to the amplification product.
  • detection of a target nucleic acid sequence of interest comprises using an isothermal amplification method and a labeled probe in combination to measure the product in real time.
  • detecting the target amplified target nucleic acid sequence comprises transferring the amplified target nucleic acid to a solid support (eg, a membrane) and detecting with a probe complementary to the amplified target nucleic acid sequence (eg, a labeled probe) membrane.
  • detecting the target amplified target nucleic acid sequence comprises hybridizing the labeled amplified target nucleic acid to a probe arranged in a predetermined array having addressable positions and complementary to the amplified target nucleic acid.
  • one or more primers are utilized in the amplification reaction.
  • Amplification of a target nucleic acid involves contacting the target nucleic acid with one or more primers that are capable of hybridizing the target nucleic acid and directing amplification of the target nucleic acid.
  • the sample is contacted with a pair of primers comprising forward and reverse primers that both hybridize to the target nucleic acid.
  • Real-time amplification monitors the fluorescence emitted during the reaction as an indicator of amplicon production as opposed to endpoint detection. The real-time progress of the reaction can be observed in some systems.
  • real-time methods involve the detection of fluorescent reporters.
  • the signal of the fluorescent reporter increases in proportion to the amount of amplification product in the reaction.
  • the amplification reaction during the exponential phase can be monitored, with the first significant increase in the amount of amplification product correlated with the initial amount of target template.
  • the fluorescently labeled probe is dependent on the fluorescence resonance energy transfer (FRET) or fluorescence emission wavelength change of the sample as a means of detecting the hybridization of the DNA probe to the amplified target nucleic acid in real time.
  • FRET fluorescence resonance energy transfer
  • the non-fluorescent quencher can discriminate the probe that specifically hybridizes to the target DNA sequence and in this way can detect the presence and/or amount of the target nucleic acid in the sample.
  • the fluorescently labeled DNA probes used to discriminate the amplification products have spectrally different emission wavelengths, whereby they can be distinguished within the same reaction tube (eg, in a multiplex reaction).
  • multiplex reactions allow simultaneous detection of amplification products of two or more target nucleic acids, even another nucleic acid (eg, a control nucleic acid).
  • a probe specific for a target nucleic acid is labeled in a detectable manner using an isotope or non-isotopic label; in an alternative embodiment, the marker amplifies the target nucleic acid.
  • the probe can be detected as an indicator of the target nucleic acid material (eg, an amplification product of the target nucleic acid material).
  • Non-isotopic labels can, for example, comprise fluorescent or luminescent molecules, or enzymes, cofactors, enzyme substrates or haptens.
  • the probe can be incubated with a single or double stranded preparation of RNA, DNA, or a mixture of the two, and hybridization is determined.
  • hybridization results in, for example, a detectable signal change of the labeled probe, such as an increase or decrease in signal.
  • detecting hybridization involves detecting a change in the signal of the labeled probe during or after hybridization relative to the signal of the label prior to hybridization.
  • the amplification product can be detected using a flow strip.
  • one detectable label produces a color and the second label is an epitope recognized by the immobilized antibody.
  • the product containing both markers will be attached to the immobilized antibody and produce a color at the location where the antibody is immobilized.
  • the analysis based on this detection method can be, for example, a test strip (dipstick) that can be applied to the entire isothermal amplification reaction. Positive amplification will create a band on the test strip as an indicator of amplification of the target nucleic acid material, while negative amplification will not produce any colored bands.
  • the amount of target nucleic acid eg, copy number
  • a known amount of target nucleic acid can be amplified in a parallel reaction and the amount of amplification product obtained from the sample can be compared to the amount of amplification product obtained in a parallel reaction.
  • several known amounts of target nucleic acid can be amplified in multiple parallel reactions and the amount of amplification product obtained from the sample can be compared to the amount of amplification product obtained in a parallel reaction. Assuming that the target nucleic acid in the sample can be utilized by the reaction component in a similar manner to the target nucleic acid in the parallel reaction, the method can be used to approximate quantify the amount of target nucleic acid in the sample.
  • the reaction components of the methods disclosed herein can be used in the form of a kit for detecting target nucleic acids.
  • an appropriate amount of one or more reactive components are provided in one or more containers or held on a substrate.
  • Nucleic acid probes and/or primers specific for the target nucleic acid can also be provided.
  • the reaction components, nucleic acid probes and/or primers can be suspended in an aqueous solution or in the form of a freeze-dried or lyophilized powder, pellet or bead.
  • the container for which the components or the like are supplied may be any conventional container capable of maintaining the supplied form, for example, a microcentrifuge tube, an ampoule, or a vial or a comprehensive test device such as a microfluidic device, a lateral flow, or the like.
  • the kit can include a labeled or unlabeled nucleic acid probe for detecting the target nucleic acid.
  • the kit can further include instructions for using the components in the methods described herein (eg, methods using crude matrices without nucleic acid extraction and/or purification).
  • one or more reaction components can be provided in a single, usually disposable tube or equivalent container in a single use amount that is pre-measured.
  • a sample of the presence of the target nucleic acid to be tested can be added to the individual tubes and the amplification performed directly.
  • the amount of the components supplied in the kit can be any suitable amount and can depend on the target market for which the product is targeted. General guidelines for determining the appropriate amount can be found in Innis et al., Sambrook et al. and Ausubel et al.
  • Example 1.0 Extraction of nucleic acids for samples of throat swabs.
  • the method of the invention is applied to real-time quantitative PCR detection of clinical samples of B-stream (RNA extraction)
  • step (3) the four PCR tubes in step (2) were placed in 95 ° C for 5 min and then placed at room temperature for 10 min;
  • step (3) Place the four PCR tubes in step (3) on the bead holder for 2 min, then use a pipette to aspirate the liquid on the opposite side of the magnetic beads, leaving only the magnetic beads.
  • RNA reverse transcription to c-DNA RNA reverse transcription to c-DNA
  • reaction solution contained upper and lower primers of a stream of 30 nmol/L, 30 nmol/L of a stream of probe, water, and a commercial one-step RT-QPCR kit (TAKARA, One Step PrimeScriptTM RT-PCR Kit, part number RR064A ) (Perfect Real Time), wherein the primers and probes were designed by the inventors themselves: the upstream primer probe is as follows: GAGTCTTATCCCAATTTG (SEQ.
  • the downstream primer probe is as follows: GTGGAATAGTATGTTATCA (SEQ. 2); the probe sequence is as follows: HEX-AAGAGCACCGATTATCACCAG-TAMRA (SEQ. 3), wherein the probe has a HEX fluorescent gene attached to the 5' end and a TAMRA quenching group attached to the 3' end, and is vortexed and shaken for 30 s after capping to ensure magnetic
  • the beads and the PCR reaction are uniformly mixed; wherein the components of the amplification reagent are as follows:
  • step (6) instantaneously centrifuging the four PCR tubes in step (5) (centrifugation at 1000-3000 rpm for 5-10 s) to make the liquid in the PCR tube at the bottom;
  • the experimental results are shown in Fig. 1.
  • the curves of No. 1, No. 2, No. 3 and No. 4 are the experimental results of No. 1, No. 2, No. 3 and No. 4 samples, respectively, and the results show that the method according to the present embodiment The operation can effectively and accurately detect the throat swab samples of the B stream.
  • No. 1 and No. 2 are positive, and Nos. 3 and No. 4 are negative, which is consistent with the confirmed sample.
  • Example 1.1 Proposal and amplification of other samples.
  • the same lysate and magnetic beads as in Example 1 were used (the magnetic beads were 500 nm diameter, 400 nm, 300 nm, 200 nm, 100 nm, 600 nm, 700 nm dissolved in sterile water, and the volume ratio of the lysate to the magnetic beads was 1: 200, 1:150; 1:100, 1:300; 1:400, etc., while the components of the lysate are treated as follows: 0.01 M NaCl, 0.1 M NaCl, 0.5 M NaCl, 1 M NaCl, 1.5 M NaCl, 1.8 M NaCl, 2.0 M NaCl, Or the above component treatment contains a surface active agent; or 0.01MNaCl+0.001MKCl, 0.1MNaCl+0.01MKCl, 0.5MNaCl+0.1MKCl, 1MNaCl+0.5MKCl, 1.5MNaCl+0.8MKCl, 1.8MNaCl+1.0MKCl, 2.0M
  • the amplification reagents for detecting these samples can be purchased commercially, if reverse transcription is required (RNA sample), adding reverse transcriptase for C-DNA synthesis, then DNA PCR amplification.), the magnetic beads can be directly subjected to PCR amplification without elution, and 50 positive results are obtained, which is consistent with the confirmed positive result detection, and the coincidence rate is 100%.
  • 50 parts of the negative sample (throat swab 30 or nasal swab sample 20 parts) were tested in parallel, and the nucleic acid-containing magnetic beads extracted by the method of the present invention also obtained a negative result, and the coincidence rate was 100% (the specific data is slightly ).
  • Example 1.2 Proposal and amplification of other samples.
  • Example 1.1 of the method of the present invention was used to carry out the sample as saliva, urine, or cerebrospinal fluid (these 150 samples were confirmed by standard methods to contain three viruses or one of them). Or negative samples) detection of 6 common human herpesviruses (DNA) such as HSV-1, HSV-2, VZV, EBV, CMV, HSV-6, etc., using well-known PCR amplification reagents and well-known primer sequences (specific data).
  • DNA common human herpesviruses
  • HSV-6 6 common human herpesviruses
  • primer sequences specific data
  • the routine amplification of the reagents contained in the column can be consistent with the results of the confirmed samples, and the coincidence rate is 100%.
  • the magnetic bead extraction method of the present invention can directly detect the target substance by using the magnetic beads adsorbed with the nucleic acid as a template without washing, and achieve the purpose of detection. Moreover, liquid, urine, or cerebrospinal fluid samples can be effectively detected.
  • Example 1.3 Proposal and amplification of other samples.
  • the sample was serum prepared by the magnetic bead extraction method in Example 1.1 of the method of the present invention (these 150 samples were confirmed by standard methods to contain three kinds of HBV, HCV).
  • One of the positive or negative samples using conventional PCR amplification reagents and well-known primer sequences (specific data omitted) to carry out routine amplification, can be consistent with the results of the confirmed samples, the coincidence rate is 100% .
  • the magnetic bead extraction method of the present invention can directly detect the target substance by using the magnetic beads adsorbed with the nucleic acid as a template without washing, and achieve the purpose of detection. Moreover, serum samples can be effectively detected.
  • Example 2 Extraction of nucleic acids for samples of throat swabs.
  • the method of the present invention applied to the real-time fluorescent quantitative PCR detection of the B-stream (RNA extraction) clinical sample has the following differences in steps 1 and 2 as compared with the example 1.0, and the other processes are the same. The differences are as follows:
  • the experimental results are similar to those in Fig. 1.
  • the curves No. 1, No. 2, No. 3, and No. 4 are the experimental results of the No. 1, No. 2, No. 3, and No. 4 samples, respectively, and the results show that the method according to the present embodiment is performed.
  • the operation can effectively and accurately detect the throat swab samples of the B flow (the specific experimental results are omitted).
  • Example 3 Extraction of nucleic acids for samples of throat swabs.
  • the method of the present invention applied to the real-time fluorescent quantitative PCR detection of the B-stream clinical sample has the following differences in steps 1 and 2 as compared with the first embodiment, and the other processes are the same. The differences are as follows:
  • the experimental results show that the amplification curve is similar to that of the first example.
