CN105349619A - Method for rapidly detecting target nucleic acid in sample and application thereof - Google Patents
Method for rapidly detecting target nucleic acid in sample and application thereof Download PDFInfo
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- CN105349619A CN105349619A CN201410414315.0A CN201410414315A CN105349619A CN 105349619 A CN105349619 A CN 105349619A CN 201410414315 A CN201410414315 A CN 201410414315A CN 105349619 A CN105349619 A CN 105349619A
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Abstract
The invention discloses a method for rapidly detecting target nucleic acid in a sample. The method of the invention comprises the following steps: A) at least a nucleic acid probe fixed on the surface of a solid phase support and biotin-labeled target nucleic acid are subjected to a one-step reaction in a hybridization solution containing alkaline phosphatase labeled-streptavidin; and B) after the reaction of the step A), the surface of the solid phase support is directly contacted with a chromogenic solution containing a substrate to carry out a chromogenic reaction so as to detect target nucleic acid in a sample. According to the method, nucleic acid hybridization and ELISA are integrated, and membrane cleaning and substrate chromogenic processes are integrated. Thus, lots of washing operational steps are canceled and lots of reaction solutions are saved. Then, reverse molecular hybridization is changed into an easy operational mode. Lots of experimental period and reagents are saved. In addition, detection throughput of target nucleic acid in the sample is also greatly raised, and high specificity of detection results can be maintained.
Description
Technical field
The invention belongs to field of nucleic acid detection, be specifically related to a kind of method of rapid detection sample target nucleic acid.
Background technology
Making nucleic acid molecular hybridization (is called for short molecular hybridization, hybridization) be a experimental technique the most basic in nucleic acids research, its ultimate principle is exactly the character of the denature and renature of application nucleic acid molecule, DNA (or RNA) fragment making source different, forms hybrid double-stranded molecule by base complementrity relation.Heteroduplex between DNA and DNA chain, also can be formed between RNA and DNA chain.At present, molecular hybridization has become one of on-radiation gene diagnosis technology the most frequently used in modern molecular biology laboratory, it not only can be used to detect the transgenation causing the transgenation of canceration and cause various heredopathia (as thalassemia), and can be used to detect cause infectious diseases bacterium, virus and parasite etc.
According to reacting, the environment carried out is different, and molecular hybridization can be divided into solid-phase hybridization and solution hybridization two type.According to different application object in laboratory, molecular hybridization can be divided into again spot (or slit) hybridization, So μ thern blot hybridization, Northern hybridization, cell and Chromosomal in situ hybridization etc.
Solid phase molecules hybridization is first fixed on solid support a nucleic acid chains of participating in reaction, and a reaction nucleic acid is free in the solution.Different according to detected sample molecule position, solid phase molecules hybridization can be divided into forward to hybridize, and (testing sample molecule is fixed on film, detection probes is in the solution) and reverse hybridized (testing sample molecule in the solution, visit detect pin be fixed on film) two kinds.Wherein, reverse hybridized refer to mark sample nucleic and unlabelled solidification nucleic acid probe hybridize.The advantage of this hybridizing method is in a hybridization, can detect the multiple nucleic acids in sample simultaneously.
Existing reverse hybridized technological operation is loaded down with trivial details, the reaction soln related to and reactions steps numerous, especially when relate to detect multiple sample time, the operating time will greatly lengthen and very easily make mistakes.Such as, a kind of method detecting nucleic acid point mutation is disclosed in Chinese patent application CN101768628A, specifically disclose that a kind of to utilize oligonucleotide probe to carry out PCR primer reverse hybridized with the method detecting nucleic acid point mutation, but the method needs the test carrying out results of hybridization by the high fluorescence detection device of price, not only complex operation, and cost is higher.Therefore, a kind of method wanting rapid detection sample target nucleic acid is needed badly.
For this consideration, the present inventor is studied, object solves the problem that association area prior art comes out, expect to provide a kind of method of short, easy to operate, rapid detection sample target nucleic acid that high-throughput, cost are low consuming time and its detecting the application in M. tuberculosis drug resistant gene.
Summary of the invention
An object of the present invention is a kind of method providing rapid detection sample target nucleic acid, reverse nucleic acid molecular hybridization process and enzyme are joined on the one hand process and unite two into one and carry out by the method, directly can form the conjugate of alkali phosphatase enzyme mark labeling nucleic acid crossbred at solid support surface; On the other hand washing and color reaction are united two into one and carry out, directly in chromophoric solution, make substrate form color products by enzymatic reaction and develop the color, thus without the need to just can realize the object that sample target nucleic acid detects fast through independent washing step after hybridization.
Another object of the present invention is to provide described method detecting the application in M. tuberculosis drug resistant gene.
According to an aspect of the present invention, the invention provides a kind of method detecting sample target nucleic acid, it comprises the steps:
A) at least one nucleic acid probe being fixed on solid support surface is carried out single step reaction with biotin labeled target nucleic acid in the hybridization solution comprising alkali phosphatase enzyme mark Streptavidin;
B) steps A) in reacted after, directly solid support surface is contacted with the chromophoric solution comprising substrate and carries out color reaction, to detect the target nucleic acid in sample.
Alkali phosphatase enzyme mark Streptavidin directly joins in hybridization solution by method of the present invention, in the process that nucleic acid probe and target nucleic acid are hybridized, synchronously achieve the cohesive process of alkali phosphatase enzyme mark Streptavidin and vitamin H, thus the conjugate of alkali phosphatase enzyme mark nucleic acid hybrids can be formed at solid support surface, eliminate after hybridization, need to carry out separately integrated enzyme reaction in conventional counter molecular hybridization method step and other multiple operation stepss, greatly shorten the consuming time of traditional technique in measuring target nucleic acid.
In the method for the invention, described alkaline phosphatase is a kind of heterodimeric protein, is a kind of metalloenzyme containing zinc.Per molecule enzyme is at least containing 2 Zn atoms.Enzyme comprises the metal-binding sites of 3 types, i.e. so-called catalyzed combination site, structure binding site and adjustment binding site.Wherein the combination in two catalyzed combination sites then only can cause the phosphorylation of a subunit, namely interacts between negative cooperation subunit.
In the method for the invention, described biology have two ring texture I and II.Wherein, I ring is imidazolone ring, is the main portions that it is combined with Streptavidin; II ring is thiphene ring, C
2on have a pentanoic acid side chain; Biotin molecule is connected with target nucleic acid of the present invention by the carboxyl of its end, thus is marked on target nucleic acid molecule.
