CN105734116A - Detection method of multiplex nucleic acid sites - Google Patents
Detection method of multiplex nucleic acid sites Download PDFInfo
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- CN105734116A CN105734116A CN201410745719.8A CN201410745719A CN105734116A CN 105734116 A CN105734116 A CN 105734116A CN 201410745719 A CN201410745719 A CN 201410745719A CN 105734116 A CN105734116 A CN 105734116A
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Abstract
The invention provides a method for detecting target bases on to-be-detected sites of two or more target nucleic acids in a sample, wherein the method comprises a step of providing an upstream outer primer and a downstream outer primer as well as two or more groups of upstream inner primers, downstream inner primers and probes to a cyclic amplification reaction mixture which contains the sample and polymerase, wherein each group of the upstream inner primer, downstream inner primer and probe is corresponding to one of the target bases on the to-be-detected sites of the two or more target nucleic acids. The invention also provides a method for detecting mutation of the two or more target nucleic acids in the sample. The invention also provides a kit for the method disclosed by the invention.
Description
Technical field
The present invention relates to field of nucleic acid detection.It is more particularly related to use the method that universal primer and many group non-universal primers detect multiple target nucleic acid site base in the sample with fluorescent probe.
Background technology
In modern molecular diagnostic field, in a small amount of detection sample, the fine difference in multiple nucleic acids content is an important job as far as possible, and wants to detect for various types of viruses or antibacterial in the program simplified and operation simultaneously.
The method using fluorescent probe monitoring nucleic acid amplification reaction is known in the art.The fluorescence signal during the available PCR process of the automated system of the analysis of PCR-based, product amplification provided detects in real time.Being critical that with the modified oligonucleotide carrying reporter group or label of this kind of method.
Detecting a small amount of nucleic acid most common method is PCR or RT-PCR.In measuring given sample, whether target sequence exists, and namely during qualitative analysis, these methods play an important role.Same, the quantitative analysis of nucleic acid uses in a large number in such as quality control, gene expression analysis, medical monitoring and diagnosis.But, there is relevant defect in using of existing double immunofluorescense probe.Such as need to use a large amount of expensive fluorescently-labeled oligonucleotide (including primer and/or probe), primer and/or probe design complicated.
Therefore, this area also needs to a kind of PCR detection method more simplified and as far as possible avoid false positive issue, and wants to detect for various types of viruses or antibacterial in as far as possible few program and operation simultaneously.The method of the present invention is by universal primer and many group non-universal primers and fluorescent probe, it is achieved to the detection of multiple target and realize double verification, it is possible to be greatly enhanced the verity checking result.Methods described herein significantly improve the degree of accuracy of the nucleic acid detection method of prior art.
Summary of the invention
The method of the present invention is by adopting universal primer and many group non-universal primers and fluorescent probe, it is achieved to the detection of multiple target and realize double verification, it is possible to be greatly enhanced the verity checking result.
Concrete, the invention provides a kind of for detecting the method for the targeting base in two or more target nucleic acids site to be measured in sample, it comprises the following steps.
Step (a), provides upstream outer primer, downstream outer primer and two groups or more upstream inner primer, downstream inner primer and probe in the cyclic amplification reaction mixture containing sample and polymerase.
In the method for the invention, described in sample, two or more site to be measured targeting base can be located on a target nucleic acid, or is positioned on different target nucleic acids.Such as, said two or two or more site to be measured can be in the different loci on a target nucleic acid, it is also possible to be in the same site on two or more target nucleic acid or different loci.
In one aspect of the invention, described method is one kettle way, and namely the described cyclic amplification reaction mixture containing sample and polymerase is a mixture (i.e. a reaction system, for instance described test occurs in a vessel).In the other side of the present invention, described method can also carry out (i.e. multiple reaction systems, for instance described test occurs in multiple containers) in two or more mixture.