  • the curves No. 1, No. 2, No. 3 and No. 4 are the experimental results of the No. 1 sample, the No. 2 sample, the No. 3 sample and the No. 4 sample.
  • the results show that The method of the embodiment operates to effectively and accurately detect the throat swab sample of the B stream. Again, the negative control did not have a visible amplification signal.
  • the above four diluted samples were subjected to real-time fluorescent PCR detection in the same manner as in Example 1 (ABI7500 was used for PCR amplification).
  • Example 5 Repeatability test for fluorescent PCR detection of the method of the invention
  • the sample was subjected to 5-well retest real-time fluorescent PCR detection (amplification by Bio-RAD CFX96) in the same manner as in the first embodiment.
  • Example 6 The method of the invention detects the B-stream and the Shanghai Zhijiang nucleic acid extraction kit to flow in the A and B types Comparison of the detection of B-stream in the susceptibility test kit
  • RNA adjuvant 6 ul of RNA adjuvant, 20 ul of magnetic beads to 500 ul of binding buffer, and mix.
  • Vortex for 10 s or repeatedly reverse for 5-10 s to evenly disperse the magnetic beads into the buffer, completely lyse the virus, and combine the RNA and the magnetic beads) and let stand for more than 3 min.
  • washing solution A to the affinity column, centrifuge at 16000 g (13,000 rpm) for 40 s; discard the collection tube waste liquid; repeat once
  • washing solution W to the affinity column, centrifuge at 16000 g (13,000 rpm) for 15 s; discard the collection tube waste liquid; repeat once
  • the affinity column was centrifuged at 16000 g (13,000 rpm) for 2 min in a centrifuge.
  • Affinity column was placed in a 1.5 ml RNAnase-free centrifuge tube, and 50 ul of 65 ° C pre-heated eluate was added and allowed to stand at room temperature for 2 min.
  • RNAnase-free centrifuge tube 16000 g (13000 rpm) was centrifuged for 2 min, and the RNA was eluted in an RNAnase-free centrifuge tube or stored at -20 ° C or -80 ° C.
  • Example 7 The method of the present invention detects RSV and RSV nucleic acid of Guangdong Huayin Pharmaceutical Technology Co., Ltd. Comparison of detection kits (including nucleic acid extraction reagents) for detecting RSV
  • Example 1 Using the negative B-stream throat swab sample in Example 1 (tested by RSV nucleic acid detection kit of Guangdong Huayin Pharmaceutical Technology Co., Ltd. as a negative), the RSV standard provided by Shaoxing CDC was used as a diluent.
  • Example 2 2.0 ⁇ 10 5 TCID 50 / L diluted, respectively 2.0 ⁇ 10 4 TCID 50 /L,2.0 ⁇ 10 3 TCID 50 /L,2.0 ⁇ 10 2 TCID 50 /L,2.0 ⁇ 10 1 TCID 50 / L four
  • One sample was extracted in the same manner as in Example 1 (Amplification by Bio-RAD CFX96) (primer and probe were replaced with primers and probes for RSV, and the same reverse transcriptase was used for C-DNA transcription. The amplification was carried out by referring to the method of Example 1.
  • the RSV nucleic acid detection kit (including nucleic acid extraction reagent) of Guangdong Huayin Pharmaceutical Technology Co., Ltd. detects four samples separately, as follows:
  • the method for nucleic acid extraction in the RSV nucleic acid detection kit of Guangdong Huayin Pharmaceutical Technology Co., Ltd. is as follows:
  • RNA extraction solution A Take 200ul RNA extraction solution A in a 0.6ml centrifuge tube, add the test sample to each tube, negative control, strong positive control and critical positive control each 50ul, mix 5-10 times and mix thoroughly. Allow to stand at room temperature for 3 minutes.
  • the expanded reagent is the same as the RSV nucleic acid detection kit of Guangdong Huayin Pharmaceutical Technology Co., Ltd., and the final volume and amplification reagent content are also the same.
  • the four curves of No. 1, No. 2, No. 3 and No. 4 in FIG. 4 are 2.0 ⁇ 10 4 TCID 50 / L, 2.0 ⁇ 10 3 TCID 50 / from top to bottom, respectively.
  • Figure 5 shows the results of the RSV nucleic acid detection kit of Guangdong Huayin Pharmaceutical Technology Co., Ltd.
  • the four curves of No. 1, No. 2, No. 3 and No. 4 in Figure 5 are 2.0 ⁇ 10 4 TCID 50 / from top to bottom respectively.
  • the amplification effect of the present invention is equivalent to the prior art, and the steps of the prior art are too cumbersome for the extraction of RNA, and the present invention uses magnetic beads to directly utilize magnetic beads for amplification, thereby realizing effective Amplification.
  • Embodiment 8 Method of the present invention and extraction method of Chinese invention patent application 201610022581.8 Comparative test
  • the nucleic acid extraction of the present invention is carried out according to the chemical method for cleavage of the sample, as follows: lysate formulation: Tris concentration 10-200 mmol, EDTA concentration 10-100 mmol, Triton X-100 concentration 1-20%, guanidine thiocyanate concentration 1-5 mol, proteinase K concentration 0.1-3 mg/ml.
  • PCR reaction solution was added (the same as the nucleic acid amplification reagent in Example 1 in Comparative Experiment 201610022581.8, the amplification conditions were the same) for PCR fluorescence. Amplification.
