CN108315325A - A kind of method and reagent for extracting nucleic acid substances using magnetic bead - Google Patents

Abstract

The invention discloses a kind of methods for extracting nucleic acid substances using magnetic bead, include the following steps：（1）Lytic reagent and magnetic bead mix are provided；The lytic reagent includes：Monovalent metal compound；（2）Sample is contacted with mixture, oscillation shakes up after mixing in 80 100 DEG C of high-temperature process 10min, after be stored at room temperature 5 10min；（3）The other materials in mixture are removed, retain the nucleic acid substances on magnetic bead and magnetic bead, while not carrying out any washing or elution to magnetic bead；（4）Magnetic bead is directly contacted with the reagent of nucleic acid amplification, provided for nucleic acid amplification it is nucleic acid-templated, to carry out the amplification of target nucleic acid.The amplification template that there is provided in this way is not only fast and convenient, highly effective, and is not required to elution or washing during nucleic acid extraction to magnetic bead, simplifies operating process, has saved time and cost, can largely handle sample, and when operation detection can prevent from polluting.

Description

A kind of method and reagent for extracting nucleic acid substances using magnetic bead

This application claims formerly China's application, application numbers：2017101221522, the applying date：March 3 in 2017, it is preferential Power.

Technical field

The invention belongs to molecular diagnostics biological technical fields, and in particular to body is related to a kind of directly using biological sample The extraction that magnetic bead is calculated, to directly allow the reagent nuclear-magnetism pearl of amplification to contact, to complete the amplification of target target nucleic acid.

Background technology

Real-Time Fluorescent Quantitative PCR Technique is to be released by Applied Biosystems companies of the U.S. for 1996, the technology Refer to that fluorophor is added in PCR reaction systems, entire PCR processes are monitored in real time using fluorescence signal accumulation, finally by Standard curve carries out quantitative analysis to unknown template.

The culture of normal person's isthmus faucium portion should have Oral normal flora, and be grown without pathogenic bacteria.Pharyngeal bacterium is all from the external world, It is not pathogenic under normal circumstances, but body whole body or local resistance decline and other external factor under can occur infecting etc. and Lead to disease.Therefore, Pharyngeal swabs Bacteria Culture can isolate pathogenic bacteria, contribute to diphtheria, suppurative tonsillitis, acute pharynx The diagnosis of laryngitis etc..Throat swab detection is a kind of medical detecting method, is with medical cotton swab, pharyngeal from human body dips on a small quantity Secretion is inoculated in custom-made Petri dish, and the process cultivated in the equipment of temperature can be controlled by being then placed within one, often It is detected using Fluorescence PCR assay, it will be appreciated that conditions of patients, mucous membrane of mouth and pharyngeal infection conditions, but need to extract nucleic acid The time of nucleic acid, PCR is long and of high cost；Sample cannot be largely handled, operation detection has the risk of pollution.

Either throat or other samples, such as saliva, urine and nose swab sample carry in the sample Virus, bacterium or fungi or other biological cell component are required for carrying out nucleic acid when carrying out detection of nucleic acids The process of extraction, extraction is exactly that release obtains nucleic acid, while removing the interfering substance in tissue or cell, can be carried out in this way The amplification of follow-up effective target nucleic acid.

Existing extracted to the nucleic acid in sample using magnetic bead is a kind of reliable method, and be may be implemented certainly Dynamicization operates, and still, the extracting method of existing magnetic bead still seems more complicated, after generally using magnetic bead adsorption of DNA or RNA, It needs to clean magnetic bead, is then eluting nucleic acid substances from magnetic bead, for expanding, or clearly remove on magnetic bead The impurity of absorption, such as albumen, polysaccharide, other nucleic acid of other non-target nucleic acid templates also want some other particle components, though Right magnetic bead passes through chemical modification, can be adsorbed to nucleic acid, and still, the absorption of magnetic bead is not selective, chemistry and object In the case that reason is adsorbed while being had both.It actually requires a great deal of time to the extraction of sample nucleic acid and reagent cost, when When needs in a short time to carrying out nucleic acid amplification detection compared with multisample, it is still desirable to which a large amount of manpower carries out, and has artificial The increase of factor can cause many uncertainties, cannot realize automatic detection well.This with regard to it is in need to the prior art into Row improves, it is desirable to the detection that can be automated.

Invention content

It is existing to overcome the purpose of the present invention is to provide a kind of method and reagent for extracting nucleic acid substances using magnetic bead The deficiency of detection technique, is throat swab, saliva, urine and nose swab and serum equal samples provide it is a kind of quickly, it is easy, Efficiently, practical detection method, should not nucleic acid extraction process, simplify operating process, saved time and cost, and can be a large amount of Sample is handled, operation can prevent from polluting when detecting.

A kind of method and reagent for extracting nucleic acid substances using magnetic bead, including following step are provided to reach above-mentioned target Suddenly：

(1) lytic reagent and nanometer magnetic bead are provided；The lytic reagent includes metallic compound；

(2) sample is contacted with the lytic reagent and magnetic bead, stand at low temperature after processing；

(3) other materials in mixture are removed, only retain magnetic bead, at the same not to magnetic bead carry out any washing or Elution is handled；

(4) magnetic bead of step (3) is directly contacted with the reagent of nucleic acid amplification, provided for nucleic acid amplification it is nucleic acid-templated, from And carry out the amplification of target nucleic acid.

Preferably, the lytic reagent and magnetic bead are mixture, preferably solution mixture.

Preferably, the metallic compound includes one or two kinds of in NaCl, KCl.

Preferably, the magnetic bead is monodispersity hydroxyl or carboxyl magnetic bead.

Preferably, wherein the lytic reagent be lytic reagent and nanometer magnetic bead solution form, the solution at subpackage It includes：The KCl or mass percent of the NaCl and 0-2mM of 0.1-2M is the surface-active agents of 0-0.3%, wherein magnetic bead A concentration of 0.00005 mg/ml.

Preferably, the volume ratio of sample and lysate is 1:3—1:10.

Preferably, wherein when mixture is solution, the volume ratio 1 of the magnetic bead and lysate:200, wherein Magnetic bead it is a concentration of：0.1-100mg/ML,.

Preferably, wherein the sample includes throat swab, nose swab, throat swab, serum, saliva, phlegm, urine, serum In one or several kinds.

Preferably, the sample is the sample after processed.

Preferably, the carrier of the throat swab, nose swab or throat swab sample is eluted by physiological saline, buffer solution Sample.

Preferably, the lytic reagent and magnetic bead are solution state, and in PCR test tubes, alternatively, the cracking Reagent and magnetic bead are the state done, and in the PCR pipe for PCR amplification.

Preferably, wherein before allowing sample to be contacted with mixture, be equipped with the mixture, mixture includes hydroxyl Magnetic bead.

Preferably, wherein the average grain diameter of the magnetic bead is less than 1000nm.

Preferably, wherein allow after magnetic bead and lysate and sample contact and either to sample or to be mixed with sample before Magnetic bead and lysate carry out high-temperature process at 60 DEG C -100 DEG C, alternatively, the low temperature is between room temperature or 20 DEG C -30 DEG C.

Preferably, wherein when the nucleic acid extracted is RNA, allow magnetic bead in step (3) simultaneously and reverse transcriptase It is expanded with nucleic acid amplification agents contact.

Preferably, wherein sample is allowed to be contacted simultaneously with lytic reagent and magnetic bead.

On the other hand, the present invention provides a kind of reagent of extraction nucleic acid, including solution, the solution include 1MNaCl, It is solution with 0.001MKCl, 0.005 milligram of magnetic bead that quality, which is 0.01%T surface-actives than content,.Preferably, described in root Magnetic bead be hydroxyl modified magnetic bead, alternatively, the coefficient of dispersion of magnetic bead be less than 5.

On the other hand, the present invention provides a kind of reagent of extraction nucleic acid, including cracking composition and magnetic bead, and described is cracked into Part includes 1MNaCl, 0.01%Triton-X, 0.001MKCl and 0.005 milligram of magnetic bead；The average grain diameter of the magnetic bead is ≤1000nm。

On the other hand, the present invention provides a kind of reagent of extraction nucleic acid, including cracked solution and 0.005 milligram of magnetic bead, The cracked solution includes that than content to be 0.01%T surface-actives be and 0.001MKCl for 1MNaCl, quality.On the other hand, originally A kind of nanometer magnetic bead containing hydroxyl modified of invention offer is as the purposes on nucleic acid extracting reagent, wherein the nanometer magnetic bead Average grain diameter be≤1000nm.

On the other hand, the method that the present invention provides the nucleic acid extraction of sample, this method include：

(1) throat swab is prepared, saliva, urine and nose swab equal samples provide lysate and PCR reaction solution；

(2) sample is added in the PCR amplification pipe containing lysate, is vibrated after shaking up mixing at 80-100 DEG C of high temperature Manage 10min, after be stored at room temperature 10min；

(3) it is added after magnetic bead in the PCR amplification pipe in step (2) and reacts 5-10min at 18-28 DEG C；

(4) it will be put into magnetic frame after PCR amplification pipe brief centrifugation, by supernatant after magnetic bead is adsorbed onto magnetic frame side It is sucked out, and retains magnetic bead, then do not do any cleaning or washing step to magnetic bead；

(5) PCR reaction solution configured is added in PCR amplification pipe, carries out real-time quantitative PCR reaction.

Preferably, the Triton-X and 0- for NaCl, 0-0.3% that the ingredient of lysate is 0-2M in the step (1) The KCl of 0.2mM.

Preferably, the high temperature in the step (2) is 100 DEG C.

Preferably, the volume ratio of step (2) sample and lysate is 1:3—1:10.

Preferably, the magnetic bead is the hydroxyl magnetic bead that commercially available nucleic acid extraction uses, i.e. superparamagnetism core and inorganic oxygen The shell of SiClx, it is preferred that average grain diameter is the≤monodispersity hydroxyl magnetic bead of 1000nm.

Preferably, all reaction steps all carry out in the same test tube.

The beneficial effects of the invention are as follows：

For sample provide it is a kind of quickly, easy, efficiently, practical detection method, should not nucleic acid extraction process, simplify behaviour Make process, saved time and cost, and can largely handle sample, operation can prevent from polluting when detecting.The method of the invention Can be manually operated and mechanical automation operation, and should not nucleic acid extraction process, simplify operating process, saved the time with And cost.