In the present invention, polymerase is archaeal dna polymerase, participates in DNA replication dna.It is mainly based on template, the polymerization of deoxo ribonucleotide.Molecule after polymerization will form template strand and further participate in pairing.The polymer that the present invention uses has the polymerase activity of 5' → 3'.Described polymerase has 5 ' → 3 ' 5 prime excision enzyme activities.5' → 3' 5 prime excision enzyme activity is exactly the DNA being hydrolyzed DNA growth chain front from 5' → 3' direction, main generation 5'-Deoxydization nucleotide.This enzymatic activity only has cutting vigor effect to matching part (double-strand) phosphodiester bond on DNA, and direction is 5' → 3'.In the one aspect of the present invention, the polymerase of use does not have 3' → 5' 5 prime excision enzyme activity.Can be used for the polymerase of the method for the present invention and include Taq DNA polymerase, TthDNA polymerase etc..
In the one aspect of the present invention, in the step (a) of the method for the invention described above, often one of them in the site to be measured targeting base of group upstream inner primer, downstream inner primer said two corresponding to probe or two or more target nucleic acid.In the one aspect of the present invention, provide one group corresponding upstream inner primer, downstream inner primer and probe for one of them in the site to be measured targeting base of said two or two or more target nucleic acid.Other side in the present invention, it is also possible to upstream inner primer, downstream inner primer and the probe of more than one group are provided for the site to be measured targeting base of a target nucleic acid.
In described often group upstream inner primer, downstream inner primer and probe:
3 ' ends of described upstream inner primer are upstream inner primer target sequence identification fragment, described target sequence identification fragment can the hybridization of specificity and corresponding target nucleic acid and described target sequence identification fragment 3 ' end ends to should the site to be measured of target nucleic acid, the base of 3 ' end ends of this target sequence identification fragment and the site described to be measured targeting base complementation (namely identical with the base in the site to be measured on another strand in double chain target acid) of target nucleic acid.The target nucleic acid that described target sequence identification fragment is not targeting base with the base in site to be measured can not be hybridized.At the one aspect of the present invention, the nucleotide sequence of described target sequence identification fragment and the sequence complete complementary of target nucleic acid.In another aspect of the present invention, the base of 3 ' end ends of described target sequence identification fragment is complementary with the site described to be measured targeting base of target nucleic acid, other base and target nucleic acid can have 1%, 2%, 3%, the mispairing of 5%, its premise is, there is the target sequence identification fragment of described mispairing can hybridize with target nucleic acid, but target nucleic acid for targeting base can not be hybridized with the base in site to be measured.
In the one aspect of the present invention, described upstream inner primer is ARMS primer, i.e. the primer according to sudden change amplification retarding system (amplificationrefractorymutationsystem, ARMS) method design.ARMS design of primers is conventional means in this area, is referred to the description in EP332435.In the present invention, the second to four nucleotide of 3 ' ends end (i.e. the target sequence identification fragment of its target sequence identification fragment) of described upstream inner primer has the mispairing of at least one nucleotide with corresponding target nucleic acid sequence, preferably, described mispairing is to hold second nucleotide of end 3 '.
It addition, 5 ' ends of described upstream inner primer are upstream outer primer overlapping fragments, the sequence of described outer primer overlapping fragments and the sequence of target nucleic acid can not be hybridized, for instance not complementary.The sequence of the outer primer overlapping fragments of described upstream inner primer is identical with the forward primer overlapping fragments that the 3 ' of upstream outer primer hold.
Described downstream inner primer includes 3 ' the downstream outer primer target sequence identification fragments held, its nucleotide sequence can be combined (such as with the complementary of target nucleic acid) with target cell specificity, 5 ' ends of described downstream inner primer are downstream outer primer overlapping fragments, the sequence of its sequence and target nucleic acid can not be hybridized, for instance not complementary.The sequence of the outer primer overlapping fragments of described downstream inner primer is identical with the primer overlapping fragments that the 3 ' of downstream outer primer hold.
In the said method of the present invention, two groups or more the sequence of upstream outer primer overlapping fragments of upstream inner primer group each with in the inner primer of downstream described is identical, and the sequence of the downstream outer primer overlapping fragments of each group is identical.