  • the extraction method of the present invention can effectively detect HBV serum samples that have been confirmed to be positive, and demonstrate that the nucleic acid extraction method of the present invention is effective.
  • the reagents used in the comparative experiments were simple and there was essentially no need to clean the beads. Further, serum samples were extracted by the method of Example 1 of the present experiment, and then amplified by the method disclosed in 201610022581.8, and the amplification reagents used were the same as the conditions, and consistent results were obtained.
  • the positive sample was diluted, and similarly, the magnetic beads extraction method (chemistry) of the present invention and the reagent of the disclosed Example 2 were used for extraction and the same amplification.
  • Embodiment 9 Method of the present invention and extraction method of Chinese invention patent application 201610022581.8 Comparative test
  • the method of the invention adopts a heating method for heating the throat swab to perform magnetic bead extraction as follows:
  • step (3) the four PCR tubes in step (2) were placed in 95 ° C for 5 min and then placed at room temperature for 10 min;
  • step (3) Place the four PCR tubes in step (3) on the bead holder for 2 min, then use a pipette to aspirate the liquid on the opposite side of the magnetic beads to retain the magnetic beads.
  • the liquid taken out here can also be automatically taken out by a straw, and the magnetic beads and other liquid components are separated, so that only the magnetic beads are retained in the PCR tube, and the separation can be carried out in various ways.
  • reagents necessary for amplifying the nucleic acid to each of the four PCR tubes.
  • the following reagents were added to the test tube: 0.5 ul (200 U/ul) unit of reverse transcriptase and 24.5 ul of reaction solution (the reaction solution contained the same concentration as the example)
  • step (6) instantaneously centrifuging the four PCR tubes in step (5) (centrifugation at 1000-3000 rpm for 5-10 s) to make the liquid in the PCR tube at the bottom;
  • RNA extraction of the same sample was carried out according to the example 1 or 2 disclosed in the application (refer to steps 1-4), and the amplification reagent added to the extracted sample was the same essential reagent as the present invention: 0.5 Ul (200 U / ul) unit of reverse transcriptase and 24.5 ul of reaction solution (as in the example), vortex and shake for 30 s after capping, to ensure uniform mixing of the magnetic beads and PCR reaction; then perform the same method of amplification .
  • results of the present invention can obtain positive results, while the control experiments cannot obtain positive results. This seems to indicate that the control method seems to be positive or positive for serum samples (DNA), but not for throat swab samples. It is feasible for DNA samples, but not for RNA.

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Abstract

本发明公开了一种利用磁珠提取核酸物质的方法，包括以下步骤： (1)提供裂解试剂和纳米磁珠；所述的裂解试剂包括金属化合物； (2)将样本与所述的裂解试剂和磁珠接触形成混合物，处理后低温静置所述的混合物； (3)去除混合物中的其它物质，仅仅保留磁珠，同时不对磁珠进行任何的洗涤或者洗脱处理； (4)将步骤(3)的磁珠直接与核酸扩增的试剂接触，为靶核酸的扩增提供核酸模板，从而进行靶核酸的扩增。

Description

一种利用磁珠提取核酸物质的方法以及试剂
本申请主张在先中国申请，申请号：2017101221522，申请日：2017年3月3，的优先权。
技术领域
本发明属于分子诊断生物学技术领域，具体涉及体涉及一种对生物样本直接采用磁珠进行核算的提取,从而直接让扩增的试剂核磁珠接触,从而完成目标靶核酸的扩增。
背景技术
实时荧光定量PCR技术是1996年由美国Applied Biosystems公司推出的，该技术是指在PCR反应体系中加入荧光基团，利用荧光信号积累实时监测整个PCR进程，最后通过标准曲线对未知模板进行定量分析。
正常人咽峡部培养应有口腔正常菌群，而无致病菌生长。咽部的细菌均来自外界，正常情况下不致病，但在机体全身或局部抵抗力下降和其他外部因素下可以出现感染等而导致疾病。因此，咽部拭子细菌培养能分离出致病菌，有助于白喉、化脓性扁桃体炎、急性咽喉炎等的诊断。咽拭子检测是一种医学检测方法，是用医用的棉签，从人体的咽部蘸取少量分泌物，接种于特制培养皿中，然后放置在一个可以控制温度的设备内进行培养的过程，常采用荧光PCR技术检测，可以了解患者病情、口腔黏膜和咽部感染情况，但是需要提取核酸核酸，PCR的时间长且成本高；不能大量处理样本，操作检测有污染的风险。
无论是咽喉部位，还是其他样本，例如唾液、尿液、以及鼻拭子样本，该样本中带有病毒、细菌、或者真菌，或者其他生物细胞成分，在进行核酸检测的时候，都需要进行核酸的提取，提取的过程就是释放获得核酸，同时去除组织或者细胞中的干扰物质，这样可以进行后续有效的靶核酸的扩增。
现有利用磁珠对样本中的核酸进行提取是一种比较可靠的方法，而且可以实现自动化操作，但是，现有磁珠的提取方法仍然显得比较复杂，一般用磁珠吸附DNA或者RNA后，需要对磁珠进行清洗，然后在从磁珠上洗脱下来核酸物质， 用来扩增，或者清晰去掉磁珠上吸附的杂质，例如蛋白、多糖，其它非靶核酸模板的其它核酸、还要一些其它的微粒成分，虽然磁珠经过化学修饰，可以对核酸进行吸附，但是，磁珠的吸附是没有选择性的，化学和物理吸附同时兼备的情况下。实际上对样本核酸的提取需要花费大量的时间和试剂成本，当需要在短时间内对较多样本进行核酸扩增检测的时候，仍然需要大量的人力进行，有人为因素的增加，会造成很多不确定性，不能很好的实现自动化检测。这就有需要对现有技术进行改进，希望可以进行自动化的检测。
发明内容
本发明的目的在于提供一种利用磁珠提取核酸物质的方法以及试剂，以克服现有检测技术的不足，为咽拭子，唾液、尿液、以及鼻拭子以及血清等样本提供一种快速、简便、高效、实用的检测方法，不要核酸提取过程，简化了操作过程，节约了时间和成本，且能大量处理样本，操作检测时可防止污染。
为达到上述目标提供了一种利用磁珠提取核酸物质的方法以及试剂，包括以下步骤：
(1)提供裂解试剂和纳米磁珠；所述的裂解试剂包括金属化合物；
(2)将样本与所述的裂解试剂和磁珠接触，处理后低温静置；
(3)去除混合物中的其它物质，仅仅保留磁珠，同时不对磁珠进行任何的洗涤或者洗脱处理；
(4)将步骤(3)的磁珠直接与核酸扩增的试剂接触，为核酸扩增提供核酸模板，从而进行靶核酸的扩增。
优选的，所述的裂解试剂和磁珠为混合物，优选的为溶液混合物。
优选的，，所述的金属化合物包括NaCl、KCl中的一种或者两种。
优选的，所述的磁珠为单分散性羟基或者羧基磁珠。
优选的，其中，所述的裂解试剂为裂解试剂和纳米磁珠溶液形式，该溶液的成分包括：0.1-2M的NaCl和0-2mM的KCl，或者质量百分比为0-0.3％的表面活性试剂，其中磁珠的浓度为0.00005毫克/毫升。
优选的，样本与裂解液的体积比为1:3—1:10。
优选的，其中，当混合物为溶液的时候，所述的磁珠和裂解液的体积比1:200，其中磁珠的浓度为：0.1-100mg/ML，。
优选的，其中，所述的样本包括咽拭子、鼻拭子、喉拭子、血清、唾液、痰、尿液、血清中的一种或者几种。
优选的，所述的样本是经过处理过后的样本。
优选的，所述的咽拭子、鼻拭子或者喉拭子样本的载体经过生理盐水、缓冲液洗脱的样本。
优选的，所述的裂解试剂和磁珠为溶液状态，并位于PCR试管中，或者，所述的裂解试剂和磁珠为干的状态，并位于用于PCR扩增的PCR管中。
优选的，其中，在让样本与混合物接触前，配备所述的混合物，混合物中包括羟基磁珠。
优选的，其中，所述的磁珠的平均粒径小于1000nm。
优选的，其中，让磁珠和裂解液和样本接触后或者之前，对样本或者混合有样本的磁珠和裂解液进行高温处理在60℃-100℃，或者，所述的低温为室温或者20℃-30℃之间。
优选的，其中，当所提取的核酸为RNA的时候，让步骤(3)中的磁珠同时与反转录酶和核酸扩增试剂接触进行扩增。
优选的，其中，让样本同时与裂解试剂和磁珠接触。