Description of the drawings

Fig. 1 is that the method for the present invention of embodiment 1 is applied to the real-time fluorescence quantitative PCR testing result of second stream clinical sample Figure；

Fig. 2 is the susceptibility results figure of the positive second stream throat swab sample of the method for the present invention detection of embodiment 4；

Fig. 3 is the repeated result figure of the positive second stream throat swab sample of the method for the present invention detection of embodiment 5；

Fig. 4 is that the method for the present invention of embodiment 7 detects the result figure of RSV；

Fig. 5 is that the RSV kit for detecting nucleic acid of the Guangdong Hua Yin Pharmaceutical Technology Co., Ltd of embodiment 7 (is carried comprising nucleic acid Take reagent) detection RSV result figure.

It is described in detail

Sample

In some embodiments, sample includes Biosample, such as the sample obtained from plant or animal individual.As herein Biosample used includes all clinical samples that can be used for detecting the nucleic acid in individual comprising but it is not limited to cell, tissue (for example, lung, liver and nephridial tissue), Bone marrow aspirates, body fluid are (for example, blood, blood derivatives and blood fraction (such as serum Or yellow layer), urine, lymph, tear, prostatic fluid, cerebrospinal fluid, tracheal aspirate, phlegm, purulence, nasopharyngeal aspirate, oropharynx aspirate, saliva Liquid), eye swab, neck swab, vaginal swab, procto swab, stool and fecal suspension liquid.Other suitable samples include from middle ear The sample that liquid, BAL fluid, tracheal aspirate, phlegm, nasopharyngeal aspirate, oropharynx aspirate or saliva obtain.In spy Determine in embodiment, Biosample is obtained from animal individual.It can get the standard technique for obtaining the sample.Referring to (for example) applying Glug (Schluger) et al., The Journal of Experimental Medicine (J.Exp.Med.) 176:1327-33(1992)；Than lattice ratio (Bigby) etc. People, U.S.'s respiratory disorder comment on (Am.Rev.Respir.Dis.) 133:515-18(1986)；Kovacs (Kovacs) et al., New England Journal of Medicine (NEJM) 318:589-93(1988)；With Ao Nibeinei (Ognibene) et al., U.S.'s respiratory disorder Comment 129:929-32(1984).In some embodiments, sample includes environmental sample, such as surface sample (for example, passing through wiping Wipe or be vacuum-treated acquisition), air sample or water sample.In some embodiments, sample includes the cell of separation, such as dynamic Object, bacterium, fungi (for example, yeast) or plant cell and/or virus.Conventional method can be used and be suitable for cultivated cell type CMC model separation cell.

Processing for sample

In some modes, sample is typically all to be sampled using carrier, such as water imbibition carrier carries out carrying sample. Carrying sample can be suction pipe, storing apparatus, sampling apparatus, these sampling apparatuses draw sample component can be filter paper, cotton Label, etc. absorptive substances draw and carry carrier.With the present invention lysate contacted can be carrier, can also be from The sample mixtures got off are washed on carrier.For example, carrier contact of the cracking liquid prior to carrying sample, to which sample is cracked With elute, then allow cracking liquid mix with magnetic bead again, alternatively, allowing the mixture of lysate and magnetic bead and carrying sample Carrier contacts, and completes cracking and elution, then gets rid of carrier and other materials, then only retain magnetic bead, not to magnetic bead Do the further processing such as any elution and washing.It is of course also possible to get out lysate and magnetic bead solution in advance, then make again With when, then the mixing of two kinds of solution is in direct contact the latter with sample and is contacted with the carrier of carrying sample, again to complete sample This processing.

Certainly, exist if cracking ingredient and magnetic bead can be dry forms, it is then right after sample contact with liquid Sample cracks or carries out the absorption of magnetic bead, completes release and absorption to nucleic acid.

Here cracking liquid can be that some solions or solion and surface-active agents mix, these Cracking can cause the destruction of cell wall either cell membrane to reach the release of nucleic acid (DNA or RNA).Certainly, in order to Accelerated release in vitro and destruction, the method that physical heating may be used carry out, for example, high-temperature heating mode, such as 60 degree or more ask Topic, temperature is higher, and the time is shorter, and temperature is lower, and the time of cost is longer.Certainly, if broken containing enough in lytic reagent The ingredient of bad cell wall or cell membrane, such as strong acid, highly basic or some enzymatic reagents, these ingredients can be directly reached and be released The purpose of nucleic acid substances is put, physical heating or the method for ultrasonication can not be contacted.Physical method is usually to heat, and is ground Mill, ultrasonication, chemical method is usually chemical reagent composition.

In some preferred modes, the present invention reaches nucleic acid substances using a small amount of chemical reagent cooperation physical method The absorption of release and magnetic bead.Subsequent direct amplification can be interfered to avoid impurity in this way.In other preferred modes, use Chemical reagent carries out the release of nucleic acid, although being both to release some non-core acid substances, such as albumen, polysaccharide, these substances can It can interfere the amplification of junior scholar's nucleic acid, but magnetic bead using the present invention carries out the absorption of nucleic acid, it, still can be with even if carrying impurity Carry out normal nucleic acid amplification.

In another way, nucleic acid RNA.Generally, the amplification of RNA is firstly the need of transcriptive process,reversed is carried out, then to anti- The DNA of transcription is expanded.Group of the present invention finds, when release is RNA, using the method for group of the present invention, profit The absorption that RNA is directly carried out with magnetic bead, then removes lysate, retains magnetic bead, does not carry out any washing or clear to magnetic bead It washes, contact of the reverse transcription of C-NDA with amplifing reagent is directly carried out, to complete to expand.And the existing amplification for RNA, one As be all to need individually to carry out reverse transcription, then the DNA of reverse transcription carries out the amplification that concentration carries out nucleic acid, the processing of such process Time is long, and there are many independent steps in centre, and material consumption is big, and in addition step is more, and the chance of cross contamination increases.Separately Outside, existing technology is complicated to the extraction process of RNA, and cumbersome, consumptive material is more.In some preferred methods, the present invention, which utilizes, to be split The mixture of solution liquid and magnetic bead cracks RNA, then removes lysate and other materials, only retains magnetic bead, then allow Magnetic bead and reverse transcriptase and amplifing reagent contact, carry out expansion sign.It can effectively be expanded using such method, Huo Zhekuo The result of increasing.

In some preferred modes, allows lytic reagent and magnetic bead with after sample contact, carry out high-temperature heating treatment, then Low temperature is kept for a period of time.Alternatively, first carrying out high-temperature process to sample, lytic reagent and magnetic bead is then allowed to contact, then low temperature Kept for a period of time.It is appreciated that high-temperature process it is not necessary to, when lytic reagent contain the ion concentration of high concentration when It waits, cell membrane or cell wall can be destroyed, high-temperature process can not had in the case of this.

Target nucleic acid

Here one of purposes of so-called " target nucleic acid " exactly distinguishes biology and other biological sample carries out Differentiate.For example, a certain virus has distinctive educational background Ei, amplification and detection to too special sequence, to confirm the spy Different virus, without causing missing inspection and false positive or false negative.That is, in a particular embodiment, target nucleic acid is to mesh Indicating body has specificity, it is, not finding target nucleic acid in other organisms or similar with target organs Target nucleic acid is not found in organism.

Target nucleic acid can be to be stored in animal (for example, mankind), plant, fungi (for example, yeast), protozoan, bacterium or disease Nucleic acid in noxious material can also be the nucleic acid substances in sample above.For example, target nucleic acid can be stored in target organs It (for example, on chromosome) or is stored on extrachromosomal nucleic acid in genome.In some embodiments, target nucleic acid is RNA, such as mRNA.Target nucleic acid can be stored in bacterium (such as Gram (Gram) positive or gram-negative bacteria).Illustrative bacterial species Including acinetobacter (Acinetobacter sp.) strains A TCC 5459, Acinetobacte rcalcoaceticus, aerococcus viridans (Aerococcus viridans), bacteroides fragilis (Bacteroides fragilis), pertussis byrd bacteria (Bordetella pertussis), Bordetella parapertussis (Bordetella parapertussis), jejunum campylobacter bar Bacterium (Campylobacter jejuni), Clestridium difficile, C.perfringens (Clostridium Perfringens), corynebacterium (Corynebacterium sp.), chlamydia pneumoniae (Chlamydia Pneumoniae), chlamydia trachomatis, citrobacter freundii category (Citrobacter freundii), clostridium perfringen (Enterobacter aerogenes), Enterococcus gallinarum category (Enterococcus gallinarum), enterococcus faecium (Enterococcus faecium), enterococcus faecalis (Enterobacter faecalis) (for example, ATCC 29212), Ai Xi Family name Escherichia coli (for example, ATCC 25927), Gardnerella vaginalis, helicobacter pylori, haemophilus influenzae (Haemophilus influenzae) (for example, ATCC 49247), klepsiella pneumoniae (Klebsiella Pneumoniae lung Legionella (Legionella pneumophila) (for example, ATCC 33495), monocyte hyperplasia), are invaded Listeria (Listeria monocytogenes) (for example, ATCC 7648), micrococcus luteus (Micrococcus sp.) bacterium Strain ATCC 14396, moraxelle catarrhalis (Moraxella catarrhalis), mycobacterium kansasii (Mycobacterium Kansasii), mycobacterium gordonae (Mycobacterium gordonae), mycobacterium fortuitum (Mycobacterium Fortuitum), mycoplasma pneumoniae, mycoplasma hominis, Neisseria meningitidis (Neisseria meningitis) (for example, ATCC 6250), Neisseria gonorrhoea, oligella urethralis (Oligella urethralis), Pasteurella multocida (Pasteurella multocida), Pseudomonas aeruginosa (Pseudomonas aeruginosa) (for example, ATCC 10145), Cuo Sore Propionibacterium (Propionibacterium acnes), proteus mirabilis (Proteus mirabilis), common variation Bacillus (Proteus vulgaris), Salmonella strains ATCC 31194, salmonella typhimurium, serratia marcesens (Serratia marcescens) (for example, ATCC 8101), staphylococcus aureus (for example, ATCC 25923), epidermis Portugal Grape coccus (Staphylococcus epidermidis) (for example, ATCC 12228), S.lugdunensis (Staphylococcus lugdunensis), staphylococcus saprophyticus (Staphylococcus saprophyticus), pneumonia Streptococcus (for example, ATCC 49619), streptococcus pyogenes (Streptococcus pyogenes), Streptococcusagalactiae (Streptococcus agalactiae) (for example, ATCC 13813), Spirochaeta pallida (Treponema Palliduma), Streptococcus viridans (Viridans group streptococci) (for example, ATCC 10556), anthrax bud Spore bacillus (Bacillus anthracis), Bacillus cercus (Bacillus cereus), clam building Francisella (Francisella philomiragia) (GAO1-2810), francisella tularensis (Francisella Tularensis) (LVSB), yersinia pseudotuberculosis (Yersinia pseudotuberculosis) (PB1/+), small intestine knot Enteritis Yersinia (Yersinia enterocolitica), 0:9 serotypes or yersinia pestis (Yersinia pestis)(P14-).In some embodiments, target nucleic acid is stored in bacterium category substance selected from the following：Acinetobacter, balloon Pseudomonas (Aerococcus), Bacteroides (Bacteroides), byrd bacteria (Bordetella), campylobacter (Campylobacter), Clostridium (Clostridium), corynebacterium (Corynebacterium), clothing are former Body (Chlamydia), Citrobacter (Citrobacter), Enterobacter (Enterobacter), enterococcus spp (Enterococcus), Escherichia (Escherichia), screw rod Pseudomonas (Helicobacter), hemophilus (Haemophilus), klebsiella (Klebsiella), Legionnella (Legionella), growth (Listeria), Micrococcus (Micrococcus), Mobiluncus (Mobilincus), Moraxella (Moraxella), point Ramibacterium (Mycobacterium), mycoplasma, neisser's coccus, Oligella (Oligella), Pasteurella (Pasteurella), prevotella (Prevotella), rufous zygosaccharomyces (Porphyromonas), pseudomonad Belong to (Pseudomonas), Propionibacterium (Propionibacterium), proteus (Proteus), Salmonella, glue Matter Serratia (Serratia), staphylococcus, streptococcus, Treponema (Treponema), Bacillus (Bacillus), Francisella category (Francisella) or yersinia's genus (Yersinia).In some embodiments, Target nucleic acid is found in A group of streptococcus or B group of streptococcus.