In the step (a) of the method for the invention described above, 3 ' and 5 ' ends of described probe marked reporter group and quenching group respectively, and the signal of described reporter group can be quenched group cancellation.Described probe can be hybridized with the fragment in the site comprising corresponding described site to be measured in the amplified production of described upstream inner primer and downstream inner primer by specificity.In the present invention, described probe comprises the site in corresponding described site to be measured.Probe the described amplified production of specific hybrid can comprise the site in corresponding described site to be measured, and as on the target nucleic acid of its template, described site to be measured is targeting base.In the one aspect of the present invention, the nucleotide sequence of described probe is identical or complementary with the amplified production of described upstream inner primer and downstream inner primer.Such as, the sequence of fragment that the 5 ' of described probe are held (5 ' end ends to the fragment in the site comprising corresponding described sudden change) is identical with the target sequence identification fragment of upstream inner primer, and namely the site of the corresponding described sudden change of described probe is identical with the 1st and the 2nd nucleotide that described inner primer 3 ' is held with the sequence of the adjacent nucleotides of the 5 ' of this site end sides.One aspect in the present invention, in described probe, the position of the base in corresponding target nucleic acid site to be measured is positioned in the middle of described probe, namely also have in the both sides of this base and the fragment of amplified production hybridization, for instance the fragment identical or complementary with the nucleotide sequence of amplified production.In the other in which of the present invention, in described probe, the position of the base in corresponding target nucleic acid site to be measured can be located at 3 ' or 5 ' end ends of described probe.In the present invention, in described probe, 5 ' ends are identical with the length of the target sequence identification fragment of described upstream inner primer to the length of the fragment (nucleotide in the site described to be measured comprising its corresponding target nucleic acid) in the site described to be measured of its corresponding target nucleic acid or lack one or more nucleotide.Thus, upstream outer primer be template with the amplified production that limited both sides by upstream outer primer (or upstream inner primer) and downstream outer primer (or downstream inner primer) prolongation reaction in, there is the probe that 5 ' → 3 ' exo-acting polymerase hydrolyzables are combined with this amplified production.
In the one aspect of the present invention, described upstream inner primer is ARMS primer, and the second to four nucleotide of its 3 ' end end has the mispairing of at least one nucleotide with corresponding target nucleic acid sequence, for instance 3 ' end second nucleotide of end are mispairing.In this case, the nucleotide sequence due to described probe is identical with the amplified production by described upstream inner primer and downstream inner primer, and therefore in the site of corresponding described mispairing, the nucleotide of described probe is different from the nucleotide of corresponding described target nucleic acid.
In the one aspect of the present invention, two groups or more upstream inner primer described in described method, downstream inner primer are different with the reporter group of each probe mark in probe.Such as in one kettle way, the reporter group of each probe mark is different, it is possible to obtain unlike signal in a reaction system, thus the targeting base in two or more target nucleic acids site to be measured in detection sample.In the other side of the present invention, two groups or more upstream inner primer described in described method, downstream inner primer can be identical with the reporter group of each probe mark in probe.
In the method for the invention, 3 ' ends of described upstream outer primer are upstream outer primer overlapping fragments, and its described outer primer overlapping fragments held with the 5 ' of aforementioned upstream inner primer is identical, and the 5 ' fragments held of described upstream outer primer are not complementary with target nucleic acid.In the method for the invention, 3 ' ends of described downstream outer primer are downstream outer primer overlapping fragments, and its described outer primer overlapping fragments held with the 5 ' of aforementioned downstream inner primer is identical, and the 5 ' fragments held of described downstream outer primer are not complementary with target nucleic acid.In the said method of the present invention, two groups or more the sequence of upstream outer primer overlapping fragments of upstream inner primer group each with in the inner primer of downstream described is identical, and the sequence of the downstream outer primer overlapping fragments of each group is identical.Therefore, in the method for the invention, it is possible to provide only a upstream outer primer and a downstream outer primer.