In the method for the invention, described Streptavidin is a kind of protein secreted by streptomycete, and its molecule is made up of 4 identical peptide chains, and every bar peptide chain can in conjunction with a vitamin H, and therefore each Streptavidin molecular energy is in conjunction with 4 biotin molecules.In addition, in the amino acid composition of every bar peptide chain, the content of glycine and L-Ala is comparatively large, and the tryptophan residue in peptide chain is the active group connecting vitamin H.The constant (K) of the affine combination of both described Streptavidin and vitamin H is 1015L/mol.In the method for the invention, while target nucleic acid and nucleic acid probe hybridization, Streptavidin and vitamin H also carry out affine combination, the alkali phosphatase enzyme mark Streptavidin molecule therefore in hybridization solution and the biotin labeled target nucleic acid molecule of nucleic acid probe molecules competitive binding in single step reaction being fixed on solid support surface.
The present inventor finds through great many of experiments and creative work, in the method for the invention, described alkali phosphatase enzyme mark Streptavidin is directly joined in hybridization solution, alkali phosphatase enzyme mark Streptavidin can be made to be combined fully with biotin labeling target nucleic acid on the one hand; Nucleic acid hybridization efficiency can also be promoted on the other hand, shorten the time of nucleic acid hybridization reaction.
According to a specific embodiment of the present invention, steps A) described in the concentration of alkali phosphatase enzyme mark Streptavidin in hybridization solution be 0.05 ~ 2 μ g/ml, preferably 0.1 ~ 1.5 μ g/ml, more preferably 0.5 ~ 1.2 μ g/ml.In the method for the invention, the concentration of described alkali phosphatase enzyme mark Streptavidin in hybridization solution can enumerated includes but not limited to: 0.05 μ g/ml, 0.06 μ g/ml, 0.07 μ g/ml, 0.08 μ g/ml, 0.09 μ g/ml, 0.1 μ g/ml, 0.2 μ g/ml, 0.3 μ g/ml, 0.4 μ g/ml, 0.5 μ g/ml, 0.6 μ g/ml, 0.7 μ g/ml, 0.8 μ g/ml, 0.9 μ g/ml, 1 μ g/ml, 1.1 μ g/ml, 1.2 μ g/ml, 1.5 μ g/ml and 2 μ g/ml.
In the method for the invention, the concentration of described alkali phosphatase enzyme mark Streptavidin in hybridization solution is importance of the present invention.The present inventor finds through lot of experiments and creative work, if the concentration of alkali phosphatase enzyme mark Streptavidin in hybridization solution is too low, so affects the sensitivity that target nucleic acid detects; If the excessive concentration of alkali phosphatase enzyme mark Streptavidin in hybridization solution, so easily brings non-specific adsorption phenomenon, affect the accuracy of target nucleic acid detected result.
According to a specific embodiment of the present invention, steps A) described in hybridization solution comprise zine ion, magnesium ion, albumen, nonionic surface active agent and cation type polymer; Wherein, described albumen is selected from albumin, casein and gelatin, and described nonionic surface active agent is selected from tween and Triton, and described cation type polymer is selected from cationic polyacrylamide, poly-lysine and polymerize aluminum chloride.
According to a specific embodiment of the present invention, in described hybridization solution, zine ion is 0.001 ~ 0.1mol/L, magnesium ion is 0.001 ~ 0.1mol/L, albumen accounts for 1% ~ 10% (w/v) of hybridization solution, nonionic surface active agent accounts for 0.01 ~ 2% (v/v) of hybridization solution, and cation type polymer accounts for 0.01 ~ 0.5% (w/v) of hybridization solution; Preferably, zine ion is 0.005 ~ 0.05mol/L, magnesium ion is 0.005 ~ 0.05mol/L, albumen accounts for 1% ~ 5% (w/v) of hybridization solution, nonionic surface active agent accounts for 0.05 ~ 1% (v/v) of hybridization solution, and cation type polymer accounts for 0.05 ~ 0.2% (w/v) of hybridization solution volume.
According to a specific embodiment of the present invention, described zine ion can be selected from the soluble salt containing zine ion.The example that can be used as the described soluble salt containing zine ion of the present invention comprises: zinc sulfate, zinc chloride and other various salt that can dissociate zine ion at solution state.
According to a specific embodiment of the present invention, described magnesium ion can be selected from the soluble salt containing magnesium ion.The example that can be used as the described soluble salt containing magnesium ion of the present invention comprises: magnesium sulfate, magnesium acetate, magnesium chloride and other various salt that can dissociate magnesium ion at solution state.
According to a specific embodiment of the present invention, described tween is selected from polysorbas20 (Tween-20), tween 21 (Tween-21), polysorbate40 (Tween-40), polysorbate60 (Tween-60), Tween61 (Tween-61), tween 80 (Tween-80), sorbimacrogol oleate100 (Tween-81) and polysorbate85 (Tween-85), and wherein polysorbas20 is particularly preferred.
According to a specific embodiment of the present invention, described Triton is selected from triton x-100 (TritonX-100), Triton X-114 (TritonX-114) and Triton X-200 (TritonX-200), wherein, triton x-100 is particularly preferred.
The present inventor finds through great many of experiments and creative work, in the method for the invention, the change of acid, alkali, salt ion or temperature condition all may change even makes alkaline phosphatase lose activity completely, therefore in order to realize an object of the present invention, the selection of hybridization solution composition is very important.In the method for the invention, contriver by adding zine ion, magnesium ion, albumen and nonionic surface active agent in hybridization solution, contribute on the one hand improving nucleic acid hybridization efficiency, prevent on the other hand the alkali phosphatase enzyme mark Streptavidin sex change because of absorption in hybridization solution, then prevent the polymerization sex change that causes because of interaction between alkali phosphatase enzyme mark Streptavidin molecule on the one hand.
The present inventor finds through great many of experiments and creative work, in the method for the invention, synergistic effect can also be there is further in described protein molecular and nonionogenic tenside in hybridization solution, the common surface being attached to solid support by Van der Waals force, effectively prevent unreacted alkali phosphatase enzyme mark Streptavidin and/or biotin labeling target nucleic acids in the non-specific adsorption of solid support surface, play good sealing process, thus the pre-hybridization step before making method of the present invention can save hybridization.
The present inventor finds through great many of experiments and creative work, in the method for the invention, described cation type polymer can produce electrostatic adsorption with biotin labeled target nucleic acid molecule (the biotin labeling target nucleic acid molecule obtained by pcr amplification), make single stranded nucleic acid molecule (being with many negative charges) bring a positive charge part, thus in nucleic acid hybridization process, be synchronously adsorbed onto the surface of solid support.Because alkali phosphatase enzyme mark Streptavidin is also with positive charge, doing so avoids the surface of alkaline phosphatase non-specific adsorption to solid support.In addition, described cation type polymer can make alkali phosphatase enzyme mark Streptavidin in hybridization solution, form even suspension, the alkaline phosphatase be marked at after hybridization is completed in nucleic acid conjugates keeps active condition, thus the conversion of the balance of alkaline phosphatase conformation is moved towards native state.