Illustrative Chlamydia target nucleic acid includes the sequence found on Chlamydia cryptic plasmid.Illustrative mycobacterium tuberculosis (M.tuberculosis) target nucleic acid is included in IS6110 (referring to US 5,731,150) and/or IS1081 (referring to Ba Hade (Bahador) et al., 2005, agro-ecology scientific research magazine (Res.J.Agr.Biol.Sci.), 1:It is found in 142-145) Sequence.Illustrative Neisseria gonorrhoea target nucleic acid is included in NGO0469 (to be tieed up strange referring to skin Caro (Piekarowicz) et al., 2007, BMC microorganisms (BMC Microbiol.) 7:66) sequence and in NGO0470 found.Example The property shown A group of streptococcus target nucleic acids be included in Spy1258 (referring to Liu (Liu) et al., 2005, microbe research (Res.Microbiol), 156:564-567), hair in Spy0193, lytA, psaA and ply (referring to US 2010/0234245) Existing sequence.Illustrative B group of streptococcus target nucleic acid be included in cfb genes (referring to other Bielski (Podbielski) of baud et al., 1994, microorganism and immune medical journal (Med.Microbiol.Immunol.), 183:The sequence found in 239-256). In some embodiments, target nucleic acid is viral nucleic acid.For example, can be in human immunodeficiency virus (HIV), influenza virus or Dengue disease Viral nucleic acid is found in poison.Illustrative HIV target nucleic acids are included in the sequence found in the regions Pol.In some embodiments, target nucleus Acid is protozoan nucleic acid.For example, can be in Plasmodium (Plasmodium spp.), leishmania (Leishmania Spp.), Trypanosoma brucei gambiense (Trypanosoma brucei gambiense), Trypanosoma brucei rhodesiense (Trypanosoma brucei rhodesiense), schizotrypanum cruzi (Trypanosoma cruzi), Entamoeba (Entamoeba spp.), toxoplasma (Toxoplasma spp.), trichomonas vaginalis (Trichomonas vaginalis) With discovery protozoan nucleic acid in intestines shape flagellate (Giardia duodenalis).In some embodiments, target nucleic acid is to feed Newborn animal (for example, mankind) nucleic acid.For example, can find that lactation is dynamic in circulating tumor cell, epithelial cell or fibroblast Object nucleic acid.In some embodiments, target nucleic acid is fungi (for example, yeast) nucleic acid.For example, can be in Mycotoruloides (Candida Spp. fungal nucleic acid) is found in (for example, Candida albicans).Here target nucleic acid can be the terminal sequence on DNA, and can To be the sequence of a terminal specific in RAN reverse transcriptions.

Magnetic bead and extraction to nucleic acid

Magnetic bead provided by the present invention can be any magnetic bead.For general magnetic bead according to different classification, magnetic bead is a kind of Nano particle is generally connected with chemical group in particle surface, and material is thus formed the magnetic beads of final products.Such magnetic bead can To be bought by commercial sources, such as from Xiamen Pu Ruimai lattice bio tech ltd (http:// Www.purimagbead.com it) buys.General magnetic bead is classified according to the size of the diameter of magnetic bead, and another kind is according to surface The chemical group of package is classified.According to the size of diameter, average grain diameter can be divided into less than 50 sodium rices, 60 sodium rices, 80 sodium Rice, 90 sodium rices, 100 sodium rices, 150 sodium rices, 200 sodium rices, 300 sodium rices, 500 sodium rices, 600 sodium rices, 700 sodium rices, 800 sodium rices, 900 Sodium rice, 1000 sodium rices, 1500 sodium rices, the magnetic beads such as 2000 sodium rices.

According to the chemical group that magnetic bead wraps up, the property according to chemical group is different, can be divided into hydroxyl magnetic bead, carboxyl or The magnetic bead of person's amino magnetic bead or mixed group package.Magnetic bead or magnetic bead shell usually containing hydroxy polymer package The a large amount of silanols of surface modification (hydroxyl).Certainly, classify according to the Shape environment of magnetic bead, can be monodispersity magnetic Pearl, the non-monodispersity magnetic bead that can also be.

In some preferred modes, hydroxyl, carboxyl either one of amino or arbitrary combination modifying magnetic bead surfaces and Form hydroxyl magnetic bead, carboxyl magnetic bead either amino magnetic bead or the magnetic bead of any two combination modification and formation.It is preferred at some Mode in, magnetic bead is the hydroxyl magnetic bead or carboxyl magnetic bead that magnetic bead shell layer surface modifies a large amount of silanols (hydroxyl).

The magnetic bead used is uniform particle diameter or polydispersity coefficient ＜ 0.2.Preferably, selection is in monodisperse magnetic bead, in this way With superparamagnetism, magnetic response time ＜ 30s.So-called grain size " uniform " does not necessarily mean that the grain size one before each magnetic bead The grain size of sample, but in the same solution, at least 30% magnetic bead it is close or 35% magnetic bead grain size it is close； The grain size of 40% magnetic bead is close；The grain size of 45% magnetic bead is close；The grain size of 50% magnetic bead is close；The grain of 55% magnetic bead Diameter is close；The grain size of 60% magnetic bead is close；The grain size of 70% magnetic bead it is close or 75% magnetic bead grain size it is close；Or 80%, the grain size of 85%, 88%, 89%, 90%, 95%, 99%, 100% magnetic bead is close or is not much different.Alternatively, not Magnetic bead with grain size is mixed, for example, the magnetic bead less than 10 sodium rices and less than 50 sodium rices mixes, alternatively, less than 50 nanometers and Magnetic bead mixing less than 100 nanometers, or the magnetic bead mixing less than 50 nanometers and less than 500 nanometers and other arbitrary grain sizes The magnetic bead mixing of nano particle carries out the absorption of nucleic acid.

Polydispersity index (polydispersity) is a key concept in polymer science, sometimes referred to as molecular weight point Cloth coefficient, non-uniform index, dispersion degree etc..It is most common to be defined as Mw/Mn (wherein Mw, Mn attaches most importance to equal, the equal molecules of number respectively Amount) this definition weigh molecular weight distribution width.Being sometimes also indicated as Lw/Ln, (wherein Lw, Ln attaches most importance to equal, the equal profiles of number respectively Length), Dw/Dn (wherein Dw, Dn attach most importance to respectively equal, the equal particle diameters of number)；Wherein the former macromolecule profile length is more Dispersibility, the latter indicate the polydispersity of macromolecule diameter.The present invention the described coefficient of dispersion refer to the latter, cardinal index Equal particle diameter.It is also the uniform sexual intercourse for indicating ionic diameter.General Decentralized coefficient ＜ 0.2, can also be and be less than 0.3, 0.4,0.5,0.6,0.7,0.8,0.9 or 1, be less than 1.5, less than 2 can.

In the conventional technology, nucleic acid extraction, either which kind of sample are carried out using magnetic bead, be required for containing core in sample Cell, tissue, the organ of acid are cracked, and the effect of cracking is release nucleic acid substances, is then adsorbed, is carried out using magnetic bead After absorption, two kinds of paths are generally divided into, one is washed from magnetic bead with the solution (such as water or other reagents) of dissolving nucleic acid Nucleic acid substances are taken off, then the solution containing nucleic acid substances is added in reagent necessary to amplification of nucleic acid again and is carried out Amplification, another is exactly that magnetic bead is isolated from cracked solution, is then washed to magnetic bead, removes cleaning solution, finally allow Magnetic bead is added in the reagent of nucleic acid amplification and is expanded (such as 200610022581 revealed side of Chinese invention patent application Method, but the patent is only for serum sample, and need the cleaning before being expanded to magnetic bead).But the prior art turns China is required for carrying out the amplification of nucleic acid after washing magnetic bead, the process of washing can be on dissolving magnetic bead anyway Nucleic acid, can also be the non-core acid substance for removing magnetic bead absorption, reduce influence of the impurity to amplification.

And the present invention is through overtesting, finds the elution or washing without carrying out any step to nucleic acid, it directly can be From the magnetic bead detached in lysate be directly appended to nucleic acid amplification reagent or reverse transcriptase in carry out reverse transcription after amplification The step of to simplify nucleic acid extraction, the extraction and inspection of full-automatic nucleic acid may be implemented in the amplification that nucleic acid can occur Survey is integrated." cleaning or washing " mentioned here can indicate the same meaning, be exactly to be needed after magnetic bead absorption nucleic acid To magnetic bead carry out after-treatment, with remove adsorption impurity or dissolving magnetic bead surfaces absorption nucleic acid substances the step of. This is because different types of nucleic acid-templated containing there are many in sample, magnetic bead absorption is generally no to nucleic acid selective, as long as Nucleic acid can all be adsorbed onto magnetic bead surfaces, and after absorption, prior art elution or washing are provided to removing protein, polysaccharide, phenols Substance, or remove the template of other non-target nucleic acids.For example, in order to detect influenza virus, but may contain in sample other Virus or bacterium, such as StrepA, RSV etc..The DNA or RNA of these viral either bacteriums can be also adsorbed on magnetic bead, though So when design amplimer, need to consider the problems of specific and sensitivity, but if purpose template content it is few or Purity is inadequate, can also influence the amplification in nucleic acid later stage, and the template of especially other non-target nucleic acids largely exists, and can also influence the later stage Amplification.