The method of the present invention also includes step (b), wherein implements multiple cyclic amplification step, and wherein each described cyclic amplification step includes hybridization step and extended nucleic acid step.Hybridization step includes the nucleic acid-templated specificity anneal making primer corresponding.Extended nucleic acid step includes under the catalysis of polymerase, the amplified production that primer extension and generation self-template derive.In step (b), also include the step of the signal of measurement report group, thus the site to be measured targeting base of target nucleic acid is detected.
In the one aspect of the present invention, wherein hybridization step described in step (b) carries out at about 45-72 DEG C, it is preferable that carry out at about 55-65 DEG C, it is most preferred that for carrying out at about 59-62 DEG C.
In step (b), when target nucleic acid is double-stranded DNA, cyclic amplification step may also include denaturing step, and wherein double-stranded DNA template annealing, becomes strand, and thus primer can with the target sequence specific hybrid in template.In the one aspect of the present invention, wherein denaturing step described in step (b) carries out at about 90-96 DEG C, it is preferable that carry out at about 94-96 DEG C, it is most preferred that for carrying out at about 95 DEG C.
In the one aspect of the present invention, wherein extended nucleic acid step described in step (b) carries out at about 55-78 DEG C, it is preferable that carry out at about 60-75 DEG C, it is most preferred that for carrying out at about 60-72 DEG C.
In the present invention, the plurality of cyclic amplification step can be divided into the first amplification stage and the second amplification stage.First amplification stage can obtain with target nucleic acid for template (such as about the 1-10 circulation), upstream inner primer and downstream inner primer the amplified production limited.The amplified production that (such as about 11-30 to 40 circulation) can obtain obtaining with the first amplification stage in the second amplification stage for template, upstream outer primer and downstream outer primer the amplified production limited.
In the method for the invention, described 3 ' and 5 ' ends marked the probe of reporter group and quenching group respectively when complete, such as it is free in reaction solution, although or with target nucleic acid template hybridization and combine, but not exo-acting by when marked the base excision of reporter group or quenching group by polymerase, energy transfer is there is so that from least partially or fully cancellation of the fluorescent emission of reporting dyes between reporter group or quenching group.In the method for the invention, in the annealing being template with the amplified production by upstream outer primer and downstream outer primer restriction both sides and prolongation reaction, when probe may identify which and combine the fragment of the amplified production in the site in corresponding described site to be measured, upstream outer primer and template annealing, extend under the effect of polymerase, now there is the probe that 5 ' → 3 ' exo-acting polymerase hydrolyzables are combined with this amplified production, thus release reporter group and/or quenching group, produce signal.In the one aspect of the present invention, wherein at the signal of described extended nucleic acid step measurement report group, thus the site to be measured targeting base of target nucleic acid is detected.
In the one aspect of the present invention, described probe has about 15-50 base, it is preferred to about 18-35 base.
In the one aspect of the present invention, wherein extended nucleic acid step described in step (b) and hybridization step carry out at the same temperature.Such as, namely it is warming up to the temperature of the denaturing step carrying out next circulation after a step of hybridization, namely this temperature-rise period achieves primer extension.
In the one aspect of the present invention, wherein said reporter group is fluorescent reporter group.One aspect in the present invention, described reporter group is selected from fluorescein(e) dye (such as FAM, JOE and HEX), rhodamine (such as R6G, TAMRA, ROX), and cyanine dye (such as Cy3, Cy3.5, Cy5, Cy5.5 and Cy7).
One aspect in the present invention, wherein said quenching group is selected from fluorescein(e) dye (such as FAM, JOE and HEX), rhodamine (such as R6G, TAMRA, ROX), and cyanine dye (such as Cy3, Cy3.5, Cy5, Cy5.5 and Cy7).