In the method for the invention, hybridization promoter can also be comprised in described hybridization solution, described hybridization promoter is known to the person skilled in the art in itself, and the example that can be used as hybridization promoter of the present invention includes but not limited to: T 500, polyoxyethylene glycol, phenol or guanidine thiocyanate.
In the method for the invention, can also comprise other compositions in described hybridization solution, in the hybridization solution that can enumerate, the example of other compositions includes but not limited to: sodium-chlor, hybridization buffer, Denhardt's solution, sarcosyl or sodium laurylsulfonate.The example that may be used for hybridization buffer of the present invention includes but not limited to: citric acid-sodium citrate buffer or Tris-hydrochloric acid buffer solution.
In the method for the invention, described hybridization solution does not comprise edetate, inorganic phosphate and thanomin.
According to a specific embodiment of the present invention, step B) described in chromophoric solution also comprise C
8~ C
18alkyl-glucoside, preferred C
9~ C
13alkyl-glucoside; Described alkyl-glucoside accounts for 0.01 ~ 0.5% (w/v) of aftertreatment fluid, preferably 0.05 ~ 0.1% (w/v).
Method of the present invention by adding alkyl-glucoside in chromophoric solution, under the prerequisite not affecting substrate for enzymatic activity reaction, chromophoric solution is made to have significant washing effect, thus the washing step that can omit before the colour developing of traditional enzyme, greatly shorten the consuming time of traditional technique in measuring target nucleic acid.
According to a specific embodiment of the present invention, step B) described in chromophoric solution comprise Tris-HCl buffered soln, the Tris-HCl buffered soln of preferred PH9.5.
The present inventor finds through great many of experiments and creative work, in the method for the invention, described Tris-HCl buffered soln can in color reaction process and the effect of alkyl-glucoside generation coordinated, realize the cleaning to solid support surface, remove non-specific adsorption at the material of solid support surface, thus need not just directly in chromophoric solution, substrate formation color products can be made by enzymatic reaction through independent film step of washing and develop the color after hybridization.
According to a specific embodiment of the present invention, step B) described in chromophoric solution to contact with described solid support surface in the mode of flowing and develop the color; The direction of described flowing is flow to up and down or the left and right flow direction; The time of described flowing is 2min ~ 30min, preferred 5min ~ 20min, more preferably 8 ~ 15min.
The present inventor finds through great many of experiments and creative work, in the method for the invention, described chromophoric solution contacts with described solid support surface in the mode of flowing, not only can promote substrate for enzymatic activity reaction efficiency, shorten developing time, improve color developing effect, and non-specific adsorption can be accelerated depart from from solid support surface at the material of solid support surface, unreacted enzyme labelling Streptavidin and biotin labeling target nucleic acid, play the effect of cleaning solid support surface.The direction of described flowing can be above-below direction, and now chromophoric solution flows through solid support surface from top to bottom; The direction of described flowing can be that left and right flows to, and now chromophoric solution is in solid support surface horizontal flow.The flow velocity of described chromophoric solution and flow can set according to actual needs, not special requirement.
According to a specific embodiment of the present invention, steps A) described in the temperature of reaction be 35 ~ 50 DEG C, the reaction times is 5 ~ 30min; Preferably, temperature of reaction is 37 ~ 42 DEG C, and the reaction times is 10 ~ 15min.
The present inventor finds through great many of experiments and creative work, in the method for the invention, owing to synchronously achieving integrated enzyme reaction process in the process of nucleic acid probe with target nucleic acid hybridization, therefore needs the time and the temperature that strictly control hybridization.If the hybridization time is shorter or hybridization temperature is lower, nucleic acid hybridization efficiency will reduce, and is difficult to form stable double-strand at solid support surface, affects the sensitivity that target nucleic acid detects; Otherwise if the hybridization time is longer or hybridization temperature is higher, alkali phosphatase enzyme mark Streptavidin is easy inactivation in hybridization solution, affect the specificity that target nucleic acid detects.The present inventor finds through great many of experiments and creative work, temperature of reaction is 35 ~ 50 DEG C, the reaction times is proper when being 5 ~ 30min, preferable reaction temperature is 37 ~ 42 DEG C, and the reaction times is 10 ~ 15min, and sensitivity and the specificity of now target nucleic acid detection are better.
According to a specific embodiment of the present invention, steps A) described in nucleic acid probe be selected from the oligonucleotide probe that length is 15 ~ 40 bases, be preferably selected from the oligonucleotide probe that length is 16 ~ 25 bases.
In the method for the invention, by adjusting the length of nucleic acid probe and composition, reduce the temperature that nucleic acid probe and biotin labeling target nucleic acid are hybridized, thus make hybridization and integrated enzyme reaction can be unified into a reaction process to carry out.The present inventor finds through great many of experiments, described nucleic acid probe is selected from when length is the oligonucleotide probe of 15 ~ 40 bases proper, be preferably selected from the oligonucleotide probe that length is 16 ~ 25 bases, now the temperature of hybridization can be 37 ~ 42 DEG C.The length of particularly preferred DNA probe comprises 16,17,18,19,20,21,22,23,24 and 25 bases.
In the method for the invention, the foundation of the selection of nucleic acid probe length is: if nucleic acid probe length is too low, although can improve the specificity of probe hybridization, significantly can reduce the sensitivity of probe hybridization; If nucleic acid probe length is long, the sensitivity of probe hybridization can be improved further, but probe hybridization specificity significantly can reduce.For long nucleic acid probe, the specificity of probe hybridization can not be improved by improving hybridization temperature, because too high temperature can make the alkali phosphatase enzyme mark Streptavidin inactivation in hybridization system.Consider that the GC content of probe is worth impact to hybridization Tm, comprehensive various factors above, the oligonucleotide probe of 16 ~ 25 bases is suitable scopes simultaneously.
According to a specific embodiment of the present invention, steps A) described in nucleic acid probe also comprise positive quality control probe and negative Quality Control probe.Wherein, described positive quality control probe can be people actin gene probe; Described negative probes can be one section of sequence of primer-design software stochastic generation, is characterized in not similar to the nucleotide sequence of any biology.
According to a specific embodiment of the present invention, in steps A) also comprise the step using the biotin labeled primer for amplifying target nucleic acid to carry out PCR reaction and the sample target nucleic acid that increases before.