另一方面，本发明提供一种提取核酸的试剂，包括溶液，所述的溶液包括1MNaCl、质量比含量为0.01％T表面活性是和0.001MKCl的溶液，0.005毫克的磁珠。优选的，根所述的磁珠为羟基修饰的磁珠，或者，磁珠的分散系数小于5。
另一方面，本发明提供一种提取核酸的试剂，包括裂解成份和磁珠，所述的裂解成份包括1MNaCl、0.01％Triton-X、0.001MKCl和0.005毫克的磁珠；所述的磁珠的平均粒径为≤1000nm。
另一方面，本发明提供一种提取核酸的试剂，包括裂解溶液和0.005毫克的磁珠， 所述的裂解溶液包括1MNaCl、质量比含量为0.01％T表面活性是和0.001MKCl。另一方面，本发明提供一种含有羟基修饰的纳米磁珠作为核酸提取试剂上的用途，其中所述的纳米磁珠的平均粒径为≤1000nm。
另一方面，本发明提供样本的核酸提取的方法，该方法包括：
(1)制备咽拭子，唾液、尿液、以及鼻拭子等样本，提供裂解液和PCR反应液；
(2)将样本加入到含有裂解液的PCR扩增管中，振荡摇匀混合后在80-100℃高温处理10min，后室温静置10min；
(3)在步骤(2)中的PCR扩增管中加入磁珠后在18-28℃反应5-10min；
(4)将PCR扩增管瞬时离心后放入到磁力架中，待磁珠吸附到磁力架一侧后将上清吸出，并保留磁珠，然后对磁珠不做任何的清洗或者洗涤步骤；
(5)在PCR扩增管内加入配置好的PCR反应液，进行实时定量PCR反应。
优选的，所述步骤(1)中裂解液的成分为0-2M的NaCl、0-0.3％的Triton-X和0-0.2mM的KCl。
优选的，所述步骤(2)中的高温为100℃。
优选的，所述步骤(2)样本与裂解液的体积比为1:3—1:10。
优选的，所述磁珠为市售的核酸提取使用的羟基磁珠，即超顺磁性核心及无机氧化硅的外壳，优选的，平均粒径为≤1000nm的单分散性羟基磁珠。
优选的，所有反应步骤都在同一个试管内进行。
本发明的有益效果是：
为样本提供一种快速、简便、高效、实用的检测方法，不要核酸提取过程，简化了操作过程，节约了时间和成本，且能大量处理样本，操作检测时可防止污染。本发明所述方法可以手动操作以及机械自动化操作，且不要核酸提取过程，简化了操作过程，节约了时间以及成本。
附图说明
图1为实施例1的本发明方法应用于乙流临床样本的实时荧光定量PCR检测结果图；
图2为实施例4的本发明方法检测阳性乙流咽拭子样本的敏感性结果图；
图3为实施例5的本发明方法检测阳性的乙流咽拭子样本的重复性结果图；
图4为实施例7的本发明方法检测RSV的结果图；
图5为实施例7的广东华银医药科技有限公司的RSV核酸检测试剂盒(包含核酸提取试剂)检测RSV的结果图。
详细说明
样本
在一些实施例中，样本包括生物试样，例如从植物或动物个体获得的试样。如本文所用生物试样包括可用于检测个体中的核酸的所有临床试样，其包括但不限于细胞、组织(例如，肺、肝和肾组织)、骨髓吸出物、体液(例如，血液、血液衍生物和血液馏份(例如血清或黄层)、尿、淋巴、泪、前列腺液、脑脊液、气管吸出物、痰、脓、鼻咽吸出物、口咽吸出物、唾液)、眼拭子、颈部拭子、阴道拭子、直肠拭子、大便和大便悬浮液。其它适宜试样包括从中耳液、支气管肺泡灌洗液、气管吸出物、痰、鼻咽吸出物、口咽吸出物或唾液获得的试样。在特定实施例中，生物试样是从动物个体获得。可获得获取所述试样的标准技术。参见(例如)施拉格(Schluger)等人，实验医学杂志(J.Exp.Med.)176:1327-33(1992)；比格比(Bigby)等人，美国呼吸疾病评论(Am.Rev.Respir.Dis.)133:515-18(1986)；科瓦克斯(Kovacs)等人，新英格兰医学杂志(NEJM)318:589-93(1988)；和奥尼贝内(Ognibene)等人，美国呼吸疾病评论129:929-32(1984)。在一些实施例中，样本包括环境试样，例如表面试样(例如，通过擦拭或真空处理获得)、空气试样或水试样。在一些实施例中，样本包括分离的细胞，例如动物、细菌、真菌(例如，酵母)或植物细胞和/或病毒。可使用常规方法和适于所培养细胞类型的条件培养分离的细胞。
对于样本的处理
在一些方式中，样本一般都是采用载体进行采样，例如吸水性载体进行承载样本。承载样本可以是吸管、存贮装置、采样装置、这些采样装置吸取样本的部件可以是滤纸、棉签、等吸水性物质来吸取和承载载体。与本发明的裂解液进行接触的可以是载体，也可是从载体上洗过下来的样本混合物。例如，裂解液体先于承载样本的载体接触，从而把样本裂解和洗脱下来，然后再让裂解液体与磁珠混合，或者，让裂解液和磁珠的混合物与承载样本的载体接触，完成裂解和洗脱，然后去除掉载体和其它物质，然后仅仅保留磁珠，并不对磁珠做任何洗脱和洗涤等进一步的处理。当然，也可以提前准备好裂解液和磁珠溶液，然后再使用的时候，两种溶液混合，然后再和样本直接接触后者与承载样本的载体接触，从而完成样本的处理。
当然，如果裂解成分和磁珠可以是干的形式存在，当与液态的样本接触后，然后对样本裂解或者进行磁珠的吸附，完成对核酸的释放和吸附。
这里的裂解液体可以是一些离子溶液，或者离子溶液和表面活性试剂混合，这些裂解都可以造成细胞壁或者细胞膜的破坏，从而达到核酸(DNA或者RNA)的释放。当然，为了加速释放和破坏，可以采用物理加热的方法进行，例如高温加热的方式，例如60度以上的问题，温度越高，时间越短，温度越低，花费的时间越长。当然，如果裂解试剂中含有足够的破坏细胞壁或者细胞膜的成分，例如强酸、强碱、或者一些酶试剂，这些成分可以直接达到释放核酸物质的目的，可以不接触物理加热或者超声破碎的方法。物理方法一般是加热，研磨，超声破碎，化学方法一般是化学试剂成分。
在一些优选的方式中，本发明采用少量的化学试剂配合物理方法达到核酸物质的释放和磁珠的吸附。这样可以避免杂质干扰后续的直接扩增。在另一些优选的方式中，采用化学试剂进行核酸的释放，虽然同是释放了一些非核酸物质，例如蛋白、多糖、这些物质可能干扰后学核酸的扩增，但是采用本发明的磁珠进行核酸的吸附，就算带有杂质，仍然可以进行正常的核酸扩增。
在另一方式中，核酸为RNA。一般，RNA的扩增首先需要进行反转录过程，然后对反转录的DNA进行扩增。本发明小组发现，当释放的为RNA的时候，采用本发明小组的方法，利用磁珠直接进行RNA的吸附，然后去掉裂解液，保留磁珠， 不对磁珠进行任何的洗涤或者清洗，直接进行C-NDA的反转录和扩增试剂的接触，从而完成扩增。而现有对于RNA的扩增，一般都是需要单独进行反转录，然后反转录的DNA进行浓缩进行核酸的扩增，这样的过程处理时间长，而且中间有很多单独的步骤，材料耗费大，另外步骤多，交叉污染的机会增加。另外，现有的技术对RNA的提取过程复杂，繁琐，耗材多。在一些优选的方法中，本发明利用裂解液和磁珠的混合物对RNA进行裂解，然后去掉裂解液和其它物质，仅仅保留磁珠，然后让磁珠和反转录酶和扩增试剂接触，进行扩征。采用这样的方法可以有效的进行扩增，或者扩增的结果。
在一些优选的方式中，让裂解试剂和磁珠与样本接触后，进行高温加热处理，然后低温保持一段时间。或者，先对样本进行高温处理，然后让裂解试剂和磁珠接触，然后低温保持一段时间。可以理解，高温处理并不是必须的，当裂解试剂含有高浓度的离子浓度的时候，可以破坏细胞膜或者细胞壁，这个情况下可以不用高温处理。
靶核酸
这里的所谓的“靶核酸”的用途之一就是对生物进行区分，和另外的生物样本进行鉴别。例如，某一种病毒具有特有的学历诶，对太特殊的序列的扩增和检测，从而确认该特殊的病毒，而不会造成漏检和假阳性或者假阴性。也就是说，在特定实施例中，靶核酸对目标有机体具有特异性，也就是，在其它有机体中未发现靶核酸或者在与目标有机体类似的有机体中未发现靶核酸。
靶核酸可为存于动物(例如，人类)、植物、真菌(例如，酵母)、原生动物、细菌或病毒物质中的核酸，也可以是来自以上样本中的核酸物质。例如，靶核酸可存于目标有机体的基因组中(例如，在染色体上)或存于染色体外核酸上。在一些实施例中，靶核酸是RNA，例如mRNA。靶核酸可存于细菌(例如格兰氏(Gram)阳性或格兰氏阴性细菌)中。例示性细菌种类包括不动杆菌属(Acinetobacter sp.)菌株ATCC 5459、乙酸钙不动杆菌属、绿浅气球菌(Aerococcus viridans)、脆弱拟杆菌(Bacteroides fragilis)、百日咳博德特菌(Bordetella pertussis)、副百日咳博德特菌(Bordetella parapertussis)、空肠弯曲杆菌(Campylobacter jejuni)、难辨梭状芽孢杆菌、产气荚膜梭菌(Clostridium perfringens)、棒 状杆菌属(Corynebacterium sp.)、肺炎衣原体(Chlamydia pneumoniae)、沙眼衣原体、弗氏柠檬酸杆菌属(Citrobacter freundii)、产气肠杆菌(Enterobacter aerogenes)、鹑鸡肠球菌属(Enterococcus gallinarum)、屎肠球菌(Enterococcus faecium)、粪肠球菌(Enterobacter faecalis)(例如，ATCC 29212)、埃希氏大肠杆菌(例如，ATCC 25927)、阴道加德纳氏菌、幽门螺杆菌、流感嗜血杆菌(Haemophilus influenzae)(例如，ATCC 49247)、肺炎克雷伯氏杆菌(Klebsiella pneumoniae)、侵肺军团菌(Legionella pneumophila)(例如，ATCC 33495)、单核细胞增生李斯忒氏菌(Listeria monocytogenes)(例如，ATCC 7648)、微球菌(Micrococcus sp.)菌株ATCC 14396、卡他莫拉菌(Moraxella catarrhalis)、堪萨斯分枝杆菌(Mycobacterium kansasii)、戈登分枝杆菌(Mycobacterium gordonae)、偶然分枝杆菌(Mycobacterium fortuitum)、肺炎支原体、人型支原体、脑膜炎奈瑟球菌(Neisseria meningitis)(例如，ATCC 6250)、奈瑟氏淋病双球菌、尿道寡源杆菌(Oligella urethralis)、多杀巴斯德菌(Pasteurella multocida)、绿脓杆菌(Pseudomonas aeruginosa)(例如，ATCC 10145)、痤疮丙酸杆菌(Propionibacterium acnes)、奇异变形杆菌(Proteus mirabilis)、普通变形杆菌(Proteus vulgaris)、沙门氏菌菌株ATCC 31194、鼠伤寒沙门氏菌、粘质沙雷菌(Serratia marcescens)(例如，ATCC 8101)、金黄色葡萄球菌(例如，ATCC 25923)、表皮葡萄球菌(Staphylococcus epidermidis)(例如，ATCC 12228)、里昂葡萄球菌(Staphylococcus lugdunensis)、腐生葡萄球菌(Staphylococcus saprophyticus)、肺炎链球菌(例如，ATCC 49619)、酿脓链球菌(Streptococcus pyogenes)、无乳链球菌(Streptococcus agalactiae)(例如，ATCC 13813)、苍白密螺旋体(Treponema palliduma)、草绿色链球菌(Viridans group streptococci)(例如，ATCC 10556)、炭疽芽孢杆菌(Bacillus anthracis)、蜡状芽孢杆菌(Bacillus cereus)、蜃楼弗朗西丝氏菌(Francisella philomiragia)(GAO1-2810)、土拉热弗朗西丝菌(Francisella tularensis)(LVSB)、假结核病耶尔森菌(Yersinia pseudotuberculosis)(PB1/+)、小肠结肠炎耶尔森菌(Yersinia enterocolitica)、0:9血清型或鼠疫耶尔森菌(Yersinia pestis)(P14-)。在一些实施例中，靶核酸存于选自以下的细菌属物质中：不动杆菌属、气球菌属(Aerococcus)、拟杆菌属(Bacteroides)、博德特菌(Bordetella)、弯曲杆菌属(Campylobacter)、梭状芽孢杆菌属 (Clostridium)、棒状杆菌属(Corynebacterium)、衣原体(Chlamydia)、柠檬酸杆菌属(Citrobacter)、肠杆菌属(Enterobacter)、肠球菌属(Enterococcus)、埃希氏菌属(Escherichia)、螺旋杆菌属(Helicobacter)、嗜血杆菌属(Haemophilus)、克雷伯氏杆菌属(Klebsiella)、军团菌属(Legionella)、李斯忒氏菌属(Listeria)、微球菌属(Micrococcus)、动弯杆菌(Mobilincus)、莫拉菌属(Moraxella)、分枝杆菌属(Mycobacterium)、支原体、奈瑟球菌、寡源杆菌属(Oligella)、巴斯德菌属(Pasteurella)、普雷沃氏菌属(Prevotella)、红棕色单胞菌属(Porphyromonas)、假单胞菌属(Pseudomonas)、丙酸杆菌属(Propionibacterium)、变形菌属(Proteus)、沙门氏菌属、粘质沙雷菌属(Serratia)、葡萄球菌属、链球菌属、密螺旋体属(Treponema)、芽胞杆菌属(Bacillus)、弗朗西丝氏菌属(Francisella)或耶尔森菌属(Yersinia)。在一些实施例中，在A组链球菌或B组链球菌中发现靶核酸。