In addition, for RNA nucleic acid, traditional nucleic acid needs to carry out the purifying and extraction of multiple steps, because of RNA ratios It is easier to degrade, so, when carrying out reverse transcription, it is required for increasing and reagent is protected to carry out the protection of RNA, prevent from dropping Solution, extraction process is cumbersome, and the more consumptive materials of step are big, and effect is bad, because extracting cycle is longer, needs that reagent is protected to carry out The protection of RNA, in addition, the final effect and bad of extraction.And after the present invention is directly adsorbed with magnetic bead, it is found surprisingly that, After directly using magnetic bead and reverse transcriptase contact, remove the reagent of reverse transcription, then allows magnetic bead directly and amplifing reagent contact, all may be used With or effective target nucleic acid amplification；Or simultaneously with reverse transcriptase and amplifing reagent basis, carry out nucleic acid Overturn ratio and Amplification, does not influence extraction effect not only, but also can reach testing goal.

And group of the present invention is found surprisingly that, after being adsorbed to nucleic acid with magnetic bead, without to magnetic bead carry out cleaning or Washing directly allows magnetic bead and amplifing reagent to contact the amplification that can realize specificity and sensitivity, no matter to blood sample, also It is swab sample or saliva, urine specimen to be all possible.

In some preferred modes, the lysate of offer nucleic acid extraction of the invention, the lysate includes a kind of the latter The compound of several metal ion species, preferably according to valence metal ion, such as potassium ion, sodium ion, calcium ion.Preferably, also May include one or several kinds of surface-active agents.In some preferred modes, the chemical combination of the monovalent metallic ion Object includes NaCl, KCl, one or more of LiCl, and surface-active agents include the surface-actives such as tween and Triton-X Substance.In some preferred modes, a concentration of 0.000001-3M of the compound of monovalent metallic ion, surface-active agents A concentration of 0.1-0.5% (mass percent).May include following component in some preferred modes：The NaCl of 0-2M, The KCl of Triton-X (mass percent) and 0-0.2mM of 0-0.3%；Either only contain one kind in NaCl KCl or Both of which contains and does not contain surface-active agents.Generally, the content of NaCl wants high, can destroy cell membrane.And KCl its tune Section act on, allow magnetic bead have maximum adsorption capacity and the ingredient in solution adjusting, it is micro can.

Include magnetic bead particles and lytic reagent ingredient in some preferred modes, in lysate.In some preferred sides In formula, certainly, herein, magnetic bead can be other volumes, for example, 0.2-5ul either 0.2-3ul or 0.1-2ul (such as A concentration of 10 milligrams every milliliter of magnetic bead).In some preferred modes, alternatively, cracking liquid in addition to contain above-mentioned metal from Further include magnetic bead except alite, the mass percent of magnetic bead can be, splitting here between 0.0000001-0.1% milligrams/ML Pure water and the mixing of magnetic bead solution of any salt can be free of by solving liquid.The content of magnetic bead and its micro, but rely on the nanometer of magnetic bead Particle adsorbs nucleic acid substances as the template expanded to adsorb.

In some preferred modes, the lytic reagent that provides is that the state of a dry powder is present in test tube, then institute The sample of the cracking needed is directly appended to be cracked in the containers such as test tube, then adds magnetic bead again.Alternatively, lytic reagent and Magnetic bead is all to exist in dry form, and sample is then directly allowed to contact to form new mixture with dry mixture, then allows magnetic Pearl is separated from mixture.There are many modes for the mode of separation magnetic bead, for example, taking-up liquid here can also be certainly certainly It is dynamic to be automatically taken out using suction pipe, allow the substance of magnetic bead and other liquid components to detach, to only retain magnetic bead in PCR pipe. Certainly, optionally, it can also be ironware using protective case, allow magnetic bead to be attracted to the outside of protective case, then protective case It is placed in PCR pipe, ironware is allowed to remove, magnetic bead is released in PCR pipe.In some preferred modes, these lytic reagents And/or magnetic bead is present in the test tube of PCR amplification.After with cracking liquid lytic cell, removes other materials, only retain magnetic Then pearl directly adds the amplification in magnetic bead to PCR test tubes or directly adding amplifing reagent progress nucleic acid in test tube, such as Isothermal duplication the latter's PCR amplification.Alternatively, these magnetic beads are adsorbed using the tool of magnetic absorption magnetic bead, or using suction iron in PCR These magnetic beads are adsorbed outside test tube, then removes cracking liquid, necessity of nucleic acid amplification is then directly added in PCR test tubes Reagent, to carry out PCR amplification.Alternatively, being inserted into PCR using the test tube (such as containing magnet in test tube) containing adsorption magnet These magnetic beads are adsorbed in pipe to the surface of test tube, and then magnetic bead is discharged and (removes magnet) into another PCR test tube, then again The necessary reagent that nucleic acid amplification is added in the PCR test tubes containing magnetic bead carries out subsequent amplification.The present invention here so-called is split The mode of solution be can carry out high-temperature heating cracking.

In some preferred methods, bead suspension and lysate mix, and are then added in PCR pipe, so The sample cracked required for being added in backward PCR pipe, such as throat swab, nose swab, sputum, cerebrospinal fluid, aqueous humor etc. are then right PCR pipe carries out heating a few minutes, then under certain temperature degree (at a temperature of room temperature or 15-30 degree) under static a few minutes Then clock lives magnetic bead from magnet adsorption, remove the liquid in PCR pipe, retains magnetic bead, nucleic acid amplification is then added into PCR pipe Necessary reagent, such as polymerase, primer, probe or other reagents.In the process, magnetic bead need not be washed It is de-, directly use the nucleic acid being adsorbed on magnetic bead as the template of amplification.

In some preferred modes, the mixture pre-selection for cracking liquid and bead suspension is present in the form of mixing In PCR test tubes, the addition sample for then carrying out step as above is mixed, then heating cracking after, allow magnetic bead and mitigate liquid Separation retains magnetic bead, any processing is not carried out to magnetic bead.

Certainly, other than the above reagent, any lyases or other chemical reagent, such as strong acid can also be added, by force Base reagent.

In other modes, the present invention can also use it is chemical by the way of carry out the release of nucleic acid (and non high temperature adds Heat), e.g. some Tris hydrochloric acid, EDTA, surface-active agents, the mixtures such as protease as cracking liquid, then these Cracking liquid and bead suspension are mixed in PCR pipe, and sample is then added (for example, throat swab, nose swab, sputum, brain ridge Liquid, aqueous humor etc.), mixing is stood；Then remove the liquid in test tube, retain magnetic bead in PCR pipe, finally directly remaining with The necessary reagent that nucleic acid amplification is added in the test tube of magnetic bead carries out the amplification of nucleic acid, such as PCR amplification.

In some preferred modes, above sample is selected in throat swab, nose swab, sputum, cerebrospinal fluid, the samples such as aqueous humor This.Preferably, these throat swabs, nose swab Sample preservation is on cotton swab, or is eluted from cotton swab using solution molten In liquid.The solution of elution is usually the virus transport liquid in the included solution of business collecting sample, such as friendly health virus sampling pipe, Or it is eluted using physiological saline.

The amplification of nucleic acid

The nucleic acid substances proposed with the method for the present invention can be used for the amplification method of multiple nucleic acids.These methods are exactly profit It is directly mixed, is then expanded, for example, by using isothermal duplication and PCR amplification with the necessary reagent of nucleic acid amplification with magnetic bead.Deng Warm method amplification is exactly reagent necessary to adding isothermal duplication, is adsorbed on the nucleic acid on magnetic bead as template.If using PCR Amplification is exactly to add the required necessary reagent of PCR amplification, and the nucleic acid being adsorbed on magnetic bead is expanded as template.

Known various equipotential nucleic acid amplification technologies include the expansion of such as recombinase polymeric enzymatic amplification (RPA), transcriptive intermediate The DNA etc. that increasing, the amplification based on nucleic acid sequence, the RNA amplification technology of signal mediation, strand displacement amplification, rolling circle amplification, ring mediate Warm amplification, isothermal multiple displacement amplification, unwinding enzyme dependent amplification, list primer isothermal duplication, ring unwinding enzyme dependent amplification with And notch and extend amplified reaction (referring to US 2009/0017453).Polymerase chain reaction is most widely known method, but area It is not that it is needed using thermal cycle to cause nucleic acid chain separation.These amplification methods and other amplification methods are discussed in following In：For example, Fan Nisi (VanNess) et al., National Academy of Sciences (PNAS) volume 2003,100, the 8th phase, the 4504th to Page 4509；Tan (Tan) et al., analytical chemistry (Anal.Chem.) 2005,77,7984-7992；Lize moral (Lizard) et al., Nature Biotechnol (Nature Biotech.) 1998,6,1197-1202；Na Fu (Notomi) et al., nucleic acids research (NAR) 2000,28,12, e63；With Kern (Kurn) et al., clinical chemistry magazine (Clin.Chem.) 2005,51:10,1973-1981. Other bibliography in relation to these common amplification techniques include such as U.S. Patent No. 7,112,423, the 5,455,166th Number, No. 5,712,124, No. 5,744,311, No. 5,916,779, No. 5,556,751, No. 5,733,733, the 5th, No. 834,202, No. 5,354,668, No. 5,591,609, No. 5,614,389, No. 5,942,391；And United States Patent (USP) Publication No. US20030082590, No. US20030138800, No. US20040058378 and US20060154286 Number.All above-mentioned files are all incorporated herein by reference.

RPA is a kind of illustrative isothermal nucleic acid amplification method.RPA can make few nucleosides using the enzyme for being referred to as recombinase Sour primer and the homologous sequence in duplex DNA are pairs of.By this method, DNA synthesis is related to defining a little in sample DNA.If There are target sequences, then use two kinds of gene-specific primer start index formula amplified reactions.Rapid reaction is in progress and 20 to 40 Specific amplification is copied to detectable level from several targets in minute.RPA methods are disclosed in (for example) US 7,270,981, US 7,399,590, in US 7,777,958, US 7,435,561, US 2009/0029421 and PCT/US2010/037611, own Case is all incorporated herein by reference.