In the method for the invention, described biotin labeling target nucleic acid can prepare by the following method: first carry out separation and Extraction to the target nucleic acid in sample, then the target nucleic acid of the biotin labeled primer pair separation and Extraction being used for amplifying target nucleic acid is used to carry out the target nucleic acid that pcr amplification obtains amplification sample, to improve the sensitivity of detection.
According to a specific embodiment of the present invention, in steps A) before be also included in the existence of biotin labeling deoxynucleotide under carry out the step of PCR reaction and the sample target nucleic acid that increases.
In the method for the invention, described biotin labeling target nucleic acid can prepare by the following method: first carry out separation and Extraction to the target nucleic acid in sample, then under the existence of biotin labeling deoxynucleotide, the target nucleic acid that pcr amplification obtains amplification sample is carried out, to improve the sensitivity of detection to the target nucleic acid of separation and Extraction.
According to a specific embodiment of the present invention, step B) described in substrate include but not limited to: nitroblue tetrazolium (NBT) (NBT) and the chloro-3-indyl-phosphoric acid (BCIP) of the bromo-4-of 5-, fast red and naphthols ASMX.Under the catalysis of alkaline phosphatase, BCIP can be hydrolyzed the product producing strong reactivity, and the meeting of this product and NBT react, and forms insoluble mazarine to hepatic NBT-formazan.
According to a specific embodiment of the present invention, described biotin labeling target nucleic acid needs to carry out sex change before carrying out hybridization, and the method for sex change can be well-known to those skilled in the art.
According to a specific embodiment of the present invention, described solid support is selected from: nylon membrane, nitrocellulose filter or polypropylene screen.Wherein, nylon membrane and nitrocellulose filter are preferred, and nitrocellulose filter is particularly preferred.
In the method for the invention, select suitable solid support treatment process and nucleic acid probe solid support surface fixing means in the knowledge of those skilled in the range.
According to another aspect of the invention, present invention also offers described method and detect the application in M. tuberculosis drug resistant gene.
Method of the present invention is used for detecting M. tuberculosis drug resistant gene, on the one hand reverse nucleic acid molecular hybridization process and enzyme are joined process to unite two into one and carry out, directly can form the conjugate of alkali phosphatase enzyme mark labeling nucleic acid crossbred at solid support surface; On the other hand washing and color reaction are united two into one and carry out, directly in chromophoric solution, make substrate form color products by enzymatic reaction and develop the color, thus without the need to just can realize the object that sample target nucleic acid detects fast through independent washing step after hybridization.
In the method for the invention, described " nucleic acid " term sums up to represent that Yeast Nucleic Acid (i.e. RNA), thymus nucleic acid (i.e. DNA), peptide nucleic acid(PNA) (i.e. PNA), methyl phosphorodithioate nucleic acid, S-are oligomeric, cDNA and cRNA, and any oligonucleotide and polynucleotide etc., nucleic acid and nucleic acid analog term, such nucleic acid can be naturally occurring, also can be synthetic.
In the method for the invention, described " nucleic acid probe " refers to and is containing with the base sequence nucleic acid of target complement sequence, be fixed on the nucleic acid fragment of solid support surface, nucleic acid probe has the sequence complementary at least partially with described target sequence, thus, can hybridize with target sequence under appropriate conditions.
In the method for the invention, described " target nucleic acid " refers to the nucleic acid with target sequence detected by method of the present invention.
In the method for the invention, described " target sequence " refers to the sequence comprised in target nucleic acid, and target sequence is for detecting target nucleic acid.
According to a specific embodiment of the present invention, in the method for the invention, conventional nucleic acid extracting method or commercially available nucleic acid extraction kit can be used from clinical sample to extract target nucleic acid.
Method of the present invention has carried out significant improvement to traditional reverse molecular hybridization method, establishes a step molecular hybridization method.Nucleic acid hybridization and integrated enzyme reaction not only unite two into one and carry out by method of the present invention, and film and substrate process color will be washed unite two into one and carry out, eliminate a large amount of washing operation steps and reaction soln, thus reverse molecular hybridization is become the pattern of an ease of Use, both for operator bring facility, experiment no longer easily makes mistakes, in turn save great many of experiments time and reagent, increase substantially the detection flux of sample target nucleic acid in addition, the specificity that detected result keeps higher can have been made.
Accompanying drawing explanation
In order to be illustrated more clearly in the technical scheme in the embodiment of the present invention, below the accompanying drawing used required in describing embodiment is done and introduce simply, obviously, accompanying drawing in brief description is only some embodiments of the present invention, for those of ordinary skill in the art, under the prerequisite not paying creative work, other accompanying drawing can also be obtained according to these accompanying drawings.
Fig. 1 represents the colour developing result figure of the embodiment of the present invention 1.
Fig. 2 represents the colour developing result figure of the embodiment of the present invention 2.
Fig. 3 represents the colour developing result figure of the embodiment of the present invention 3.
Fig. 4 represents the colour developing result figure of the embodiment of the present invention 4.
Fig. 5 represents the colour developing result figure of the embodiment of the present invention 5.
Fig. 6 represents the colour developing result figure of the embodiment of the present invention 6.
Fig. 7 represents the colour developing result figure of the embodiment of the present invention 7.
Fig. 8 represents the colour developing result figure of the embodiment of the present invention 8.
Embodiment
Below in conjunction with embodiment, embodiment of the present invention are described in detail, but it will be understood to those of skill in the art that the following example only for illustration of the present invention, and should not be considered as limiting scope of the present invention.Unreceipted actual conditions person in embodiment, the condition of conveniently conditioned disjunction manufacturers suggestion is carried out.Agents useful for same or the unreceipted production firm person of instrument, being can by the conventional products of commercial acquisition.
In the examples below, the concentration of bovine serum albumin (hereinafter referred to as BSA), cationic-type polyacrylamide (hereinafter referred to as CPAM), alkali phosphatase enzyme mark Streptavidin (hereinafter referred to as SA-AP), poly-lysine (hereinafter referred to as PLL), PEG 8000 and alkyl-glucoside is quality volume percent (w/v) concentration in g/ml; The concentration of Tween-20 and TritonX-100 refers to volume percent (v/v) concentration.
Embodiment 1:
(1) nucleic acid probe fixing at solid support surface
1.1 experiment materials:
Nucleic acid probe: the M. tuberculosis drug resistant gene abrupt climatic change probe of nucleotide sequence as shown in SEQIDNOs:1 ~ 2, for detecting 533 site mutations of mycobacterium tuberculosis rpoB gene; The positive control probe of nucleotide sequence as shown in SEQIDNO:3; The negative control probe of nucleotide sequence as shown in SEQIDNO:4.Above-mentioned four kinds of nucleic acid probes are added water respectively and is mixed with 100 μMs (i.e. 100pmol/ μ l), for subsequent use.