例示性衣原体靶核酸包括在衣原体隐性质粒上发现的序列。例示性结核分枝杆菌(M.tuberculosis)靶核酸包括于IS6110(参见US 5,731,150)和/或IS1081(参见巴哈德(Bahador)等人，2005，农业生物科学研究杂志(Res.J.Agr.Biol.Sci.)，1:142-145)中发现的序列。例示性奈瑟氏淋病双球菌靶核酸包括于NGO0469(参见皮耶卡罗维奇(Piekarowicz)等人，2007，BMC微生物(BMC Microbiol.)7:66)和NGO0470中发现的序列。例示性A组链球菌靶核酸包括于Spy1258(参见刘(Liu)等人，2005，微生物研究(Res.Microbiol)，156:564-567)、Spy0193、lytA、psaA和ply(参见US 2010/0234245)中发现的序列。例示性B组链球菌靶核酸包括于cfb基因(参见波德别尔斯基(Podbielski)等人，1994，微生物与免疫医学杂志(Med.Microbiol.Immunol.)，183:239-256)中发现的序列。在一些实施例中，靶核酸是病毒核酸。例如，可在人类免疫缺陷病毒(HIV)、流感病毒或登革病毒中发现病毒核酸。例示性HIV靶核酸包括于Pol区域中发现的序列。在一些实施例中，靶核酸是原生动物核酸。例如，可在疟原虫属(Plasmodium spp.)、利什曼原虫属(Leishmania spp.)、冈比亚布氏锥虫(Trypanosoma brucei gambiense)、罗德西亚布氏锥虫(Trypanosoma brucei rhodesiense)、克氏锥虫(Trypanosoma cruzi)、内阿米巴属(Entamoeba spp.)、弓形虫属(Toxoplasma spp.)、阴道毛滴虫(Trichomonas vaginalis)和肠形 鞭毛虫(Giardia duodenalis)中发现原生动物核酸。在一些实施例中，靶核酸是哺乳动物(例如，人类)核酸。例如，可在循环肿瘤细胞、上皮细胞或成纤维细胞中发现哺乳动物核酸。在一些实施例中，靶核酸是真菌(例如，酵母)核酸。例如，可在念珠菌属(Candida spp.)(例如，白色念珠菌)中发现真菌核酸。这里的靶核酸可以是DNA上的一端序列，也是可以是RAN反转录上的一端特异的序列。
磁珠以及对核酸的提取
本发明所提供的磁珠可以是任何的磁珠。一般磁珠按照不同的分类，磁珠是一种纳米颗粒，在颗粒表面一般连接有化学基团，这样就形成了最终产品的磁珠。这样的磁珠可以通过商业途径进行购买，例如从厦门普睿迈格生物科技有限公司(http://www.purimagbead.com)购买。一般磁珠按照磁珠的直径的大小进行分类，另一种按照表面包裹的化学基团进行分类。按照直径的大小，可以分为平均粒径小于50钠米、60钠米、80钠米、90钠米、100钠米、150钠米、200钠米、300钠米、500钠米、600钠米、700钠米、800钠米、900钠米、1000钠米，1500钠米，2000钠米等磁珠。
按照磁珠包裹的化学基团，按照化学基团的性质不同，可以分为羟基磁珠，羧基或者氨基磁珠，或者混合基团包裹的磁珠。一般是含有羟基聚合物包裹的磁珠或者磁珠壳层表面修饰大量硅烷醇基团(羟基)。当然，按照磁珠的形态环境进行分类，可以是单分散性磁珠，也可以是的非单分散性磁珠。
在一些优选的方式中，羟基、羧基或者氨基之一或者任意组合来修饰磁珠表面而形成羟基磁珠、羧基磁珠或者氨基磁珠，或者任意两个组合修饰而形成的磁珠。在一些优选的方式中，磁珠为磁珠壳层表面修饰大量硅烷醇基团(羟基)的羟基磁珠或者羧基磁珠。
使用的磁珠为粒径均一或者多分散系数＜0.2。优选的，选择呈单分散磁珠，这样具有超顺磁性，磁响应时间＜30s。所谓的粒径“均一”并不一定表示每个磁珠之前的粒径一样，而是在同一个溶液中，至少有30％的磁珠的粒径接近，或者35％的磁珠的粒径接近；40％的磁珠的粒径接近；45％的磁珠的粒径接近；50％的磁珠的粒径接近；55％的磁珠的粒径接近；60％的磁珠的粒径接近；70％的磁珠 的粒径接近或75％的磁珠的粒径接近；或者80％、85％、88％、89％、90％、95％、99％、100％的磁珠的粒径接近或则相差不大。或者，不同粒径的磁珠进行混合，例如，小于10钠米和小于50钠米的磁珠混合，或者，小于50纳米和小于100纳米的磁珠混合，或者小于50纳米和小于500纳米的磁珠混合，以及其它任意粒径纳米颗粒的磁珠混合进行核酸的吸附。
多分散指数(polydispersity)是高分子科学中的一个重要概念，有时称分子量分布系数、非均匀指数、分散度等。最常见的定义为Mw/Mn(其中Mw、Mn分别为重均、数均分子量)该定义衡量分子量分布的宽度。有时也表示为Lw/Ln(其中Lw、Ln分别为重均、数均轮廓长度)，Dw/Dn(其中Dw、Dn分别为重均、数均粒子直径)；其中前者表示高分子轮廓长度的多分散性，后者表示高分子直径的多分散性。本发明所所说的分散系数指的是后者，主要指数均粒子直径。也是表示离子直径的均匀性关系。一般分散系数＜0.2，也可是是小于0.3,0.4,0.5,0.6,0.7,0.8,0.9，或者1，小于1.5，小于2的都可以。
在传统技术中，利用磁珠进行核酸提取，无论是哪种样本，都需要对样本中含有核酸的细胞、组织、器官进行裂解，裂解的作用是释放核酸物质，然后利用磁珠进行吸附，进行吸附后，一般分为两种路径，一种是用溶解核酸的溶液(例如水或者其它试剂)从磁珠上洗脱下来核酸物质，然后再把含有核酸物质的溶液进行添加到扩增核酸所必要的试剂中进行扩增，另外一种就是从裂解溶液中分离出磁珠，然后对磁珠进行洗涤，去掉洗涤液，最终让磁珠添加到核酸扩增的试剂中进行扩增(例如中国发明专利申请200610022581所揭示的方法，但是该专利仅仅是针对血清样本，而且需要对磁珠做扩增前的清洗)。但是，现有技术转中国，无论如何，都需要对磁珠进行洗涤后进行核酸的扩增，洗涤的过程可以是溶解磁珠上的核酸，也可以是去掉磁珠吸附的非核酸物质，减少杂质对扩增的影响。
而本发明经过试验，发现无需对核酸进行任何步骤的洗脱或者洗涤，直接可以把从裂解液里分离的磁珠直接添加到核酸扩增的试剂或者反转录酶中进行反转录后的扩增就可以发生核酸的扩增，从而简化了核酸提取的步骤，可以实现全自动化核酸的提取和检测合为一体。这里所说的“清洗或者洗涤”可以表示同一个意思，就是磁珠吸附核酸后，需要对磁珠进行二次处理，以去除表面吸附的杂质或者溶解磁珠表面吸附的核酸物质的步骤。这是因为，样本中含有多种不同种 类的核酸模板，磁珠吸附一般对核酸没有选择性，只要是核酸，都会吸附到磁珠表面，吸附后，现有技术洗脱或者洗涤都是为了去除蛋白、多糖、酚类物质，或者去掉其它非靶核酸的模板。例如，为了检测流感病毒，但是样本中可能含有其它病毒或者细菌，例如StrepA,RSV等。这些病毒或者细菌的DNA或者RNA也会吸附在磁珠上，虽然设计扩增引物的时候，需要考虑特异性和灵敏度的问题，但是如果目的模板含量少或者纯度不够，也会影响核酸后期的扩增，特别是其它非靶核酸的模板大量存在，也会影响后期的扩增。
另外，对于RNA核酸来讲，传统的核酸需要进行多个步骤的纯化和提取，因为RNA比较容易降解，所以，在进行反转录的时候，都需要增加保护试剂来进行RNA的保护，防止降解，提取过程繁琐，步骤多耗材大，而且效果不好，因为提取周期越长，需要保护试剂进行RNA的保护，另外，提取的最终效果并不好。而本发明直接用磁珠进行吸附后，意外的发现，直接用磁珠和反转录酶接触后，去掉反转录的试剂，然后让磁珠直接和扩增试剂接触，都可以或者有效的靶核酸的扩增；或者同时与反转录酶和扩增试剂基础，进行核酸的反转率和扩增，不仅不影响提取效果，而且可以达到检测目的。
而本发明小组意外的发现，用磁珠对核酸进行吸附后，不用对磁珠进行清洗或者洗涤，直接让磁珠和扩增试剂接触就可以实现特异性和灵敏度的扩增，无论对血液样本，还是拭子样本，还是唾液、尿液样本都是可以的。
在一些优选的方式中，本发明的提供核酸提取的裂解液，该裂解液包括一种后者几种金属离子的化合物，优选的为依价金属离子，例如钾离子，钠离子，钙离子。优选的，还可以包括一种或者几种表面活性试剂。在一些优选的方式中，所述的一价金属离子的化合物包括NaCl，KCl，LiCl中的一种或者多种，表面活性试剂包括吐温和Triton-X等表面活性物质。在一些优选的方式中，一价金属离子的化合物的浓度为0.000001-3M，表面活性试剂的浓度为0.1-0.5％(质量百分比)。在一些优选的方式中，可以包括如下组分：0-2M的NaCl、0-0.3％的Triton-X(质量百分比)和0-0.2mM的KCl；或者只是含有NaCl，KCl中的一种或者两种都含有而不含有表面活性试剂。一般，NaCl的含量要高些，可以破坏细胞膜。而KCl其调节作用，让磁珠具有最大的吸附能力和溶液里的成分的调节，微量就可以了。
在一些优选的方式中，裂解液里包括磁珠颗粒和裂解试剂成分。在一些优选的方式中，当然，在这里，磁珠可以是其它体积，例如0.2-5ul，或者0.2-3ul，或者0.1-2ul(例如磁珠的浓度为10毫克每毫升)。在一些优选的方式中，或者，裂解液体除了含有上述金属离子盐之外，还包括磁珠，磁珠的质量百分比可以是0.0000001-0.1％毫克/ML之间，这里的裂解液可以不含任何盐的纯水和磁珠溶液混合。磁珠的含量及其微量，而是依靠磁珠的纳米颗粒来吸附吸附核酸物质作为扩增的模板。
在一些优选的方式中，提供的裂解试剂是一干粉的状态存在于试管中，然后把所需要的裂解的样本直接添加到试管等容器中进行裂解，然后再添加磁珠。或者，裂解试剂和磁珠都是以干的形式存在，然后直接让样本与干的混合物接触形成新的混合物，然后让磁珠从混合物中分离出来。分离磁珠的方式有多种方式，例如，当然这里的取出液体也可以自动采用吸管自动取出，让磁珠和其它液体成分的物质分离，从而仅仅保留磁珠在PCR管中。当然，可选的，也可以是采用保护套的铁器，让磁珠被吸附在保护套的外侧，然后把保护套放置在PCR管中，让铁器移开，磁珠被释放在PCR管中。在一些优选的方式中，这些裂解试剂和/或磁珠存在于PCR扩增的试管里。当用裂解液体裂解细胞后，去掉其它物质，仅仅保留磁珠在试管里，然后直接添加磁珠到PCR试管中或者直接添加扩增试剂进行核酸的扩增，比如等温扩增后者PCR扩增。或者，采用磁性吸附磁珠的工具吸附这些磁珠，或者采用吸铁在PCR试管外吸附这些磁珠，然后去掉裂解液体，然后直接在PCR试管中进行添加核酸扩增的必要试剂，从而进行PCR扩增。或者，利用含有吸附磁铁的试管(例如试管内含有磁铁)插入到PCR管中吸附这些磁珠到试管的表面，然后把磁珠释放(去掉磁铁)到另一个PCR试管中，然后再在含有磁珠的PCR试管中添加核酸扩增的必要试剂进行后续的扩增。本发明这里的所谓裂解的方式是可以是进行高温加热裂解。