RPA reactions support the active other factors for recombinating element and the support of system containing protein with required The admixture of the factor synthesized from the ends the 3' DNA of the oligonucleotides pairs of with complementary substrate.The key protein group of recombination system Part is recombinase itself, may originate from protokaryon, virus or eukaryotic source.In addition, however, it is necessary to single-stranded DNA binding protein matter with Make nucleic acid stability during the various exchange transaction carried out in the reaction.Since the feature of many substrates is still partial double helix, So especially needing the polymerase with strand displacement feature.It can be from some embodiments of the nucleic acid amplification of trace level reacting In, it includes the in vitro condition for using crowding agent (for example, polyethylene glycol) and loading albumen that can be used.It has reported comprising phagocytosis Body T4UvsX recombinases, bacteriophage T4 UvsY supported reagents, bacteriophage T4gp32 and bacillus subtilis (Bacillus Subtilis) the illustrative system of polymerase I large fragments.

The component of isothermal amplification can be provided in the form of solution and/or drying (for example, freeze-drying).It is carrying in a dry form When for one or more kinds of components, settling flux or reconstitution buffer also can be used.

Specific type based on amplified reaction, reaction mixture can contain buffer solution, salt, nucleotide and carry out reacting required Other components.Reaction mixture can be cultivated under the specific temperature for being suitable for reaction.In some embodiments, dimension is maintained In 80 DEG C or hereinafter, for example, 70 DEG C or less, 60 DEG C or less, 50 DEG C or less, 40 DEG C or less, 37 DEG C or less or 30 DEG C or less.In some embodiments, reaction mixture is maintained at room temperature.In some embodiments, in entire reaction In the Celsius' thermometric scale temperature of mixture changed be less than 25% (for example, less than 20%, less than 15%, less than 10% or be less than 5%) and/or within the entire reaction time by the temperature of mixture change and be less than 15 DEG C (for example, being less than 10 DEG C, being less than 5 DEG C, small In 2 DEG C or less than 1 DEG C).

The amplification of nucleic acid can also be any other PCR method, for example, Real-Time Fluorescent Quantitative PCR Technique be 1996 by What Applied Biosystems companies of the U.S. released, which refers to that fluorophor is added in PCR reaction systems, and utilization is glimmering Optical signal accumulation monitors entire PCR processes in real time, and quantitative analysis is carried out to unknown template finally by standard curve.

The detection of nucleic acid

Detection amplified production generally includes to use labeled probe, it is complementary enough and with the amplification production corresponding to target nucleic acid Object hybridizes.Therefore, can be expanded to detect by making the labeled probe (such as fluorescence labeling probe) with amplified production complementation hybridize Increase production existence, amount and/or the characteristic of object.In some embodiments, the detection of target target nucleic acid sequence includes being applied in combination Warm amplification method and labeled probe, to measure product in real time.In another embodiment, the inspection of target amplification target nucleic acid sequence Survey include target nucleic acid will be expanded to be transferred to solid carrier (such as film), and with amplifying target nucleic acid sequence complementation probe (such as Labeled probe) detection membrane.In another embodiment, the detection of target amplification target nucleic acid sequence includes by labeled amplification target nucleus Acid hybridize with probe, the probe with addressable point predetermined array arrange and with expand complementary target.In general, expanding Increase and utilizes one or more kinds of primers in reaction.The amplification of target nucleic acid is related to that target nucleic acid is made to connect with one or more kinds of primers It touches, the primer can make target nucleus acid hybridization and guide target nucleic acid amplification.In some embodiments, sample is made to be connect with pair of primers It touches, the primer includes the forward and reverse primer with target nucleus acid hybridization.The fluorescence emitted during real-time amplification monitoring reaction The indicant generated as the amplicon opposite with end point determination.Can observing response in some systems progresses in real time.In general, Real-time method is related to the detection of fluoreporter.In general, the signal of fluoreporter is directly proportional to the amount of amplified production in reaction Example increases.The amount of fluorescent emission when by recording each cycle, amplified reaction that can be during the Monitoring Index phase, wherein amplified production Amount dramatically increase for the first time it is related to the primary quantity of target template.The starting copy number of nucleic acid target is higher, observes sooner Fluorescence dramatically increases.

In some embodiments, the probe of fluorescent marker depends on the fluorescence resonance energy transfer (FRET) or fluorescence of sample Variation in emission wavelength, the method as real-time detection DNA probe and amplification target nucleus acid hybridization.For example, in different probe (example Such as, using HybProbes) on fluorescent marker between or same probe (for example, using molecular beacon orProbe) on Fluorogen and non-fluorescent quencher between the FRET that occurs can distinguish with target dna sequence specific hybrid and can examine by this method The probe of the existence and/or amount of target nucleic acid in test specimens.In some embodiments, the fluorescent marker for distinguishing amplified production DNA probe there is SPECTRAL DIVERSITY launch wavelength, thus (such as multiplexing reaction in) it can be subject in same reaction tube It distinguishes.For example, multiple reaction allows to detect two or more target nucleic acid, even another nucleic acid (such as control nucleic acid) simultaneously Amplified production.

In some embodiments, marked with detectable mode using isotope or nonisotopic labels has spy to target nucleic acid Anisotropic probe；In alternative embodiments, label amplification target nucleic acid.Probe can detect as target nucleus acid substance (such as target nucleic acid object The amplified production of matter) indicant.Nonisotopic labels can (for example) include fluorescence or light emitting molecule or enzyme, co-factor, enzyme bottom Object or haptens.Probe can be cultivated together with the single-stranded or double-stranded preparation of RNA, DNA or the mixture of the two, and measured miscellaneous It hands over.In some instances, hybridization causes the detectable signal intensity of (for example) labeled probe, such as signal increasing to add deduct It is few.Therefore, signal of the detection hybridization comprising the labeled probe during or after detection hybridization is relative to the label before hybridization Signal variation.

In certain methods, test strips (flow strip) can be used to detect amplified production.In some embodiments, a kind of It is the epitope identified by sessile antibody that detectable label, which generates color and the second label,.Containing there are two types of the products of label to be attached to Sessile antibody and the generation color at the position of sessile antibody.Analysis based on this detection method can be whole can be (for example) applied to The test strips (dip rod) of a isothermal amplification.Positive amplification will generate band in test strips, be expanded as target nucleus acid substance Indicant, and negative amplification will not generate any color ribbon.In some embodiments, the method disclosed herein pair can be used The amount (for example, copy number) of target nucleic acid carries out almost quantitative.For example, can make in parallel reaction the target nucleic acid amplification of known quantity and The amount of the amount and the amplified production obtained in parallel reaction of the comparable amplified production obtained from sample.In some embodiments In, the amount of the target nucleic acid amplification of several known quantities and the comparable amplified production obtained from sample can be made in multiple parallel reaction With the amount of the amplified production obtained in parallel reaction.It is assumed that the target nucleic acid in sample with the target nucleic acid in parallel reaction with similar Mode can be utilized by reaction component, then it is almost quantitative the method to can be used to carry out the amount of the target nucleic acid in sample.

The reaction component of methods disclosed herein can be used for detecting the form supply of the kit of target nucleic acid.In the examination In agent box, one or more kinds of reaction components of appropriate amount are provided in one or more containers or are retained on substrate On.The nucleic acid probe and/or primer that there is specificity to target nucleic acid can also be provided.For example, reaction component, nucleic acid probe and/or Primer can be suspended in aqueous solution or in freeze-drying or freeze-dried powder, pellet or bead form.Supply the appearance of described component etc. Device can by can keep supply form any conventional vessel, for example, microcentrifugal tube, ampoule bottle or bottle or comprehensive surveying Trial assembly is set, such as microfluidic device, lateral flow or other similar devices.Kit may include labeled or un-marked core Acid probe, for detecting target nucleic acid.In some embodiments, kit can further comprise methods described herein (for example, Using thick matrix without nucleic acid extraction and/or the method for purifying) the middle specification using the component.

In some applications, the first use amount that one or more kinds of reaction components can measure in advance is provided in a Not, in usually disposable pipe or equivalent container.Using the arrangement, the existence of target nucleic acid to be tested can be added into a don't bother about Sample and directly implement amplification.The amount for the component supplied in kit can be any appropriate amount, and may depend on product institute needle To target market.General guideline for measuring appropriate amount can be found in sound Nice (Innis) et al., Pehanorm Brooker And Ao Subaier (Ausubel) et al. (Sambrook) et al..

Specific implementation mode

Below in conjunction with the drawings and specific embodiments, invention is further described in detail, and the scheme that embodiment provides is Preferred embodiment is to understand the side for how realizing the present invention as those of ordinary skill in the art marrow according to the invention Case, but not as the restriction to the application, scope of the present application embodies in the claims.Explanation：In following embodiment PCR amplification instrument used is Bio-RAD CFX96 or ABI 7500.

Embodiment 1.0：Extraction for the nucleic acid of the sample of throat swab.

The real-time fluorescence quantitative PCR that the method for the present invention is applied to second stream (extraction of RNA) clinical sample detects

(1), it is configured to be divided into the lysate 100ml of 1MNaCl, 0.01%Triton-X and 0.001MKCl, according to magnetic bead With lysate 1:200 volume ratio adds 0.5ul (0.005 milligram, a concentration of 0.00005 milligram/ml), and (wherein, magnetic bead is magnetic bead The 500nm diameters of sterile water dissolution, magnetic core Fe3O4, shell are the hydroxyl Superparamagnetic beads of silica, and magnetic bead is a concentration of 10mg/ml, purchase is from Ying Ruichengsheng biochemical technologies Co., Ltd) to mixing in the above-mentioned lysate prepared, take 4 it is identical specially The lysate for being mixed with magnetic bead configured with PCR pipe, often pipe packing 80ul.Certainly, herein, magnetic bead can be other volumes, 0.2-5ul either 0.2-3ul or 0.1-2ul；Alternatively, lysate here can be free of any salt pure water and magnetic bead it is molten Liquid mixes.

(2), (four in the 2 second streams positives provided from Shaoxing Disease Control and Prevention Center and 2 second stream feminine gender four samples of throat swab Sample is (buying from friend Kang Heng industry biotechnology (Beijing) Co., Ltd, viral sampling pipe, article No. MT0301- for friendly Kandy confession 1) throat swab or nose swab sample, the throat swab or nose swab of sampling of viral sampling pipe acquisition are put into the friendly Kanggong department of 3ml In the sampling pipe of the virus transport liquid of offer) it takes in four PCR pipes that 20ul is added separately in step (1) respectively as 1 Number sample, No. 2 samples, No. 3 samples and No. 4 samples, cover pipe lid；In this way, the total volume of each test tube is 100ul.Here Virus transport liquid can be physiological saline or some buffer solutions, PBS buffer solution.In four samples, what is had been acknowledged has 2 Sample is the positive, and 2 samples are feminine gender.