Solid support: nitrocellulose filter, Millipore Corp. (Millipore) produces, and 0.45 μm of aperture specification, is cut into the film being of a size of 2cm × 1cm for subsequent use
Terminal deoxynucleotidyl transferase (TdT enzyme): 5 μ/μ l, 100 μ l are purchased from Jiang Lai bio tech ltd, Shanghai.
10 × TdT damping fluid: support the use with TdT enzyme, by Shanghai, Jiang Lai bio tech ltd provides.
DTTP:100mmol/L, purchased from promega company
1.2 experimental procedures:
1.2.1 nucleic acid probe tailing: to four kinds of nucleic acid probe solution (100 μMs), get 2 μ l, the i.e. nucleic acid probe amount of 200pmol, joins 100 μ l and contains in the dTTP solution of 60 μ TdT enzymes, 1 × TdT reaction buffer, 100nmol/L, 37 DEG C of incubation 60min; Then the 10mmol/LEDTA termination reaction (now probe final concentration is 1pmol/ μ l) of 100 μ l is added.
1.2.2 fixing at solid support surface of nucleic acid probe: nucleic acid probe (1pmol/ μ l) after to tailing), positive control probe and negative control probe solution, get 1 μ l (the probe amount containing 1pmol) respectively to put on nitrocellulose filter, film is placed on the paper soaked with TE, fixes through 254nm UV-irradiation 10min.
Nucleic acid probe is described in table 1 below at the distributing order on nitrocellulose filter surface:
Table 1
| T533C wild-type probe | T533C mutant probe | Positive control probe | Negative control probe |
(2) amplification of target nucleic acid
2.1 experiment materials:
Target nucleic acid: the human genome nucleic acid of concentration to be mycobacterium tuberculosis (be confirmed that it is rpoB gene by order-checking and there occurs T533C sudden change) genomic nucleic acids and the concentration of 0.01ng/ μ l be 50ng/ μ l
The primer pair of amplification mycobacterium tuberculosis rpoB gene, wherein the nucleotide sequence of upstream primer is as shown in SEQIDNO:5, the nucleotide sequence of downstream primer is as shown in SEQIDNO:6, and 5 ' end biotin labeling of upstream primer, the concentration of upstream primer and downstream primer is 0.2 μM.
The primer pair of amplification people actin gene, wherein the nucleotide sequence of upstream primer is as shown in SEQIDNO:7, the nucleotide sequence of downstream primer as shown in SEQIDNO:8,5 ' end biotin labeling of upstream primer.
GoTaq enzyme: 5 μ/μ l, purchased from promega company.
10 × Taq enzyme reaction buffered soln: purchased from promega company.
MgCl
2: 25mM, purchased from promega company.
DNTPsMix:10mM, purchased from promega company
2.2 operation stepss:
2.2.1 PCR reaction system (using the PCR amplification system of following 50 μ l, with M. tuberculosis genes group nucleic acid and human genome nucleic acid for template carries out pcr amplification) is prepared:
GoTaq enzyme: 1U;
Enzyme reaction buffered soln: 1 ×;
Upstream primer SEQIDNO:5:0.2 μM;
Downstream primer SEQIDNO:6:0.2 μM;
Upstream primer SEQIDNO:7:0.2 μM;
Downstream primer SEQIDNO:8:0.2 μM;
MgCl
2：2.0mM；
dNTPsMix：0.2mM；
Template 1:1 μ l M. tuberculosis genes group nucleic acid;
Template 2:1 μ l human genome nucleic acid;
Surplus is water.
2.2.2PCR amplified reaction: first 95 DEG C of denaturation 5min; Then 95 DEG C of 1.5min; 55 DEG C of 1.5min; 72 DEG C, 1min, 35 circulations; Last 72 DEG C extend 5min.
2.2.3PCR the sex change of product: first hatch 10min at 95 DEG C, then ice bath 5min.
(3) single step reaction
3.1 experiment materials:
20 × SSC solution: 3.0mol/LNaCl, 0.3mol/L Trisodium Citrate, pH7.0.
Alkali phosphatase enzyme mark Streptavidin: purchased from Gibcol company.
Zinc chloride (ZnCl
2): purchased from Sangon Biotech (Shanghai) Co., Ltd..
Magnesium chloride hexahydrate: purchased from Sangon Biotech (Shanghai) Co., Ltd..
Bovine serum albumin: purchased from Sigma-Aldrich.
Tween-20: purchased from Sangon Biotech (Shanghai) Co., Ltd..
Poly-lysine (PLL): purchased from Sangon Biotech (Shanghai) Co., Ltd..
PEG 8000: purchased from Sangon Biotech (Shanghai) Co., Ltd..
3.2 operation stepss:
3.2.1 shown hybridization solution composed as follows is prepared: PH7.0,3 × SSC, 1 μ g/mlSA-AP, 10mMZnCl
2, 10mMMgCl
2, 2%BSA, 0.5%Tween-20,0.1%PLL, 2% PEG 8000, surplus is water.
3.2.2 single step reaction: the nitrocellulose filter that the surface obtained in step () is fixed with nucleic acid probe is put into 1ml hybridization solution, add the PCR primer after sex change in the step (two) of 10 μ l, in 37 DEG C of reaction 15min after mixing simultaneously.。
(4) color reaction
4.1 experiment materials:
Nitroblue tetrazolium (NBT) (NBT): purchased from Sangon Biotech (Shanghai) Co., Ltd..
The chloro-3-indyl-phosphoric acid (BCIP) of the bromo-4-of 5-: purchased from Sangon Biotech (Shanghai) Co., Ltd..
Tris: purchased from Sangon Biotech (Shanghai) Co., Ltd..
NaCl: purchased from Sangon Biotech (Shanghai) Co., Ltd..
Ndodecyl glucoside: purchased from Sangon Biotech (Shanghai) Co., Ltd..
4.2 operation stepss:
4.2.1 the chromophoric solution that formulation components is as follows: PH9.5,0.1mol/LTris-HCl, 50mMMgCl
2, 0.33mg/mlNBT, 0.17mg/mlBCIP and 0.07% ndodecyl glucoside, surplus is water.
4.2.2 color reaction: clamp reacted nitrocellulose filter in step (three) with tweezers, allow be fixed with nucleic acid probe nitrocellulose filter surface upwards, tilt film a little, makes film one end higher than the other end.Get chromophoric solution with pipettor and continue drippage chromophoric solution from the top of the upper end of film, make chromophoric solution (instill from the upper end of film, flow out drippage from lower end) from top to bottom, continue to flow through the surface of nitrocellulose filter, flowing time is 10min, observes colour developing result.