One aspect in the present invention, it is provided that be used for detecting in sample by the method for the invention described above the sudden change to be measured of two or more target nucleic acids, it comprises the following steps:
A () provides upstream outer primer, downstream outer primer and two groups or more upstream inner primer, downstream inner primer and probe in the cyclic amplification reaction mixture containing sample and polymerase, wherein often organize one of them in the sudden change to be measured of upstream inner primer, downstream inner primer said two corresponding to probe or two or more target nucleic acid
Wherein, often organize in upstream inner primer, downstream inner primer and probe:
3 ' ends of described upstream inner primer are upstream inner primer target sequence identification fragment, described target sequence identification fragment can 3 ' end ends of the hybridization of specificity and corresponding target nucleic acid and described target sequence identification fragment to should the mutational site to be measured of target nucleic acid, the base of 3 ' end ends of described target sequence identification fragment is complementary with the mutant bases in the described mutational site of corresponding target nucleic acid, additionally, 5 ' ends of described upstream inner primer are upstream outer primer overlapping fragments, and the sequence of described outer primer overlapping fragments is not complementary with the sequence of corresponding target nucleic acid；
3 ' ends of described downstream inner primer are for downstream primer target sequence identification fragment, and the complementary of its nucleotide sequence and corresponding target nucleic acid, the sequence that 5 ' ends are downstream outer primer overlapping fragments, its sequence and target nucleic acid of described downstream primer is not complementary；
3 ' and 5 ' ends of wherein said probe marked wild-type reporter group and quenching group respectively, the signal of described reporter group can be quenched group cancellation, described probe can specificity and the amplified production of the described upstream inner primer that the target nucleic acid being mutating alkali yl with described site to be measured is template and downstream inner primer comprises corresponding described site to be measured fragment hybridization, such as complete complementary, and in described probe, the length of the fragment in its 5 ' site described to be measured held to its corresponding target nucleic acid is identical with the length of the target sequence identification fragment of described upstream inner primer or lacks one or more nucleotide；
B () implements multiple cyclic amplification step, described cyclic amplification step includes hybridization step and extended nucleic acid step；The signal of measurement report group, thus detects the sudden change to be measured of multiple target nucleic acids.
In the described detection sample of the present invention in the method for two or more target nucleic acids sudden change to be measured, one or more groups upstream inner primer of the wild type of corresponding described target nucleic acid also can be provided in the cyclic amplification reaction mixture containing sample and polymerase, downstream inner primer and probe, upstream inner primer at the wild type of described corresponding target nucleic acid, in downstream inner primer and probe, 3 ' ends of described upstream inner primer are upstream inner primer target sequence identification fragment, described target sequence identification fragment can hold the base of ends complementary with the wild-type base in the described mutational site of corresponding target nucleic acid with the 3 ' of the hybridization of corresponding target nucleic acid and described target sequence identification fragment by specificity；3 ' and 5 ' ends of wherein said probe marked wild-type reporter group and quenching group respectively, the signal of described reporter group can be quenched group cancellation, described probe can specificity and the amplified production of the described upstream inner primer that the target nucleic acid being wild-type base with described site to be measured is template and downstream inner primer comprises corresponding described site to be measured fragment hybridization, for instance complete complementary.Described probe can labelling and corresponding described site to be measured be the different signal of the probe mark signal of variation base, and namely described saltant type reporter group is different with the reporter group of wild-type reporter group.
This is used for detecting in the method for the sudden change of sample target nucleic acid in the method for other condition such as aforementioned present invention definition or limits.
Present invention also offers the test kit of method for implementing the aforementioned present invention.