(3), four PCR pipes in step (2) are placed in after heating 5min in 95 DEG C and are placed at room temperature for 10min；

(4), four PCR pipes in step (3) are put on magnetic bead frame and stand 2min, then use pipettor in magnetic bead pair Side, which is inhaled, abandons liquid, only retains magnetic bead.

(5), reagent necessary to amplification of nucleic acid is respectively added in the PCR pipe with magnetic bead at four.In the present example, In order to expand influenza B, following reagent is added in test tube：Reverse transcriptase (the RNA reverse transcriptions of 0.5ul (200U/ul) unit For c-DNA) and 24.5ul reaction solution (reaction solution include a concentration of 30nmol/L second stream primer up and down, 30nmol/ (article No. of TAKARA is the One Step of RR064A to the one-step method RT-QPCR kits of the probes of L second streams, water and business PrimeScript^TMRT-PCR Kit (Perfect Real Time), wherein primer and probe designs for the present inventor oneself： Sense primer probe is as follows：GAGTCTTATCCCAATTTG(SEQ.1)；Downstream primer probe is as follows： GTGGAATAGTATGTTATCA(SEQ.2)；Probe sequence is as follows：HEX-AAGAGCACCGATTATCACCAG-TAMRA (SEQ.3), wherein 5 ' ends of the probe are connected with HEX fluorogenes, 3 ' ends are connected with TAMRA quenching groups), whirlpool after capping Rotation oscillation shakes up 30s, it is ensured that magnetic bead and PCR reactions are uniformly mixed；The wherein ingredient of amplifing reagent such as following table：

(6) four PCR pipe brief centrifugations (centrifuging 5-10s under 1000-3000rmp) in step (5) are made into liquid in PCR pipe Body is all in bottom；

(7) four PCR pipes that above-mentioned steps (6) obtain are placed on PCR amplification instrument (selection is Bio-RAD CFX96 it is expanded in), 45 DEG C, 10min；95 DEG C, 10min；95 DEG C of progress 40-45 cycles, 15S and 60 DEG C, 45S, 60 DEG C detection fluorescence signal.

Experimental result as shown in Figure 1, No. 1, No. 2, No. 3 and No. 4 curve be respectively No. 1 sample, No. 2 samples, No. 3 samples and The experimental result of No. 4 samples, the results showed that, it is operated according to the method for the present embodiment, effectively can accurately detect second The throat swab sample of stream, No. 1 and No. 2 is the positive, and 3 and No. 4 are feminine gender, consistent with the result of the sample of confirmation.

Examples of implementation 1.1：The it is proposed and amplification of other samples.

Equally, use identical lysate in examples of implementation 1 and magnetic bead (magnetic bead for the 500nm diameters of sterile water dissolution, The volume ratio of 400nm, 300nm, 200nm, 100nm, 600nm, 700nm, lysate and magnetic bead is 1:200,1:150；1:100, 1:300；1:400 equal processing, meanwhile, the component of lysate is handled as follows：0.01MNaCl, 0.1MNaCl, 0.5MNaCl, 1MNaCl, 1.5MNaCl, 1.8MNaCl, 2.0MNaCl, or the processing of the above component contain surface-active agents；Or 0.01MNaCl+0.001MKCl, 0.1MNaCl+0.01MKCl, 0.5MNaCl+0.1MKCl, 1MNaCl+0.5MKCl, 1.5MNaCl+0.8MKCl 1.8MNaCl+1.0MKCl, 2.0MNaCl+1.5MKCl) carry out throat swab or nose swab sample Nucleic acid (the RNA or DNA) extraction of (being previously confirmed as positive 50 parts of throat swabs of sample and 20 parts of nose swabs), the core of absorption The magnetic bead of acid directly carries out detection (wherein, the amplification of Flu-A, StrepA, StrepB without washing or clean Primer and probe oneself can design, and can also buy.The amplifing reagent for detecting these samples can be commercially available, if needed Reverse transcription (RNA samples), addition reverse transcriptase is wanted to carry out the synthesis of C-DNA, then carry out PCR amplification to DNA.), magnetic bead is not required to PCR amplification can directly be carried out by carrying out elution, obtain 50 positive findings, consistent with the positive findings detection confirmed, symbol Conjunction rate is 100%.Equally, parallel that negative sample 50 parts (20 parts of throat swab 30 or nose swab samples) is tested, this The magnetic bead containing nucleic acid of the method extraction of invention also obtains negative findings, and coincidence rate is 100% (specific data summary).

Examples of implementation 1.2：The it is proposed and amplification of other samples.

In addition to throat swab, using with the magnetic bead extracting method in the method for the present invention examples of implementation 1.1, be saliva to sample, Urine or cerebrospinal fluid carry out that (these 150 samples all pass through standard method and confirm containing whether containing there are three types of virus or wherein one Kind or negative sample) HSV-1, HSV-2, VZV, EBV, CMV, the detection (DNA) of 6 kinds of common herpes virus hominis such as HSV-6, profit Conventional amplification is carried out with the contained reagent of well known PCR amplification reagent and well known primer sequence (specific data summary) row, it can It is consistent with the result of the sample of confirmation, coincidence rate 100%.This absolutely proves, magnetic bead extracting method of the invention, without washing It washs and directly allows the magnetic bead for being adsorbed with nucleic acid that can successfully detect target substance as template, achieve the purpose that detection.And And liquid, urine or CSF sample can be detected effectively.

Examples of implementation 1.3：The it is proposed and amplification of other samples.

Other than the sample in examples of implementation 1 and 1.2 and 1.2, using with the magnetic bead in the method for the present invention examples of implementation 1.1 Extracting method, to sample be serum carry out (whether these 150 samples all pass through standard method and confirm containing containing there are three types of HBV, A kind of or negative sample in HCV), contained by well known PCR amplification reagent and well known primer sequence (specific data summary) row Some reagents carry out conventional amplification, can be consistent with the result of the sample of confirmation, coincidence rate 100%.This absolutely proves, this hair Bright magnetic bead extracting method directly allows the magnetic bead for being adsorbed with nucleic acid as template without washing, can successfully detect mesh Substance is marked, achievees the purpose that detection.Moreover, can effectively be detected for serum sample.

Examples of implementation 2:Extraction for the nucleic acid of the sample of throat swab.

The method of the present invention is applied to the real-time fluorescence quantitative PCR detection and embodiment of second stream (extraction of RNA) clinical sample Son 1.0 is compared, and has following difference in step 1 and 2, other processes are all.Difference is as follows：

(1), it is configured to be divided into the lysate 100ml of 1MNaCl, 0.01%Triton-X and 0.001MKCl, according to magnetic bead With lysate 1:200 volume ratio adds 0.5ul (0.005 milligram) magnetic bead mixed solution, takes 4 identical special PCR pipes, often manages The lysate for being mixed with magnetic bead that packing 80ul has been configured；Then vacuum freeze-drying is carried out, is saved backup.

(2), (four in the 2 second streams positives provided from Shaoxing Disease Control and Prevention Center and 2 second stream feminine gender four samples of throat swab Sample is the throat swab or nose swab sample that the viral sampling pipe of friend Kang Heng industry biotechnology (Beijing) Co., Ltd acquires, sampling Throat swab or nose swab be put into the sampling pipe of virus transport liquid (physiological saline) of 3ml.) 20ul is taken to be added separately to Respectively as No. 1 sample, No. 2 samples, No. 3 samples and No. 4 samples in four PCR pipes in step (1), (four, pipe lid is covered In sample, 2 samples that have having been acknowledged are the positive, and 2 samples are feminine gender.)；It is sufficiently mixed, then adds 80 microlitres Sterile water, carry out other steps in examples of implementation 1, such as carry out step 3-7.

Experimental result such as Fig. 1 is similar, No. 1, No. 2, No. 3 and No. 4 curve be respectively No. 1 sample, No. 2 samples, No. 3 samples and The experimental result of No. 4 samples, the results showed that, it is operated according to the method for the present embodiment, effectively can accurately detect second The throat swab sample (summary of specific experiment result) of stream.

Examples of implementation 3:Extraction for the nucleic acid of the sample of throat swab.

The real-time fluorescence quantitative PCR that the method for the present invention is applied to second stream clinical sample detects

Compared with examples of implementation 1, there is following difference in step 1 and 2, other processes are all.Difference is as follows：

(1), it is configured to be divided into the lysate 100ml of 1MNaCl, 0.01%Triton-X and 0.001MKCl, according to magnetic bead With lysate 1:200 volume ratio adds 1.0ml (0.01 milligram) magnetic bead mixed solution, takes 4 identical special PCR pipes, often manages The lysate for being mixed with magnetic bead that packing 80ul has been configured；Then vacuum freeze-drying is carried out, is preserved.Before use, with 80 microlitres of nothing Bacterium water (being free of any inorganic organic substance) dissolving, makes its fully dispersed and dissolves；

(2), (four in the 2 second streams positives provided from Shaoxing Disease Control and Prevention Center and 2 second stream feminine gender four samples of throat swab Sample is that the throat swab of friendly health virus sampling pipe acquisition or nose swab sample, the throat swab or nose swab of sampling are put into 3ml's In the sampling pipe of virus transport liquid (physiological saline)) it takes in four PCR pipes that 20ul is added separately in step (1) and makees respectively For No. 1 sample, No. 2 samples, No. 3 samples and No. 4 samples, pipe lid is covered；It is sufficiently mixed, carries out its in examples of implementation 1 Its step, such as carry out step 3-7.

The experimental results showed that amplification curve is similar with examples of implementation 1, No. 1, No. 2, No. 3 and No. 4 curve is respectively No. 1 sample The experimental result of sheet, No. 2 samples, No. 3 samples and No. 4 samples, the results showed that, it is operated according to the method for the present embodiment, it can Effectively accurately to detect the throat swab sample of second stream.And it is same, negative control does not have visible amplified signal.