The colour developing result of the present embodiment as shown in Figure 1.
Result shows: T533C mutant probe point is strong positive, and T533C wild-type probe point is negative, thus can determine that the rpoB gene of this M. tuberculosis genes group there occurs the nucleic acid base variation of T → C in 533 sites.Positive control point display is simultaneously positive, and the display of negative control point is negative, shows that the detected result that this is tested is normal.
Embodiment 2:
With embodiment 1, difference is the composition of hybridization solution in embodiment 1 to be adjusted to: 3 × SSC, 0.1 μ g/mlSA-AP, 5mMZnCl
2, 5mMMgCl
2, 0.5%BSA, 0.03%Tween-20,0.03%PLL and 2% PEG 8000, surplus is water.By the adjustment of formula of chromophoric solution be: PH9.5,0.1mol/LTris-HCl, 50mMMgCl simultaneously
2, 0.33mg/mlNBT, 0.17mg/mlBCIP and 0.03% n-hexadecyl glucoside, surplus is water.Other conditions are constant.
The colour developing result of the present embodiment as shown in Figure 2.
Embodiment 3:
With embodiment 1, difference is hybridization solution composition in embodiment 1 to be adjusted to: 3 × SSC, 1.5 μ g/mlSA-AP, 50mMZnCl
2, 50mMMgCl
2, 7%BSA, 1.5%Tween-20,0.3%CPAM and 4% PEG 8000, surplus is water.By the adjustment of formula of chromophoric solution be: PH9.5,0.1mol/LTris-HCl, 50mMMgCl simultaneously
2, 0.33mg/mlNBT, 0.17mg/mlBCIP and 0.2% n-Octylglucoside, surplus is water.Other conditions are constant.
The colour developing result of the present embodiment as shown in Figure 3.
Embodiment 4:
With embodiment 1, difference is hybridization solution composition in embodiment 1 to be adjusted to: 3 × SSC, 0.5 μ g/mlSA-AP, 20mMZnCl
2, 20mMMgCl
2, 1%BSA, 0.05%Tween-20,0.05%CPAM, 4% PEG 8000, surplus is water.By the adjustment of formula of chromophoric solution be: PH9.5,0.1mol/LTris-HCl, 50mMMgCl simultaneously
2, 0.33mg/mlNBT, 0.17mg/mlBCIP and 0.05% ndodecyl glucoside, surplus is water.Other conditions are constant.
The colour developing result of the present embodiment as shown in Figure 4.
Embodiment 5:
With embodiment 1, difference is hybridization solution composition in embodiment 1 to be adjusted to: 3 × SSC, 1.2 μ g/mlSA-AP, 40mMZnCl
2, 40mMMgCl
2, 5%BSA, 1%TritonX-100,0.2%PLL, 4% PEG 8000, surplus is water.By the adjustment of formula of chromophoric solution be: PH9.5,0.1mol/LTris-HCl, 50mMMgCl simultaneously
2, 0.33mg/mlNBT, 0.17mg/mlBCIP and 0.1% ndodecyl glucoside, surplus is water.Other conditions are constant.
The colour developing result of the present embodiment as shown in Figure 5.
Embodiment 6:
With embodiment 1, difference is the single step reaction condition of step 3.2.2 in embodiment 1 to become: 42 DEG C of reaction 10min.Other conditions are constant.
The colour developing result of the present embodiment as shown in Figure 6.
Embodiment 7:
With embodiment 1, difference is that the time chromophoric solution in step 4.2.2 color reaction in embodiment 1 being continued to flow through from top to bottom nitrocellulose filter surface is adjusted to 5min.Other conditions are constant.
The colour developing result of the present embodiment as shown in Figure 7.
The detection of embodiment 8 clinical sample
Clinical sample in the present embodiment is from sputum sample to the clinical tuberculosis patient of rifampin-resistance this (carry out PCR order-checking experiment confirm that its rpoB gene to there occurs the nucleic acid base variation of T → C in 533 sites through extracting nucleic acid).
(1) nucleic acid probe fixing at solid support surface
1.1 experiment materials:
Nucleic acid probe: the M. tuberculosis drug resistant gene abrupt climatic change probe of nucleotide sequence as shown in SEQIDNOs:1 ~ 2, for detecting 533 site mutations of mycobacterium tuberculosis rpoB gene; The positive control probe of nucleotide sequence as shown in SEQIDNO:3; The negative control probe of nucleotide sequence as shown in SEQIDNO:4.Above-mentioned four kinds of nucleic acid probes are added water respectively and is mixed with 100 μMs (i.e. 100pmol/ μ l), for subsequent use;
Solid support: nitrocellulose filter, Millipore Corp. (Millipore) produces, and 0.45 μm of aperture specification, is cut into the film being of a size of 2cm × 1cm for subsequent use;
Terminal deoxynucleotidyl transferase (TdT): 5 μ/μ l, 100 μ l are purchased from Jiang Lai bio tech ltd, Shanghai;
10 × TdT damping fluid: supporting with TdT enzyme, by Shanghai, Jiang Lai bio tech ltd provides;
DTTP:100nmol/L, purchased from Promega company.
1.2 experimental procedures:
1.2.1 nucleic acid probe tailing: to four kinds of nucleic acid probe solution (100 μMs), the nucleic acid probe amount of getting 2 μ l and 200pmol joins 100 μ l and contains in the dTTP solution of 60 μ TdT enzymes, 1 × TdT reaction buffer, 100nmol/L, 37 DEG C of incubation 60min; Then the 10mmol/LEDTA termination reaction (now probe final concentration is 1pmol/ μ l) of 100 μ l is added.
1.2.2 fixing at solid support surface of nucleic acid probe: nucleic acid probe (1pmol/ μ l) after to tailing), positive control probe and negative control probe solution, get 1 μ l (the probe amount containing 1pmol) respectively to put on nitrocellulose filter, film is placed on the paper soaked with TE, fixes through 254nm UV-irradiation 10min.
Nucleic acid probe is described in table 1 below at the distributing order on nitrocellulose filter surface:
Table 1
| T533C wild-type probe | T533C mutant probe | Positive control probe | Negative control probe |
(2) extraction of target nucleic acid
Get the NaOH solution that 1ml sputum sample is originally 4M with 1ml concentration to mix, room temperature places 30min liquefy sputum, after mixing, getting 1ml to be added in centrifuge tube with the centrifugal 2min of 12000rpm rotating speed, abandons supernatant, adds 1ml physiological saline and suspend, again with the centrifugal 2min of 12000rpm rotating speed, abandon supernatant.Remaining steps carries out with reference to the method for patent EP1407051B1: in centrifuge tube, add 200 μ lTris-EDTA damping fluids (i.e. TE damping fluid, PH8.0) vibration suspend; Add two kinds of specification glass pearls (200 μm of diameters and 900 μm of diameters, 4: 1 mass ratios) more respectively; Vortex oscillation 5min, then heats 10min the water-bath that the centrifuge tube crossed through oscillation treatment is placed in 90 DEG C, centrifugal with 12000 revs/min of rotating speeds, gets supernatant for subsequent use.