在一些优选的方法中，磁珠悬浮液和裂解液混合在一起，然后添加到PCR管中，然后向PCR管中添加所需要裂解的样本，例如咽拭子，鼻拭子，痰液，脑脊液，房水等，然后对PCR管进行加热几分钟，然后在一定的温度度下(室温或者15-30度的温度下)下静止几分钟，然后从磁铁吸附住磁珠，去掉PCR管内的液体，保留磁珠，然后向PCR管中添加核酸扩增所必要的试剂，例如聚合酶、引 物、探针或者其他试剂。在此过程中，不需要对磁珠进行洗脱，直接用吸附在磁珠上的核酸作为扩增的模板。
在一些优选的方式中，裂解液体和磁珠悬浮液的混合物预选以混合的形式存在于PCR试管中，然后进行如上步骤的添加样本进行混合，随后加热裂解后，让磁珠和缓和液体分离，保留磁珠，不对磁珠进行任何的处理。
当然，除了以上试剂外，还可以添加任何裂解酶，或者其它化学试剂，例如强酸，强碱试剂。
在另一些方式中，本发明也可以采用化学的方式进行核酸的释放(而非高温加热)，例如是一些Tris盐酸，EDTA，表面活性试剂，蛋白酶等混合体作为裂解液体，然后这些裂解液体和磁珠悬浮液混合于PCR管中，然后加入样本(例如，咽拭子，鼻拭子，痰液，脑脊液，房水等)，混匀，静置；然后去掉试管中的液体，保留磁珠在PCR管中，最后直接在保留有磁珠的试管中添加核酸扩增的必要试剂进行核酸的扩增，例如PCR扩增。
在一些优选的方式中，以上的样本选在咽拭子，鼻拭子，痰液，脑脊液，房水等样本。优选的，这些咽拭子，鼻拭子样本保存于棉签上，或者采用溶液从棉签上洗脱下来的溶液中。洗脱的溶液一般是商业采集样本自带的溶液，例如友康病毒采样管中的病毒运输液，或者使用生理盐水进行洗脱。
核酸的扩增
用本发明的方法提出的核酸物质可以用于多种核酸的扩增方法。这些方法就是利用磁珠直接和核酸扩增必要的试剂混合，然后进行扩增，例如采用等温扩增和PCR扩增。等温方法扩增就是添加等温扩增所必要的试剂，吸附在磁珠上的核酸作为模板。如果采用PCR扩增就是添加PCR扩增所需要的必要试剂，吸附在磁珠上的核酸作为模板进行扩增。
已知各种等位核酸扩增技术，包括例如重组酶聚合酶扩增(RPA)、转录介导的扩增、基于核酸序列的扩增、信号介导的RNA扩增技术、链置换扩增、滚环扩增、环介导的DNA等温扩增、等温多重置换扩增、解链酶依赖性扩增、单引物等 温扩增、环解链酶依赖性扩增以及切口和延长扩增反应(参见US 2009/0017453)。聚合酶链反应是最广泛已知的方法，但区别在于其需要使用热循环以引起核酸链分离。这些扩增方法和其它扩增方法论述于以下中：例如，凡尼思(VanNess)等人，美国科学院院报(PNAS)2003，第100卷，第8期，第4504至4509页；谭(Tan)等人，分析化学(Anal.Chem.)2005，77，7984-7992；利泽德(Lizard)等人，自然生物技术(Nature Biotech.)1998，6，1197-1202；纳富(Notomi)等人，核酸研究(NAR)2000，28，12，e63；和克恩(Kurn)等人，临床化学杂志(Clin.Chem.)2005，51:10，1973-1981。有关这些常用扩增技术的其它参考文献包括例如美国专利第7,112,423号、第5,455,166号、第5,712,124号、第5,744,311号、第5,916,779号、第5,556,751号、第5,733,733号、第5,834,202号、第5,354,668号、第5,591,609号、第5,614,389号、第5,942,391号；和美国专利公开案第US20030082590号、第US20030138800号、第US20040058378号和第US20060154286号。所有上述文件都以引用方式并入本文中。
RPA是一种例示性等温核酸扩增方法。RPA采用称作重组酶的酶，其能够使寡核苷酸引物与双螺旋DNA中的同源序列成对。以此方式，DNA合成涉及试样DNA中的界定点。如果存在靶序列，则使用两种基因特异性引物开始指数式扩增反应。反应快速进展且在20到40分钟内从数个靶拷贝特异性扩增到可检测水平。RPA方法揭示于(例如)US 7,270,981、US 7,399,590、US 7,777,958、US 7,435,561、US 2009/0029421和PCT/US2010/037611中，所有案件都以引用方式并入本文中。
RPA反应含有蛋白质与所需用以支持系统的重组元件的活性的其它因子以及支持从与互补底物成对的寡核苷酸的3'末端DNA合成的因子的掺合物。重组系统的关键蛋白组份是重组酶自身，其可源自原核、病毒或真核来源。另外，然而，需要单链DNA结合蛋白质以在各种在反应中进行的交换交易期间使核酸稳定。由于许多底物的特征仍为部分双螺旋，所以特别需要具有链置换特征的聚合酶。在反应能够从痕量水平的核酸扩增的一些实施例中，可使用包括使用拥挤试剂(例如，聚乙二醇)和负载蛋白的活体外条件。已报告包含噬菌体T4UvsX重组酶、噬菌体T4UvsY负载试剂、噬菌体T4gp32和枯草芽孢杆菌(Bacillus subtilis)聚合酶I大片段的例示性系统。
可以溶液和/或干燥(例如，冻干)形式提供等温扩增反应的组份。在以干燥形式提供一种或一种以上组份时，还可使用再悬浮或重构缓冲液。
基于扩增反应的特定类型，反应混合物可含有缓冲液、盐、核苷酸和进行反应必需的其它组份。可在适合于反应的特定温度下培育反应混合物。在一些实施例中，将维度维持于80℃或以下，例如，70℃或以下、60℃或以下、50℃或以下、40℃或以下、37℃或以下或30℃或以下。在一些实施例中，将反应混合物维持于室温下。在一些实施例中，在整个反应时间内将混合物的摄氏温标温度改变小于25％(例如，小于20％、小于15％、小于10％或小于5％)和/或在整个反应时间内将混合物的温度改变小于15℃(例如，小于10℃、小于5℃、小于2℃或小于1℃)。
核酸的扩增也可以是任意其它的PCR方法，例如实时荧光定量PCR技术是1996年由美国Applied Biosystems公司推出的，该技术是指在PCR反应体系中加入荧光基团，利用荧光信号积累实时监测整个PCR进程，最后通过标准曲线对未知模板进行定量分析。
核酸的检测
检测扩增产物通常包括使用经标记探针，其足够互补且与对应于靶核酸的扩增产物杂交。因此，可通过使与扩增产物互补的经标记探针(例如荧光标记探针)杂交来检测扩增产物的存在性、量和/或特性。在一些实施例中，目标靶核酸序列的检测包括组合使用等温扩增方法和经标记探针，以便实时测量产物。在另一实施例中，目标扩增靶核酸序列的检测包括将扩增靶核酸转移到固体载体(例如膜)，和用与扩增靶核酸序列互补的探针(例如经标记探针)探测膜。在又一实施例中，目标扩增靶核酸序列的检测包括将经标记扩增靶核酸与探针杂交，所述探针以具有可寻址位置的预定阵列排列且与扩增靶核酸互补。通常，扩增反应中利用一种或一种以上引物。靶核酸的扩增涉及使靶核酸与一种或一种以上引物接触，所述引物能够使靶核酸杂交并引导靶核酸扩增。在一些实施例中，使试样与一对引物接触，所述引物包括均与靶核酸杂交的正向和反向引物。实时扩增监测反应期间发射的荧光作为与终点检测相反的扩增子产生的指示物。可在一些系统中观察反应的实时进展。通常，实时方法涉及荧光报告子的检测。通常，荧光报 告子的信号与反应中扩增产物的量成正比例增加。通过记录每一循环时荧光发射的量，可监测指数期期间的扩增反应，其中扩增产物的量的首次显著增加与靶模板的初始量相关。核酸靶标的起始拷贝数越高，就越快观察到荧光显著增加。
在一些实施例中，荧光标记的探针取决于试样的荧光共振能量转移(FRET)或荧光发射波长变化，其作为实时检测DNA探针与扩增靶核酸杂交的方法。例如，在不同探针(例如，使用HybProbes)上的荧光标记间或在相同探针(例如，使用分子信标或
Figure PCTCN2018077797-appb-000001
探针)上的荧光团与非荧光猝灭剂间发生的FRET可辨别与目标DNA序列特异性杂交且以此方式可检测试样中靶核酸的存在性和/或量的探针。在一些实施例中，用于辨别扩增产物的荧光标记的DNA探针具有光谱差异发射波长，由此可在同一反应管内(例如在多工反应中)对其加以区分。例如，多重反应容许同时检测两种或两种以上靶核酸、甚至另一核酸(例如对照核酸)的扩增产物。
在一些实施例中，利用同位素或非同位素标记以可检测方式标记对靶核酸具有特异性的探针；在替代实施例中，标记扩增靶核酸。探针可检测为靶核酸物质(例如靶核酸物质的扩增产物)的指示物。非同位素标记可(例如)包含荧光或发光分子、或酶、辅因子、酶底物或半抗原。可将探针与RNA、DNA的单链或双链制剂或二者的混合物一起培育，并测定杂交。在一些实例中，杂交导致(例如)经标记探针的可检测的信号变化，例如信号增加或减少。因此，检测杂交包含检测杂交期间或之后的经标记探针的信号相对于杂交之前的标记的信号的变化。
在一些方法中，可使用试纸条(flow strip)检测扩增产物。在一些实施例中，一种可检测标记产生颜色且第二标记是由固定抗体识别的表位。含有两种标记的产物将附接到固定抗体且在固定抗体的位置处产生颜色。基于此检测方法的分析可为(例如)可施加到整个等温扩增反应的试纸条(浸量尺)。阳性扩增将在试纸条上产生带，作为靶核酸物质扩增的指示物，而阴性扩增将不产生任何彩色带。在一些实施例中，可使用本文所揭示的方法对靶核酸的量(例如，拷贝数)进行近似定量。例如，可在平行反应中使已知量的靶核酸扩增且可比较从试样获得的扩增产物的量与在平行反应中获得的扩增产物的量。在一些实施例中，可在多重平行反应中使若干已知量的靶核酸扩增且可比较从试样获得的扩增产物的量与在平行反应中获得的扩增产物的量。假定试样中的靶核酸与平行反应中的靶 核酸以类似方式可为反应组份所利用，那么可使用所述方法对试样中的靶核酸的量进行近似定量。
本文所揭示方法的反应组份可以用于检测靶核酸的试剂盒的形式供应。在所述试剂盒中，适当量的一种或一种以上反应组份提供于一个或一个以上容器中或固持在衬底上。还可提供对靶核酸具有特异性的核酸探针和/或引物。例如，反应组份、核酸探针和/或引物可悬浮于水溶液中或呈冷冻干燥或冻干粉末、丸粒或珠粒形式。供应所述组份等的容器可为能够保持所供应形式的任何常规容器，例如，微量离心管、安瓿瓶，或小瓶或综合测试装置，例如微流体装置、侧向流动或其它类似装置。试剂盒可包括经标记或未经标记的核酸探针，以用于检测靶核酸。在一些实施例中，试剂盒可进一步包括在本文所述方法(例如，使用粗基质而不进行核酸提取和/或纯化的方法)中使用所述组份的说明书。
在一些应用中，一种或一种以上反应组份可以预先测量的一次使用量提供于个别、通常一次性管或等效容器中。利用所述布置，可向个别管中添加待测试靶核酸的存在性的试样并直接实施扩增。试剂盒中供应的组份的量可为任何适当量，且可取决于产品所针对的目标市场。用于测定适当量的一般指南可参见音尼斯(Innis)等人、萨姆布鲁克(Sambrook)等人和奥苏伯尔(Ausubel)等人。
具体实施方式
以下结合附图和具体实施方式对本发明作进一步详细说明，实施例提供的方案为优选方案，是作为本领域的一般技术人员按照本发明的精髓来理解如何实现本发明的方案，但不作为对本申请的限定，本申请的范围体现在权利要求中。说明：在以下实施例子中所用的PCR扩增仪器为Bio-RAD CFX96或ABI 7500。
实施例1.0：对于咽拭子的样本的核酸的提取。
本发明方法应用于乙流(RNA的提取)临床样本的实时荧光定量PCR检测
(1)、配置成分为1MNaCl、0.01％Triton-X和0.001MKCl的裂解液100ml，按照磁珠和裂解液1:200的体积比加0.5ul(0.005毫克，浓度为0.00005毫克/ml)磁珠(其中，磁珠为无菌水溶解的500nm直径，磁核为Fe3O4，壳层为氧化硅的羟基超顺磁性磁珠，磁珠浓度为10mg/ml，购买自英芮诚生生化科技有限 公司)到上述配好的裂解液中混匀，取4个相同的专用PCR管，每管分装80ul配置好的混有磁珠的裂解液。当然，在这里，磁珠可以是其它体积，0.2-5ul，或者0.2-3ul，或者0.1-2ul；或者，这里的裂解液可以不含任何盐的纯水和磁珠溶液混合。
(2)、从绍兴疾控中心提供的2例乙流阳性和2例乙流阴性咽拭子四个样本中(四个样本为友康提供的(购买自友康恒业生物科技(北京)有限公司，病毒采样管，货号MT0301-1)病毒采样管采集的咽拭子或鼻拭子样本，采样的咽拭子或鼻拭子放入到3ml的友康公司提供的病毒运输液的采样管中)均取20ul分别加入到步骤(1)中的四个PCR管中分别作为1号样本、2号样本、3号样本和4号样本，盖好管盖；这样，每个试管的总体积为100ul。这里的病毒运输液可以是生理盐水，或者一些缓冲液，PBS缓冲液。四个样本中，已经确认的有2个样本为阳性，2个样本为阴性。