Embodiment 4：The sensitivity tests of the method for the present invention

Use the negative second stream throat swab sample in 1 step of embodiment (2) as dilution (being RNA), it willChinese drug Biological products assay instituteFirst and second stream national standards in second stream minimum detection limit reference material S2 2.0 × 10⁶TCID₅₀/ L is carried out Dilution obtains 2.0 × 10 respectively⁴TCID₅₀/ L, 2.0 × 10³TCID₅₀/ L, 2.0 × 10²TCID₅₀/ L, 2.0 × 10¹TCID₅₀/L Four samples, using with implement 1 identical method to above four diluted samples carry out real-time fluorescence PCR detection (select ABI7500 carries out PCR amplification).

Experimental result is as shown in Figure 2：No. 1, No. 2, No. 3 and No. 4 curve represents a concentration of 2.0 successively from top to bottom × 10⁴TCID₅₀/ L, 2.0 × 10³TCID₅₀/ L, 2.0 × 10²TCID₅₀/ L, 2.0 × 10¹TCID₅₀The sample of/L, the results showed that, this The minimum concentration for inventing detectable second stream is 2.0 × 10¹TCID₅₀/ L meets the requirement lowest detection 2.0 of National reference ×10³TCID₅₀The concentration requirement of/L.

Embodiment 5：The reperformance test of the fluorescent PCR detection of the method for the present invention

Use the negative second stream throat swab sample in embodiment 1 as dilution, it willNat'l Pharmaceutical ＆ Biological Products Control Institute First and second stream national standards in second stream minimum detection limit reference material S2 2.0 × 10⁶TCID₅₀/ L is diluted, and acquisition 2.0 × 10³TCID₅₀The sample of/L.5 holes reinspection real-time fluorescence PCR detection (Bio- is carried out to sample using with 1 identical method of implementation RAD CFX96 are expanded).

(No. 1 to No. 5 curve in Fig. 3 is 2.0 × 10 by testing result such as Fig. 3³TCID₅₀The second stream sample of/L) and it is as follows Table：

The result shows that the detection of the method for the present invention is repeatable.

Embodiment 6：The method of the present invention is detected second stream and is joined with first, influenza B virus with Shanghai Zhijiang River nucleic acid extraction kit Detection second stream in assay kit is closed to compare

The second stream throat swab sample for taking acknowledged 2 that Shaoxing Disease Control and Prevention Center is given positive is respectively as No. 5 samples and 6 Number sample, using method identical with embodiment 1.0 carry out nucleic acid extraction (Bio-RAD CFX96 are expanded) and Shanghai it The nucleic acid extracting reagent of river bio tech ltd extracts, and the sample of extraction all uses the limited public affairs of Shanghai Zhijiang River biotechnology First, the influenza B virus combined test kit of department carries out Parallel testing (article No. Z-ME-0010/z-ME-0025).

One, it to same sample, is extracted using the nucleic acid extracting reagent of Shanghai Zhijiang Biological Science Co., Ltd, side Method is as follows：

1. configuration combines liquid：The RNA settling agents of 6ul, the magnetic bead of 20ul are added to 500ul combination buffers, mixing.

2. 140ul samples are added to 526ul combination liquid.

A. it takes in 526ul combinations liquid to 1.5m centrifuge tubes.

B. 140ul samples are added in above-mentioned centrifuge tube, suction nozzle is lightly immersed and is combined in liquid, to prevent liquid from splashing out Caused cross contamination.

C. vortex oscillation 10s or repeatedly overturn 5-10s (keep magnetic bead evenly dispersed to buffer solution, complete lytic virus, and make RNA and magnetic bead are combined), stand 3min or more.

3. drawing the above-mentioned static system 666ul of mixing with affinity column, 16000g (13000rpm) centrifuges 60s；Discard receipts Collector waste liquid.

4. 500ul cleaning solutions A, 16000g (13000rpm) are added in affinity column centrifuges 40s；Discard collecting pipe waste liquid；Weight It is multiple primary

5. 500ul cleaning solutions W, 16000g (13000rpm) are added in affinity column centrifuges 15s；Discard collecting pipe waste liquid；Weight It is multiple primary

6. affinity column is put into 16000g in centrifuge (13000rpm) centrifugations 2min.

7.50ul elution RNA

A. affinity column is put into centrifuge tubes of the 1.5ml without RNAnase, and 50ul65 DEG C of preheating eluent is added, is placed at room temperature for 2min。

B.16000g (13000rpm) centrifuges 2min, and RNA is eluted spare in the centrifuge tube of no RNAnase or is protected It is stored in -20 DEG C or -80 DEG C.

Two and the extracting method of the present invention is shown in that the extraction step 1-4 in examples of implementation 1.0 obtains magnetic bead, sample is same Sample.

Three：Using first, the influenza B virus combined test kit of the offer of Shanghai Zhijiang Biological Science Co., Ltd (including the reverse transcriptase used in amplification and amplifing reagent and primer, polymerase etc.) is detected and amplification condition one It causes, the result of acquisition is as follows：

As a result such as following table：

The result shows that the result that the result of this method detection is detected with Shanghai Zhijiang River kit is without significant difference.But The extraction process of RNA, reagent of the present invention, and also the time spent is short, and consumptive material is few, simple and quick.

Embodiment 7：The method of the present invention detects the RSV nucleic acid detection reagents of RSV and Guangdong Hua Yin Pharmaceutical Technology Co., Ltd The comparison of box (including nucleic acid extracting reagent) detection RSV

With throat swab sample (the RSV cores through Guangdong Hua Yin Pharmaceutical Technology Co., Ltd of the negative second stream in embodiment 1 Acid detection kit detect RSV be feminine gender) be used as dilution, the RSV standard items that Shaoxing Disease Control and Prevention Center is provided 2.0 × 10⁵TCID₅₀/ L is diluted, and obtains 2.0 × 10 respectively⁴TCID₅₀/ L, 2.0 × 10³TCID₅₀/ L, 2.0 × 10²TCID₅₀/ L, 2.0×10¹TCID₅₀Four samples of/L are carried out using the identical method with embodiment 1 (Bio-RAD CFX96 are expanded) (wherein primer and probe changes the primer and probe of RSV into, and the transcription of C-DNA, reference are carried out using identical reverse transcriptase for extraction The method of examples of implementation 1 is expanded).

The RSV kit for detecting nucleic acid (including nucleic acid extracting reagent) of Guangdong Hua Yin Pharmaceutical Technology Co., Ltd is to four samples This is detected respectively, and method is as follows：

Wherein, in the RSV kit for detecting nucleic acid of Guangdong Hua Yin Pharmaceutical Technology Co., Ltd to the method for nucleic acid extraction such as Under：

1. 200ulRNA is taken to extract A liquid in 0.6ml centrifuge tubes, often pipe is separately added into sample to be checked, negative control, Qiang Yang Property control and each 50ul of critical positive control, reverse mixing makes it from mixing well 5-10 times, is stored at room temperature 3 minutes.

2. 200ulRNA, which is added, extracts B liquid, reverse mixing makes for 5-10 time it from mixing well, 10 points of 13000rpm centrifugations Clock removes supernatant.

3. 200ulRNA, which is added, extracts C liquid, mixing for several times is overturned, 13000rpm is centrifuged 5 minutes, and supernatant is abandoned in suction, is uncapped Room temperature or 55 DEG C are placed and are dried for 2-5 minute (to without apparent liquid until, but cannot be too dry, otherwise influence RNA dissolves).

4. 25ul Sample dilutions, which are added, fully dissolves sediment, of short duration centrifugation is that liquid falls within bottom.

Expand the reagent of sign as the RSV kit for detecting nucleic acid of Guangdong Hua Yin Pharmaceutical Technology Co., Ltd, final volume It is same with the content of amplifing reagent.

As a result such as Fig. 4, Fig. 5 and following table：

Fig. 4 be the method for the present invention detection as a result, No. 1, No. 2, No. 3 and No. 4 four curves in Fig. 4 from being that top to bottm is divided It is not 2.0 × 10⁴TCID₅₀/ L, 2.0 × 10³TCID₅₀/ L, 2.0 × 10²TCID₅₀/ L, 2.0 × 10¹TCID₅₀The detection of/L samples As a result.Fig. 5 is the testing result of the RSV kit for detecting nucleic acid of Guangdong Hua Yin Pharmaceutical Technology Co., Ltd, No. 1 in Fig. 5,2 Number, No. 3 and No. 4 four curves are 2.0 × 10 respectively from top to bottom⁴TCID₅₀/ L, 2.0 × 10³TCID₅₀/ L, 2.0 × 10²TCID₅₀/ L, 2.0 × 10¹TCID₅₀The testing result of/L samples..

Illustrating, expanding effect of the invention is that existing technology and effect are suitable, but for the extraction of RNA, existing skill The step of art, is excessively cumbersome, and the present invention uses magnetic bead, is directly expanded using magnetic bead, it can be achieved that effective amplification.

Examples of implementation 8：The ratio of the method for the present invention and the extracting method of Chinese invention patent application 201610022581.8 Compared with experiment

The nucleic acid extraction of the present invention is specific as follows according to the cracking for carrying out sample using chemical method：Crack formula of liquid： Tris concentration 10-200mmol, EDTA concentration 10-100mmol, triton x-100 concentration 1-20%, guanidine thiocyanate concentration 1- 5mol, proteinase K concentration 0.1-3mg/ml.

One, operating procedure of the invention is as follows：

In the lysate of 1.100ul be added 0.5ul magnetic bead, oscillation mixing be added 20ul HBV DNA quantitatively detect after (2X10¹The HBV serum samples of IU/ML) serum sample mixing, be stored at room temperature 10min；

2. above-mentioned pipe is added in magnetic frame, stands 1min or magnetic bead is all adsorbed on tube wall, is abandoned with pipettor suction Supernatant；

3. above-mentioned pipe is taken out, 75% ethyl alcohol of 100ul is added, is placed on after mixing in magnetic frame and stands 1min or magnetic Pearl is all adsorbed on tube wall, and supernatant is abandoned with pipettor suction.

PCR reaction solution is added afterwards (with contrast test 4. being stored at room temperature and placing 2-3min (or 50-60 DEG C of placement 1min) Nucleic acid expansion sign reagent in examples of implementation 1 in 201610022581.8 is the same, and amplification condition is the same) carry out PCR amplified fluorescences.

5.201610022581.8 the extracting method nucleic acid that carries out same sample according to example 1 is applied disclosed in real this application Extraction and amplification.

6. Comparative result is as follows：

The extracting method of the present invention can effectively detect, by the positive HBV serum samples of confirmation, to illustrate, of the invention Method for extracting nucleic acid is effective.Relative contrast's experiment reagent used is simple, and does not have to cleaning magnetic bead substantially.In addition, using this The method of EXPERIMENTAL EXAMPLE 1 extracts serum sample, and then the method disclosed in 201610022581.8 is expanded, Amplifing reagent used is as condition, it is also possible to obtain consistent result.