(3) amplification of target nucleic acid
3.1 experiment materials:
Target nucleic acid: this nucleic acid extraction of sputum sample liquid of the clinical tubercular extracted in step (two).
The primer pair of amplification mycobacterium tuberculosis rpoB gene, wherein the nucleotide sequence of upstream primer is as shown in SEQIDNO:5, the nucleotide sequence of downstream primer is as shown in SEQIDNO:6, and 5 ' end biotin labeling of upstream primer, the concentration of upstream primer and downstream primer is 0.2 μM.
The primer pair of amplification people actin gene, wherein the nucleotide sequence of upstream primer is as shown in SEQIDNO:7, the nucleotide sequence of downstream primer as shown in SEQIDNO:8,5 ' end biotin labeling of upstream primer.
GoTaq enzyme: 5 μ/μ l, purchased from promega company.
10 × Taq enzyme reaction buffered soln: purchased from promega company.
MgCl
2: 25mM, purchased from promega company.
DNTPsMix:10mM, purchased from promega company.
2.2 operation stepss:
2.2.1 PCR reaction system (using the PCR amplification system of following 50 μ l, with M. tuberculosis genes group nucleic acid and human genome nucleic acid for template carries out pcr amplification) is prepared:
GoTaq enzyme: 1 μ;
Enzyme reaction buffered soln: 1 × Taq;
Upstream primer SEQIDNO:5:0.2 μM;
Downstream primer SEQIDNO:6:0.2 μM;
Upstream primer SEQIDNO:7:0.2 μM;
Downstream primer SEQIDNO:8:0.2 μM;
MgCl
2：2.0mM；
dNTPsMix：0.2mM；
Template is 1 μ l M. tuberculosis genes group nucleic acid;
Surplus is water.
2.2.2PCR amplified reaction: first 95 DEG C of denaturation 5min; Then 95 DEG C of 1.5min; 55 DEG C of 1.5min; 72 DEG C, 1min, 35 circulations; Last 72 DEG C extend 5min.
2.2.3PCR the sex change of product: first hatch 10min at 95 DEG C, then ice bath 5min.
(4) single step reaction
4.1 experiment materials:
20 × SSC solution: 3.0mol/LNaCl, 0.3mol/L Trisodium Citrate, pH7.0.
Alkali phosphatase enzyme mark Streptavidin: purchased from Gibcol company.
Zinc chloride: purchased from Sangon Biotech (Shanghai) Co., Ltd..
Magnesium chloride hexahydrate: purchased from purchased from Sangon Biotech (Shanghai) Co., Ltd..
Bovine serum albumin: purchased from Sigma-Aldrich.
Tween-20: purchased from purchased from Sangon Biotech (Shanghai) Co., Ltd..
Poly-lysine: purchased from purchased from Sangon Biotech (Shanghai) Co., Ltd..
PEG 8000: purchased from purchased from Sangon Biotech (Shanghai) Co., Ltd..
4.2 operation stepss:
4.2.1 shown hybridization solution composed as follows is prepared: PH7.0,3 × SSC, 1 μ g/mlSA-AP, 10mMZnCl
2, 10mMMgCl
2, 2%BSA, 0.5%Tween-20,0.1%PLL, 2% PEG 8000, surplus is water.
4.2.2 single step reaction: the nitrocellulose filter that the surface obtained in step () is fixed with nucleic acid probe is put into 1ml hybridization solution, add the PCR primer after sex change in the step (three) of 10 μ l, in 37 DEG C of reaction 15min after mixing simultaneously.。
(5) color reaction
5.1 experiment materials:
Nitroblue tetrazolium (NBT) (NBT): purchased from Sangon Biotech (Shanghai) Co., Ltd..
The chloro-3-indyl-phosphoric acid (BCIP) of the bromo-4-of 5-: purchased from Sangon Biotech (Shanghai) Co., Ltd..
Tris: purchased from Sangon Biotech (Shanghai) Co., Ltd..
NaCl: purchased from Sangon Biotech (Shanghai) Co., Ltd..
Ndodecyl glucoside: purchased from Sangon Biotech (Shanghai) Co., Ltd..
5.2 operation stepss:
5.2.1 the chromophoric solution that formulation components is as follows: PH9.5,0.1mol/LTris-HCl, 50mMMgCl
2, 0.33mg/mlNBT, 0.17mg/mlBCIP and 0.07% ndodecyl glucoside, surplus is water.
5.2.2 color reaction: clamp reacted nitrocellulose filter in step (three) with tweezers, allow be fixed with nucleic acid probe nitrocellulose filter surface upwards, tilt film a little, makes one end, the film left side higher than one end, the right.Get chromophoric solution with pipettor and continue drippage chromophoric solution from the top of the upper end of film, make chromophoric solution (instill from the upper end of film, flow out drippage from lower end) from top to bottom, continue to flow through the surface of nitrocellulose filter, flowing time is 10min, observes colour developing result.
Result shows: T533C mutant probe point is strong positive, and the wild-type probe in this site corresponding is negative, thus determines that the rpoB gene of this M. tuberculosis genes group there occurs the nucleic acid base variation of T → C in 533 sites.Positive control point display is simultaneously positive, and the display of negative control point is negative, shows that the detected result that this is tested is normal.Due to the genomic nucleic acids of tubercular simultaneously containing mycobacterium tuberculosis and human body, so detect the positive findings that a sample can show rpoB gene and human body actin gene simultaneously.
Can see from embodiment 8, not only hybridization check process included by method of the present invention quick but also succinct, mainly contains only 2 reactions steps: (1) single step reaction: while the hybridization of generation between single nucleic acid strands and the nitrocellulose filter being fixed with a nucleic acid probe association reaction of biotin labeled PCR primer, Streptavidin and vitamin H association reaction is there occurs between alkali phosphatase enzyme mark Streptavidin and biotin labeling PCR primer strand, just can be formed the conjugate of alkali phosphatase enzyme mark nucleic acid hybrids like this on nitrocellulose filter surface by single step reaction, (2) color reaction: directly drip on the nitrocellulose filter after chromophoric solution to single step reaction, chromophoric solution rinses out at the PCR primer combined by unreacted and alkali phosphatase enzyme mark Streptavidin and vitamin H and generates colour developing precipitation, observations simultaneously.Therefore, the present invention has short, easy to operate, feature that high-throughput, cost are low consuming time, by the application greatly promoting solid phase molecules hybridization technique in clinical detection.