(3)、将步骤(2)中的四个PCR管放入在95℃中加热5min后室温放置10min；
(4)、将步骤(3)中的四个PCR管放入到磁珠架上静置2min，然后用移液器在磁珠对侧吸弃液体，仅仅保留磁珠。
(5)、在四个带有磁珠的PCR管中均分别加入扩增核酸所必要的试剂。在本例子中，为了扩增乙型流感，在试管中添加如下试剂：0.5ul(200U/ul)单位的逆转录酶(RNA反转录为c-DNA)和24.5ul的反应液(所述反应液包含浓度为30nmol/L的乙流的上下引物、30nmol/L乙流的探针、水和商业的一步法RT-QPCR试剂盒(TAKARA的货号为RR064A的One Step PrimeScript TMRT-PCR Kit(Perfect Real Time)，其中，引物和探针为本发明人自己设计：上游引物探针如下：GAGTCTTATCCCAATTTG(SEQ.1)；下游引物探针如下：GTGGAATAGTATGTTATCA(SEQ.2)；探针序列如下：HEX-AAGAGCACCGATTATCACCAG-TAMRA(SEQ.3)，其中所述探针的5’端连接有HEX荧光基因，3’端连接有TAMRA淬灭基团)，封盖后涡旋振荡摇匀30s，确保磁珠和PCR反应混合均匀；其中扩增试剂的成分如下表：
Figure PCTCN2018077797-appb-000002
Figure PCTCN2018077797-appb-000003
(6)将步骤(5)中的四个PCR管瞬时离心(1000-3000rmp下离心5-10s)使PCR管中液体都在底部；
(7)将上述步骤(6)得到的四个PCR管放置在PCR扩增仪器(选用的为Bio-RAD CFX96)中进行扩增，45℃，10min；95℃，10min；进行40-45循环的95℃，15S和60℃，45S，在60℃检测荧光信号。
实验结果如图1所示，1号、2号、3号和4号曲线分别为1号样本、2号样本、3号样本和4号样本的实验结果，结果表明，按照本实施例的方法进行操作，可以有效准确的检测出乙流的咽拭子样本，1号和2号为阳性，3和4号为阴性，与确认的样本的结果一致。
实施例子1.1：其它样本的提出和扩增。
同样，采用实施例子1中的相同的裂解液和磁珠(磁珠为无菌水溶解的500nm直径、400nm、300nm、200nm、100nm、600nm、700nm，裂解液与磁珠的体积比为1:200,1:150；1:100,1:300；1:400等处理，同时，裂解液的组分如下处理：0.01MNaCl，0.1MNaCl，0.5MNaCl，1MNaCl，1.5MNaCl，1.8MNaCl，2.0MNaCl，或者以上组分处理含有表面活性试剂；或者0.01MNaCl+0.001MKCl，0.1MNaCl+0.01MKCl，0.5MNaCl+0.1MKCl，1MNaCl+0.5MKCl，1.5MNaCl+0.8MKCl，1.8MNaCl+1.0MKCl，2.0MNaCl+1.5MKCl)进行咽拭子或者鼻拭子样本(先前确认为阳性的样本50份咽拭子，和20份鼻拭子)的核酸(RNA或者DNA)提取，吸附的核酸的磁珠不进行洗涤或者清洗，而直接进行甲型流感、StrepA、StrepB的检测(其中，扩增的引物和探针可以自己设计，也可以购买。检测这些样本的扩增试剂都可以商业购买，如果需要反转录(RNA样本)，添加反转录酶进行C-DNA的合成，然后对DNA进行PCR扩增。)，磁珠不需要进行洗脱就可以直接进行PCR扩增，获得50个阳性结果，和确认的阳性结果检测一致，符合率为100％。同样，平行对于阴性样本50份(咽拭子30或者鼻拭子样本20份)进行试验，本发明的方法提取的含有核酸的磁珠也获得了阴性结果，符合率为100％(具体数据略)。
实施例子1.2：其它样本的提出和扩增。
除了咽拭子，采用与本发明方法实施例子1.1中的磁珠提取方法，对样本为唾液，尿液，或脑脊液进行(这些150样本都经过标准方法确认含有是否含有三种病毒或者其中一种或者阴性样本)HSV-1，HSV-2，VZV，EBV,CMV,HSV-6等6种常见人疱疹病毒的检测(DNA)，利用公知的PCR扩增试剂和公知的引物序(具体数据略)列所含有的试剂进行常规扩增，都能和确认的样本的结果一致，符合率为100％。这充分说明，本发明的磁珠提取方法，不经过洗涤而直接让吸附有核酸的磁珠作为模板，都能成功检测出目标物质，达到检测的目的。而且，对于 液，尿液，或脑脊液样本都可以有效检测出。
实施例子1.3：其它样本的提出和扩增。
除了实施例子1和1.2和1.2中的样本外，采用与本发明方法实施例子1.1中的磁珠提取方法，对样本为血清进行(这些150样本都经过标准方法确认含有是否含有三种HBV，HCV中一种或者阴性样本)，利用公知的PCR扩增试剂和公知的引物序(具体数据略)列所含有的试剂进行常规扩增，都能和确认的样本的结果一致，符合率为100％。这充分说明，本发明的磁珠提取方法，不经过洗涤而直接让吸附有核酸的磁珠作为模板，都能成功检测出目标物质，达到检测的目的。而且，对于血清样本都可以有效检测出。
实施例子2:对于咽拭子的样本的核酸的提取。
本发明方法应用于乙流(RNA的提取)临床样本的实时荧光定量PCR检测与实施例子1.0相比,在步骤1和2中具有如下区别，其它过程都一样。区别如下：
(1)、配置成分为1MNaCl、0.01％Triton-X和0.001MKCl的裂解液100ml，按照磁珠和裂解液1:200的体积比加0.5ul(0.005毫克)磁珠混合溶液，取4个相同的专用PCR管，每管分装80ul配置好的混有磁珠的裂解液；然后进行真空冻干，保存备用。
(2)、从绍兴疾控中心提供的2例乙流阳性和2例乙流阴性咽拭子四个样本中(四个样本为友康恒业生物科技(北京)有限公司的病毒采样管采集的咽拭子或鼻拭子样本，采样的咽拭子或鼻拭子放入到3ml的病毒运输液(生理盐水)的采样管中。)均取20ul分别加入到步骤(1)中的四个PCR管中分别作为1号样本、2号样本、3号样本和4号样本，盖好管盖(四个样本中，已经确认的有2个样本为阳性，2个样本为阴性。)；进行充分混合，然后添加80微升的无菌水，进行实施例子1中的其它步骤，例如进行步骤3-7。
实验结果如图1类似，1号、2号、3号和4号曲线分别为1号样本、2号样本、3号样本和4号样本的实验结果，结果表明，按照本实施例的方法进行操作，可以有效准确的检测出乙流的咽拭子样本(具体实验结果略)。
实施例子3:对于咽拭子的样本的核酸的提取。
本发明方法应用于乙流临床样本的实时荧光定量PCR检测与实施例子1相比,在步骤1和2中具有如下区别，其它过程都一样。区别如下：
(1)、配置成分为1MNaCl、0.01％Triton-X和0.001MKCl的裂解液100ml，按照磁珠和裂解液1:200的体积比加1.0ml(0.01毫克)磁珠混合溶液，取4个相同的专用PCR管，每管分装80ul配置好的混有磁珠的裂解液；然后进行真空冻干，保存。在使用前，用80微升的无菌水(不含任何无机有机物质)溶解，让其充分分散和溶解；
(2)、从绍兴疾控中心提供的2例乙流阳性和2例乙流阴性咽拭子四个样本中(四个样本为友康病毒采样管采集的咽拭子或鼻拭子样本，采样的咽拭子或鼻拭子放入到3ml的病毒运输液(生理盐水)的采样管中)均取20ul分别加入到步骤(1)中的四个PCR管中分别作为1号样本、2号样本、3号样本和4号样本，盖好管盖；进行充分混合，进行实施例子1中的其它步骤，例如进行步骤3-7。
实验结果表明，扩增曲线和实施例子1类似，1号、2号、3号和4号曲线分别为1号样本、2号样本、3号样本和4号样本的实验结果，结果表明，按照本实施例的方法进行操作，可以有效准确的检测出乙流的咽拭子样本。而同样，阴性对照没有可见的扩增信号。
实施例4：本发明方法的敏感性测试
用实施例1步骤(2)中的阴性的乙流咽拭子样本作为稀释液(是RNA)，将 中国药品生物制品检定所的甲乙流国家标准中的乙流最低检测限参考品S22.0×10 6TCID 50/L进行稀释，分别获得2.0×10 4TCID 50/L，2.0×10 3TCID 50/L，2.0×10 2TCID 50/L，2.0×10 1TCID 50/L的四个样本，利用与实施1相同的方法对以上四个稀释样本进行实时荧光PCR检测(选用ABI7500进行PCR扩增)。
实验结果如图2所示：1号，2号，3号和4号曲线从上至下依次代表浓度为2.0×10 4TCID 50/L，2.0×10 3TCID 50/L，2.0×10 2TCID 50/L，2.0×10 1TCID 50/L的样本，结果表明，本发明可检测出的乙流的最低浓度为2.0×10 1TCID 50/L，符合国 家参考品的要求最低检测2.0×10 3TCID 50/L的浓度要求。
实施例5：本发明方法的荧光PCR检测的重复性测试
用实施例1中的阴性的乙流咽拭子样本作为稀释液，将 中国药品生物制品检 定所的甲乙流国家标准中的乙流最低检测限参考品S2 2.0×10 6TCID 50/L进行稀释，获得2.0×10 3TCID 50/L的样本。利用与实施1相同的方法对样本进行5孔复检实时荧光PCR检测(Bio-RAD CFX96进行扩增)。
检测结果如图3(图3中的1号到5号曲线为2.0×10 3TCID 50/L的乙流样本)和如下表：
Figure PCTCN2018077797-appb-000004
结果表明，本发明方法的检测可重复。
实施例6：本发明方法检测乙流与上海之江核酸提取试剂盒以甲、乙型流 感病毒联合测定试剂盒中检测乙流比较
取绍兴疾控中心给予的确认过的2例阳性的乙流咽拭子样本分别作为5号样本和6号样本，利用与实施例1.0相同的方法进行核酸的提取(Bio-RAD CFX96进行扩增)和上海之江生物科技有限公司的核酸提取试剂进行提取，提取的样本都用上海之江生物科技有限公司的甲、乙型流感病毒联合测定试剂盒进行 平行检测(货号Z-ME-0010/z-ME-0025)。
一，对同样的样本，采用上海之江生物科技有限公司的核酸提取试剂进行提取，方法如下：
1.配置结合液：6ul的RNA助沉剂、20ul的磁珠加入至500ul结合缓冲液，混匀。
2.加入140ul样本至526ul结合液。
a.取526ul结合液至1.5m离心管中。
b.在上述离心管中加入140ul样本，将吸头轻轻地浸入结合液中，以防止液体溅出导致的交叉污染。
c.涡旋振荡10s或反复颠倒5-10s(使磁珠均匀分散至缓冲液，完全裂解病毒，并使RNA和磁珠相结合)，静置3min以上。
3.吸取上述混匀静止体系666ul与亲和柱中，16000g(13000rpm)离心60s；弃去收集管废液。
4.亲和柱中加入500ul洗涤液A，16000g(13000rpm)离心40s；弃去收集管废液；重复一次
5.亲和柱中加入500ul洗涤液W，16000g(13000rpm)离心15s；弃去收集管废液；重复一次
6.亲和柱放入离心机中16000g(13000rpm)离心2min。
7. 50ul洗脱液洗脱RNA
a.亲和柱放入1.5ml无RNAnase的离心管中，加入50ul65℃预热洗脱液，室温放置2min。
b.16000g(13000rpm)离心2min，RNA被洗脱于无RNAnase的离心管中备用或将其保存于-20℃或-80℃。
二、而本发明的提取方法见实施例子1.0中的提取步骤1-4获得磁珠，样本为同样的样本。
三：采用上海之江生物科技有限公司的提供的甲、乙型流感病毒联合测定试剂盒(包括扩增所用的反转录酶以及扩增试剂以及引物、聚合酶等)进行检测以及扩增条件一致，获得的结果如下：
结果如下表：
Figure PCTCN2018077797-appb-000005
结果表明，本方法检测的结果与上海之江试剂盒检测的结果无显著差异。但是，RNA的提取过程，本发明试剂，而且花费的时间短，耗材少，简单快速。
实施例7：本发明方法检测RSV与广东华银医药科技有限公司的RSV核酸 检测试剂盒(包含核酸提取试剂)检测RSV的比较