Two, positive sample is diluted, equally, disclosed in magnetic bead extracting method (chemistry) using the present invention and comparison Reagent in examples of implementation 2 is extracted and is similarly expanded.

As a result as follows：

Sample	The present invention	Control experiment
			2X101IU/ML	It is positive	It is positive
20IU/ML	It is positive	It is positive
			10IU/ML	It is positive	It does not detect
5IU/ML	It is positive	It does not detect

Pass through the dilution to sample, it is found that the detection threshold of method of the invention reaches 5IU/ML, and the existing skill compared Art cannot detect.

Examples of implementation 9：The ratio of the method for the present invention and the extracting method of Chinese invention patent application 201610022581.8 Compared with experiment

The method of the present invention is used carries out magnetic bead extraction to throat swab this progress heating means, and method is as follows：

It (is configured to be divided into the lysate 100ml of 1MNaCl, 0.01%Triton-X and 0.001MKCl, according to magnetic bead and split Solve liquid 1:200 volume ratio adds 0.5uL magnetic beads, and (magnetic bead is the 500nm diameters of sterile water dissolution, and magnetic core Fe3O4, shell is oxygen The hydroxyl Superparamagnetic beads of SiClx, a concentration of 10mg/ml of magnetic bead) to mixing in the above-mentioned lysate prepared, take 4 it is identical Special PCR pipe, the lysate for being mixed with magnetic bead that often pipe packing 80ul has been configured；

(2), (four in the 2 second streams positives provided from Shaoxing Disease Control and Prevention Center and 2 second stream feminine gender four samples of throat swab Sample is the throat swab or nose swab sample of the viral sampling pipe acquisition of friendly health (with 1 sample of examples of implementation), the throat swab or nose of sampling Swab is put into the sampling pipe of virus transport liquid of 3ml) it takes in four PCR pipes that 20ul is added separately in step (1) Respectively as No. 1 sample, No. 2 samples, No. 3 samples and No. 4 samples, pipe lid is covered；

(3), four PCR pipes in step (2) are placed in after heating 5min in 95 DEG C and are placed at room temperature for 10min；

(4) four PCR pipes in step (3) are put on magnetic bead frame and stand 2min, then use pipettor in magnetic bead pair Side, which is inhaled, abandons liquid, retains magnetic bead.Certainly taking-up liquid here can also be automatically taken out using suction pipe automatically, allow magnetic bead and other The substance of liquid component detaches, and to only retain magnetic bead in PCR pipe, the mode of separation may be used a variety of.

(5) reagent necessary to amplification of nucleic acid is added in four PCR pipes.In the present example, in order to expand B-mode stream Pipe, adds following reagent in test tube：Reaction solution (the reaction of the reverse transcriptase and 24.5ul of 0.5ul (200U/ul) unit Liquid include the second stream of a concentration of 30nmol/L as examples of implementation primer up and down and the probes of 30nmol/L second streams, water and The necessary reagent of the one-step method RT-QPCR kits of business, specific expansion sign reagent are identical as examples of implementation 1), it is vortexed and shakes after capping It swings and shakes up 30s, it is ensured that magnetic bead and PCR reactions are uniformly mixed)；

(6) four PCR pipe brief centrifugations (centrifuging 5-10s under 1000-3000rmp) in step (5) are made into liquid in PCR pipe Body is all in bottom；

(7) four PCR pipes that above-mentioned steps (6) obtain are placed on PCR amplification instrument (selection is Bio-RAD CFX96 it is expanded in), 45 DEG C, 10min；95 DEG C, 10min；95 DEG C of progress 40-45 cycles, 15S and 60 DEG C, 45S, 60 DEG C detection fluorescence signal.

Contrast method according to 201610022581.8 extracting method according to applied disclosed in real this application example 1 or 2 into The RNA of the same sample of row extracts the amplifing reagent (with reference to step 1-4), being added in the sample of extraction and same necessity of the invention Reagent：The reverse transcriptase of 0.5ul (200U/ul) unit and the reaction solution (as examples of implementation) of 24.5ul, are vortexed after capping Oscillation shakes up 30s, it is ensured that magnetic bead and PCR reactions are uniformly mixed；Then the amplification of same method is carried out.

Results contrast：The result of the present invention can obtain positive as a result, and control experiment cannot obtain positive result. This seems to illustrate, the method for control for serum sample (DNA) seem can or it is positive as a result, still for throat swab sample This really cannot.It is feasible for DNA sample, it cannot but lacked for RNA.

The all patents and publications mentioned in description of the invention all indicates that these are the public technology of this field, this hair It is bright to use.All patents referred to herein and publication are all equally listed in bibliography, with each publication It is specific to be individually referenced equally.The present invention described here can lack any type element or multiple element, and one It is realized in the case of kind limitation or a variety of limitations, this limitation here is not particularly illustrated.Such as art in each example here Language "comprising", " essence by ... form " and " by ... form " can be replaced with remaining 2 term of one of both.Here it adopts Describing mode carried out by terms and expressions mode, and be not limited except as, also indicate that this book describes without any intention here These terms and explain and eliminate any equivalent feature, but it is recognised that can be in the model of the present invention and claim Any suitable be altered or modified is done in enclosing.Preferably implement it is appreciated that examples of implementation described in the invention are all some Example and feature, any those of ordinary skill in the art can be changed and become according to some are done under the marrow of the invention that describe Change, these are changed and variation is recognized as belonging to the scope of the present invention and independent claims and appended claims are limited In the range of.

Claims (24)

1. a kind of method for extracting nucleic acid substances using magnetic bead, includes the following steps：

(1) lytic reagent and nanometer magnetic bead are provided；The lytic reagent includes metallic compound；

(2) sample is contacted to form mixture with the lytic reagent and magnetic bead, the mixture after processing described in stand at low temperature；

(3) other materials in mixture are removed, only retain magnetic bead, while not carrying out any washing or elution to magnetic bead Processing；

(4) magnetic bead of step (3) is directly contacted with the reagent of nucleic acid amplification, for target nucleic acid amplification provide it is nucleic acid-templated, from And carry out the amplification of target nucleic acid.

2. according to the method described in claim 1, wherein, the lytic reagent and magnetic bead are mixture, preferably solution Mixture.

3. according to claim 1 or claim 2, wherein the metallic compound includes one kind in NaCl, KCl Or two kinds.

4. according to the method described in one of claim 1-3, wherein the magnetic bead is monodispersity hydroxyl or carboxyl magnetic Pearl.

5. according to the method described in one of claim 1-4, wherein the lytic reagent be solution form, the solution at Divide and includes：The KCl or mass percent of the NaCl and 0-2mM of 0.1-2M are the surface-active agents of 0-0.3%.

6. according to the method described in one of claim 1-5, wherein the volume ratio of sample and lysate is 1:3—1:10.

7. according to the method described in one of claim 1-6, wherein when lytic reagent and nanometer magnetic bead are solution, institute The volume ratio 1 of the magnetic bead and lysate stated:200, wherein magnetic bead is a concentration of：0.1-100mg/ML.

8. according to the method described in one of claim 1-7, wherein the sample include throat swab, nose swab, throat swab, One or several kinds in serum, saliva, phlegm, urine, serum.

9. according to the method described in claim 8, wherein, the sample is the sample after processed.

10. according to the method described in claim 9, wherein, the carrier of the throat swab, nose swab or throat swab sample passes through Cross physiological saline, the sample that buffer solution elutes.

11. according to the method described in claim 1, wherein, the lytic reagent and magnetic bead are solution state, and are located at PCR In test tube, alternatively, the lytic reagent and magnetic bead is dry state, and in the PCR pipe for PCR amplification.

12. according to the method for claim 11, wherein before allowing sample to be contacted with mixture, it is equipped with the mixture, Mixture includes hydroxyl magnetic bead.

13. according to the method described in claim 1, wherein, the average grain diameter of the magnetic bead is less than 1000nm.

14. according to the method described in claim 1, wherein, allow after magnetic bead and lysate and sample contact or before, to sample Or be mixed with sample magnetic bead and lysate carry out high-temperature process at 60 DEG C -100 DEG C, alternatively, the low temperature be room temperature or Between 20 DEG C -30 DEG C of person.

15. according to the method described in claim 1, wherein, when the nucleic acid extracted is RNA, magnetic in step (3) is allowed Pearl contacts with reverse transcriptase and nucleic acid amplification agents simultaneously carries out PCR amplification.

16. according to the method described in claim 1, wherein, sample is allowed to be contacted simultaneously with lytic reagent and magnetic bead.

It is described when lytic reagent and magnetic bead are solution mixture 17. according to the method described in claim 1, wherein The content of magnetic bead is 0.005 milligram, and the lysate exists with the state of solution, and the ingredient of the lysate is as follows： The KCl or mass percent of the NaCl and 0-2mM of 0.1-2M are the surface-active agents of 0-0.3%, the wherein volume of solution It is 80 microlitres.

18. a kind of reagent of extraction nucleic acid, including solution, it than content is 0.01%T tables that the solution, which includes 1MNaCl, quality, Face activity is and the solution of 0.001MKCl and 0.005 milligram of magnetic bead.

19. reagent according to claim 16, wherein the magnetic bead is the magnetic bead of hydroxyl modified, alternatively, point of magnetic bead It dissipates coefficient and is less than 5.

20. a kind of reagent of extraction nucleic acid, including cracking composition and magnetic bead, the cracking composition include 1MNaCl, 0.01% Triton-X, 0.001MKCl and 0.005 milligram of magnetic bead；The average grain diameter of the magnetic bead is≤1000nm.

21. a kind of reagent of extraction nucleic acid, including cracked solution and 0.005 milligram of magnetic bead, the cracked solution include 1MNaCl, quality are 0.01%T surface-actives than content, when solution is 100 milliliters, magnetic bead Content is 0.005 milligram, alternatively, a concentration of 0.00005 milligram every milliliter of the magnetic bead.

22. a kind of nanometer magnetic bead containing hydroxyl modified is as the purposes on nucleic acid extracting reagent, wherein the nanometer magnetic bead Average grain diameter be≤1000nm.

23. purposes according to claim 21, the nucleic acid is RNA.

24. purposes according to claim 21, wherein after nucleic acid and magnetic bead contact, before magnetic bead is contacted with expansion sign reagent, The processing steps such as any washing, cleaning are not carried out to magnetic bead.