It should be noted that above-described embodiment only for explaining the present invention, not forming any limitation of the invention.By referring to exemplary embodiments, invention has been described, but to should be understood to word wherein used be descriptive and explanatory vocabulary, instead of limited vocabulary.Can modify the present invention by the scope being defined in the claims in the present invention, and the present invention be revised not deviating from scope and spirit of the present invention.Although the present invention wherein described relates to specific method, material and embodiment, and do not mean that the present invention is limited to particular case disclosed in it, on the contrary, easily extensible of the present invention is to other all methods and applications with identical function.
Claims (11)
1. a method for rapid detection sample target nucleic acid, it comprises the steps:
A) at least one nucleic acid probe being fixed on solid support surface is carried out single step reaction with biotin labeled target nucleic acid in the hybridization solution comprising alkali phosphatase enzyme mark Streptavidin;
B) steps A) in reacted after, directly solid support surface is contacted with the chromophoric solution comprising substrate and carries out color reaction, to detect the target nucleic acid in sample.
2. method according to claim 1, is characterized in that, steps A) described in the concentration of alkali phosphatase enzyme mark Streptavidin in hybridization solution be 0.05 ~ 2 μ g/ml, preferably 0.1 ~ 1.5 μ g/ml, more preferably 0.5 ~ 1.2 μ g/ml.
3. method according to claim 1 and 2, is characterized in that, steps A) described in hybridization solution comprise zine ion, magnesium ion, albumen, nonionic surface active agent and cation type polymer; Wherein, albumen is selected from albumin, casein and gelatin, and nonionic surface active agent is selected from tween and Triton, and cation type polymer is selected from cationic polyacrylamide, poly-lysine and polymerize aluminum chloride.
4. according to the method in claims 1 to 3 described in any one, it is characterized in that, in described hybridization solution, zine ion is 0.001 ~ 0.1mol/L, magnesium ion is 0.001 ~ 0.1mol/L, albumen accounts for 1% ~ 10% (w/v) of hybridization solution, and nonionic surface active agent accounts for 0.01 ~ 2% (v/v) of hybridization solution, and cation type polymer accounts for 0.01 ~ 0.5% (w/v) of hybridization solution; Preferably, zine ion is 0.005 ~ 0.05mol/L, magnesium ion is 0.005 ~ 0.05mol/L, albumen accounts for 1% ~ 5% (w/v) of hybridization solution, nonionic surface active agent accounts for 0.05 ~ 1% (v/v) of hybridization solution, and cation type polymer accounts for 0.05 ~ 0.2% (w/v) of hybridization solution volume.
5. according to the method in Claims 1 to 4 described in any one, it is characterized in that, step B) described in chromophoric solution also comprise C
8~ C
18alkyl-glucoside, preferred C
9~ C
13alkyl-glucoside; Described alkyl-glucoside accounts for 0.01 ~ 0.5% (w/v) of aftertreatment fluid, preferably 0.05 ~ 0.1% (w/v).
6. according to the method in Claims 1 to 5 described in any one, it is characterized in that, step B) described in chromophoric solution to contact with described solid support surface in the mode of flowing and develop the color; The direction of described flowing is flow to up and down or the left and right flow direction; The time of described flowing is 2min ~ 30min, preferred 5min ~ 20min, more preferably 8 ~ 15min.
7. according to the method in claim 1 ~ 6 described in any one, it is characterized in that, steps A) described in reaction temperature be 35 ~ 50 DEG C, the reaction times is 5 ~ 30min; Preferably, temperature of reaction is 37 ~ 42 DEG C, and the reaction times is 10 ~ 15min.
8. according to the method in claim 1 ~ 7 described in any one, it is characterized in that, steps A) described in nucleic acid probe be selected from the oligonucleotide probe that length is 15 ~ 40 bases, be preferably selected from the oligonucleotide probe that length is 16 ~ 25 bases.
9. according to the method in claim 1 ~ 8 described in any one, it is characterized in that, in steps A) also comprise the step using the biotin labeled primer for amplifying target nucleic acid to carry out PCR reaction and the sample target nucleic acid that increases before.
10. according to the method in claim 1 ~ 8 described in any one, it is characterized in that, in steps A) before be also included in the existence of biotin labeling deoxynucleotide under carry out the step of PCR reaction and the sample target nucleic acid that increases.
In 11. claims 1 ~ 10, method described in any one is detecting the application in mycobacterium tuberculosis and drug-tolerant gene mutation.
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| PCT/CN2015/087681 WO2016026453A2 (en) | 2014-08-20 | 2015-08-20 | Test kit and method for testing target nucleic acid in sample |
| US15/504,764 US10494665B2 (en) | 2014-08-20 | 2015-08-20 | Test kit and method for testing target nucleic acid in sample |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN112955568A (en) * | 2018-08-15 | 2021-06-11 | Illumina公司 | Compositions and methods for improved library enrichment |
| CN115125291A (en) * | 2022-08-04 | 2022-09-30 | 山东鲁抗好丽友生物技术开发有限公司 | Nucleic acid immobilization method and application thereof |
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|---|---|---|---|---|
| CN102312001A (en) * | 2011-09-14 | 2012-01-11 | 四川大学 | Method for detecting drug resistance of Mycobacterium tuberculosis with multiple PCR-reverse dot blot technique |
| CN103667520A (en) * | 2013-03-04 | 2014-03-26 | 中国检验检疫科学研究院 | Primers, probes and kit for classification diagnosis of foot and mouth disease viruses, and using method of kit |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN102312001A (en) * | 2011-09-14 | 2012-01-11 | 四川大学 | Method for detecting drug resistance of Mycobacterium tuberculosis with multiple PCR-reverse dot blot technique |
| CN103667520A (en) * | 2013-03-04 | 2014-03-26 | 中国检验检疫科学研究院 | Primers, probes and kit for classification diagnosis of foot and mouth disease viruses, and using method of kit |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN112955568A (en) * | 2018-08-15 | 2021-06-11 | Illumina公司 | Compositions and methods for improved library enrichment |
| CN115125291A (en) * | 2022-08-04 | 2022-09-30 | 山东鲁抗好丽友生物技术开发有限公司 | Nucleic acid immobilization method and application thereof |
| CN115125291B (en) * | 2022-08-04 | 2023-03-10 | 山东鲁抗好丽友生物技术开发有限公司 | Nucleic acid immobilization method and application thereof |
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