用实施例1中的阴性的乙流的咽拭子样本(经广东华银医药科技有限公司的RSV核酸检测试剂盒检测RSV为阴性)作为稀释液，将绍兴疾控中心提供的RSV标准品的2.0×10 5TCID 50/L进行稀释，分别获得2.0×10 4TCID 50/L，2.0×10 3TCID 50/L，2.0×10 2TCID 50/L，2.0×10 1TCID 50/L的四个样本，利用与实施例1(Bio-RAD CFX96进行扩增)相同的方法进行提取(其中引物和探针换成RSV的引物和探针，采用相同的反转录酶进行C-DNA的转录，参考实施例子1的方法进行扩增)。
广东华银医药科技有限公司的RSV核酸检测试剂盒(包含核酸提取试剂)对四个样本分别检测，方法如下：
其中，广东华银医药科技有限公司的RSV核酸检测试剂盒中对核酸提取的方法如下：
1.取200ulRNA抽提A液于0.6ml离心管中，每管分别加入待检样本，阴性对照、强阳性对照和临界阳性对照各50ul，颠倒混匀5-10次使其从充分混匀，室温静 置3分钟。
2.加入200ulRNA抽提B液，颠倒混匀5-10次使其从充分混匀，13000rpm离心10分钟，去上清。
3.加入200ulRNA抽提C液，颠倒数次混匀，13000rpm离心5分钟，吸弃上清，开盖在室温或55℃放置2-5分钟晾干(到无明显液体为止，但不能太干，否则影响RNA溶解)。
4.加入25ul样本稀释液充分溶解沉淀物，短暂离心，是液体落于底部。
扩征的试剂与广东华银医药科技有限公司的RSV核酸检测试剂盒一样，最终体积和扩增试剂的含量也一样。
结果如图4、图5和下表：
Figure PCTCN2018077797-appb-000006
图4为本发明方法检测的结果，图4中的1号，2号，3号和4号四条曲线从是上到下分别是2.0×10 4TCID 50/L，2.0×10 3TCID 50/L，2.0×10 2TCID 50/L，2.0×10 1TCID 50/L样本的检测结果。图5为广东华银医药科技有限公司的RSV核酸检测试剂盒的检测结果，图5中的1号，2号，3号和4号四条曲线从上到下分别是2.0×10 4TCID 50/L，2.0×10 3TCID 50/L，2.0×10 2TCID 50/L，2.0×10 1TCID 50/L样本的检测结果。。
说明，本发明的扩增效果是现有的技术和效果相当，但是对于RNA的提取， 现有技术的步骤过于繁琐，而本发明采用磁珠，直接利用磁珠进行扩增，可实现有效的扩增。
实施例子8：本发明的方法与中国发明专利申请201610022581.8的提取方法的 比较试验
本发明的核酸提取按照采用化学方法进行样本的裂解，具体如下：裂解液配方：Tris浓度10-200mmol，EDTA浓度10-100mmol，曲拉通X-100浓度1-20％，硫氰酸胍浓度1-5mol，蛋白酶K浓度0.1-3mg/ml。
一、本发明的操作步骤如下：
1. 100ul的裂解液中加入0.5ul的磁珠，振荡混匀加入20ul的HBV DNA定量检测后(2X10 1IU/ML的HBV血清样本)的血清样本混匀，室温静置10min；
2.将上述管加入到磁力架中，静置1min或磁珠全部吸附在管壁上，用移液器吸弃上清；
3.将上述管取出，加入100ul的75％的乙醇，混匀后放置在磁力架中静置1min或磁珠全部吸附在管壁上，用移液器吸弃上清。
4.室温静置放置2-3min(或50-60℃放置1min)后加入PCR反应液(与对比试验的201610022581.8中的实施例子1中的核酸扩征试剂一样，扩增条件一样)进行PCR荧光扩增。
5. 201610022581.8的提取方法按照实该申请公开的施例子1进行同样样本的核酸提取和扩增。
6.结果对比如下：
本发明的提取方法可以有效检测出经过确认阳性的HBV血清样本，说明，本发明的核酸提取方法有效。相对对比实验所用的试剂简单，而且基本不用清洗磁珠。另外，采用本实验实施例子1的方法对血清样本进行提取，然后用201610022581.8公开的方法进行扩增，所用的扩增试剂和条件一样，也可以获得一致的结果。
二、对阳性样本进行稀释，同样，采用本发明的磁珠提取方法(化学)和对比公 开的实施例子2中的试剂进行提取和进行同样的扩增。
结果如下：
样本 本发明 对照实验
2X10 1IU/ML 阳性 阳性
20IU/ML 阳性 阳性
10IU/ML 阳性 未检测出
5IU/ML 阳性 未检测出
通过对样本的稀释，发现，本发明的方法的检测阀值达到5IU/ML，而对比的现有技术不能检测出。
实施例子9：本发明的方法与中国发明专利申请201610022581.8的提取方法的 比较试验
本发明的方法采用对咽拭子本进行加热方法进行磁珠提取，方法如下：
(配置成分为1MNaCl、0.01％Triton-X和0.001MKCl的裂解液100ml，按照磁珠和裂解液1:200的体积比加0.5uL磁珠(磁珠为无菌水溶解的500nm直径，磁核为Fe3O4，壳层为氧化硅的羟基超顺磁性磁珠，磁珠浓度为10mg/ml)到上述配好的裂解液中混匀，取4个相同的专用PCR管，每管分装80ul配置好的混有磁珠的裂解液；
(2)、从绍兴疾控中心提供的2例乙流阳性和2例乙流阴性咽拭子四个样本中(四个样本为友康(与实施例子1样)病毒采样管采集的咽拭子或鼻拭子样本，采样的咽拭子或鼻拭子放入到3ml的病毒运输液的采样管中)均取20ul分别加入到步骤(1)中的四个PCR管中分别作为1号样本、2号样本、3号样本和4号样本，盖好管盖；
(3)、将步骤(2)中的四个PCR管放入在95℃中加热5min后室温放置10min；
(4)将步骤(3)中的四个PCR管放入到磁珠架上静置2min，然后用移液器在磁珠对侧吸弃液体，保留磁珠。当然这里的取出液体也可以自动采用吸管自 动取出，让磁珠和其它液体成分的物质分离，从而仅仅保留磁珠在PCR管中，分离的方式可以采用多种。
(5)在四个PCR管中均加入扩增核酸所必要的试剂。在本例子中，为了扩增乙型流管，在试管中添加如下试剂：0.5ul(200U/ul)单位的逆转录酶和24.5ul的反应液(所述反应液包含与实施例子一样的浓度为30nmol/L的乙流的上下引物和30nmol/L乙流的探针、水和商业的一步法RT-QPCR试剂盒的必要试剂，具体扩征试剂与实施例子1相同)，封盖后涡旋振荡摇匀30s，确保磁珠和PCR反应混合均匀)；
(6)将步骤(5)中的四个PCR管瞬时离心(1000-3000rmp下离心5-10s)使PCR管中液体都在底部；
(7)将上述步骤(6)得到的四个PCR管放置在PCR扩增仪器(选用的为Bio-RAD CFX96)中进行扩增，45℃，10min；95℃，10min；进行40-45循环的95℃，15S和60℃，45S，在60℃检测荧光信号。
对照方法按照201610022581.8的提取方法按照实该申请公开的施例子1或者2进行同样样本的RNA提取(参照步骤1-4)，提取的样本中加入的扩增试剂与本发明同样的必要试剂：0.5ul(200U/ul)单位的逆转录酶和24.5ul的反应液(与实施例子一样)，封盖后涡旋振荡摇匀30s，确保磁珠和PCR反应混合均匀；然后进行同样方法的扩增。
结果比较：本发明的结果可以获得阳性的结果，而对照实验不能得到阳性的结果。这似乎说明，对照的方法对于血清样本(DNA)似乎可以或者阳性的结果，但是对于咽拭子样本确不能。对于DNA样本可行，但是对于RNA缺不能。
本发明说明书中提到的所有专利和出版物都表示这些是本领域的公开技术，本发明可以使用。这里所引用的所有专利和出版物都被同样列在参考文献中，跟每一个出版物具体的单独被参考引用一样。这里所述的本发明可以在缺乏任何一种元素或多种元素，一种限制或多种限制的情况下实现，这里这种限制没有特别说明。例如这里每一个实例中术语“包含”，“实质由……组成”和“由……组成”可以用两者之一的其余2个术语代替。这里采用的术语和表达方式所为描述方式，而不受其限制，这里也没有任何意图来指明此书描述的这些术语和解释排除了任 何等同的特征，但是可以知道，可以在本发明和权利要求的范围内做任何合适的改变或修改。可以理解，本发明所描述的实施例子都是一些优选的实施例子和特点，任何本领域的一般技术人员都可以根据本发明描述的精髓下做一些更改和变化，这些更改和变化也被认为属于本发明的范围和独立权利要求以及附属权利要求所限制的范围内。

Claims (24)

  1. 一种利用磁珠提取核酸物质的方法，包括以下步骤：
    (1)提供裂解试剂和纳米磁珠；所述的裂解试剂包括金属化合物；
    (2)将样本与所述的裂解试剂和磁珠接触形成混合物，处理后低温静置所述的混合物；
    (3)去除混合物中的其它物质，仅仅保留磁珠，同时不对磁珠进行任何的洗涤或者洗脱处理；
    (4)将步骤(3)的磁珠直接与核酸扩增的试剂接触，为靶核酸的扩增提供核酸模板，从而进行靶核酸的扩增。
  2. 根据权利要求1所述的方法，其中，所述的裂解试剂和磁珠为混合物，优选的为溶液混合物。
  3. 根据权利要求1或者2所述的方法，其中，所述的金属化合物包括NaCl、KCl中的一种或者两种。
  4. 根据权利要求1-3之一所述的方法，其中，所述的磁珠为单分散性羟基或者羧基磁珠。
  5. 根据权利要求1-4之一所述的方法，其中，所述的裂解试剂为溶液形式，该溶液的成分包括：0.1-2M的NaCl和0-2mM的KCl，或者质量百分比为0-0.3％的表面活性试剂。
  6. 根据权利要求1-5之一所述的方法，其中，样本与裂解液的体积比为1:3—1:10。
  7. 根据权利要求1-6之一所述的方法，其中，当裂解试剂和纳米磁珠为溶液的时候，所述的磁珠和裂解液的体积比1:200，其中磁珠的浓度为：0.1-100mg/ML。
  8. 根据权利要求1-7之一所述的方法，其中，所述的样本包括咽拭子、鼻拭子、喉拭子、血清、唾液、痰、尿液、血清中的一种或者几种。
  9. 根据权利要求8所述的方法，其中，所述的样本是经过处理过后的样本。
  10. 根据权利要求9所述的方法，其中，所述的咽拭子、鼻拭子或者喉拭子样本的载体经过生理盐水、缓冲液洗脱的样本。
  11. 根据权利要求1所述的方法，其中，所述的裂解试剂和磁珠为溶液状态，并位于PCR试管中，或者，所述的裂解试剂和磁珠为干的状态，并位于用于PCR扩增的PCR管中。
  12. 根据权利要求11所述的方法，其中，在让样本与混合物接触前，配备所述的混合物，混合物中包括羟基磁珠。
  13. 根据权利要求1所述的方法，其中，所述的磁珠的平均粒径小于1000nm。
  14. 根据权利要求1所述的方法，其中，让磁珠和裂解液和样本接触后或者之前，对样本或者混合有样本的磁珠和裂解液进行高温处理在60℃-100℃，或者，所述的低温为室温或者20℃-30℃之间。
  15. 根据权利要求1所述的方法，其中，当所提取的核酸为RNA的时候，让步骤(3)中的磁珠同时与反转录酶和核酸扩增试剂接触进行PCR扩增。
  16. 根据权利要求1所述的方法，其中，让样本同时与裂解试剂和磁珠接触。
  17. 根据权利要求1所述的方法，其中，当裂解试剂和磁珠为溶液混合物的时候，所述的磁珠的含量为0.005毫克，而所述的裂解液以溶液的状态存在，所述的裂解液的成分如下：0.1-2M的NaCl和0-2mM的KCl，或者质量百分比为0-0.3％的表面活性试剂，其中溶液的体积为80微升。
  18. 一种提取核酸的试剂，包括溶液，所述的溶液包括1MNaCl、质量比含量为0.01％T表面活性是和0.001MKCl的溶液和0.005毫克的磁珠。
  19. 根据权利要求16所述的试剂，其中，所述的磁珠为羟基修饰的磁珠，或者，磁珠的分散系数小于5。
  20. 一种提取核酸的试剂，包括裂解成份和磁珠，所述的裂解成份包括1MNaCl、0.01％Triton-X、0.001MKCl和0.005毫克的磁珠；所述的磁珠的平均粒径为≤1000nm。
  21. 一种提取核酸的试剂，包括裂解溶液和0.005毫克的磁珠，所述的裂解溶液包括1MNaCl、质量比含量为0.01％T表面活性是和0.001MKCl，当溶液为100毫升的时候，磁珠的含量为0.005毫克，或者，所述的磁珠的浓度为0.00005毫克 每毫升。
  22. 一种含有羟基修饰的纳米磁珠作为核酸提取试剂上的用途，其中所述的纳米磁珠的平均粒径为≤1000nm。
  23. 根据权利要求21所述的用途，所述的核酸为RNA。
  24. 根据权利要求21所述的用途，其中，当核酸与磁珠接触后，磁珠与扩征试剂接触前，不对磁珠进行任何的洗涤、清洗等处理步骤。
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