Wherein one side in the present invention, provide the test kit of the above-mentioned site to be measured targeting base for detecting sample target nucleic acid, comprising: upstream outer primer, downstream outer primer and two groups or more upstream inner primer, downstream inner primer and probe, wherein often organize one of them in the site to be measured targeting base of upstream inner primer, downstream inner primer said two corresponding to probe or two or more target nucleic acid
Wherein, often organize in upstream inner primer, downstream inner primer and probe:
3 ' ends of described upstream inner primer are upstream inner primer target sequence identification fragment, described target sequence identification fragment can 3 ' end ends of the hybridization of specificity and corresponding target nucleic acid and described target sequence identification fragment to should the site to be measured of target nucleic acid, the base of 3 ' end ends of target sequence identification fragment is complementary with the site described to be measured targeting base of corresponding target nucleic acid, additionally, 5 ' ends of described upstream inner primer are upstream outer primer overlapping fragments, and the sequence of described outer primer overlapping fragments is not complementary with the sequence of corresponding target nucleic acid；
3 ' ends of described downstream inner primer are for downstream primer target sequence identification fragment, and the complementary of its nucleotide sequence and corresponding target nucleic acid, the sequence that 5 ' ends are downstream outer primer overlapping fragments, its sequence and target nucleic acid of described downstream primer is not complementary；
Two groups or more the sequence of upstream outer primer overlapping fragments of upstream inner primer group each with in the inner primer of downstream wherein said is identical, and the sequence of the downstream outer primer overlapping fragments of each group is identical；
3 ' and 5 ' ends of wherein said probe marked reporter group and quenching group respectively, the signal of described reporter group can be quenched group cancellation, described probe can specificity and the amplified production of described upstream inner primer and downstream inner primer comprises corresponding described site to be measured fragment hybridization, such as complete complementary, and in described probe, the length of the fragment in its 5 ' site described to be measured held to its corresponding target nucleic acid is identical with the length of the target sequence identification fragment of described upstream inner primer or lacks one or more nucleotide
And,
3 ' ends of wherein said upstream outer primer are forward primer overlapping fragments, and the described upstream outer primer overlapping fragments that it is held with the 5 ' of upstream inner primer is identical, and the 5 ' fragments held of described upstream outer primer are not complementary with target nucleic acid；
3 ' ends of described downstream outer primer are downstream primer overlapping fragments, and it is identical with the downstream outer primer overlapping fragments that the 5 ' of downstream inner primer hold, and 5 ' ends of described downstream outer primer are not complementary with target nucleic acid.
The one aspect of the present invention, described test kit also include have 5 ' → 3 ' exo-acting polymerases.In the one aspect of the present invention, described polymerase does not have 3' → 5' 5 prime excision enzyme activity.
The test kit of the present invention can be used for detecting nucleic acid mutation or microorganism typing.Described microorganism is selected from virus or antibacterial, for instance HBV, HPV etc..
Definition that this is used for detecting in the test kit of the site to be measured targeting base of sample target nucleic acid other composition in the method for the aforementioned present invention or limit.
Definition:
Term " primer " is generally referred to as can as the oligonucleotide of the starting point synthesized along complementary strand under the condition (such as have the template of polymerase and complementary series, and in suitable temperature) with the complementary primer extension product synthesis of catalysis and nucleic acid chains.In polymerization reaction system, primer can be one or more.Term " primer pair " represents one group or pair of primers, it include with amplification nucleotide sequence 5' end complementary sequence hybridization 5' sense primer (being sometimes referred to as " forward " or " upstream ") and with amplification sequence 3' end hybridization 3' antisense primer (being sometimes referred to as " reversely " or " downstream ").
Term " base pairing " or " complementation " refer to known Watson-Crick base pairing, including base pair adenine in double-stranded DNA-between thymus pyrimidine and guanine-cytosine pair, or the pairing of adenine in RNA/DNA hybrid molecule-between uracil and guanine-cytosine pair, and the similar combination between unconventional nucleotide or derivant.The complement of term nucleotide sequence refers to when corresponding with nucleotide sequence so that the 5' end of a sequence and another 3' end are in the oligonucleotide of " antiparallel combination " when matching.Such as, sequence 5'-AGTTC-3' and sequence 5'-GAACT-3' is complementary.Term " complete complementary " etc. refers to the complementary series between antiparallel strand with perfect Watson-Crick base pairing (not having mispairing in polynucleotide Double helix).But, complementation is not completely sometimes, for instance can comprise base mismatch to or do not mate base.Term " partial complementarity ", " partial complementarity ", " non-fully complementary " or " non-fully complementary " etc. refer to any base ratio between antiparallel polynucleotide chain to less than 100% completely (such as, polynucleotide Double helix exists at least one mispairing or does not mate base), but remain to be formed the situation of more stable duplex structure.Such as, the base ratio between antiparallel polynucleotide chain to can be at least 99%, 95%, 90%, or between any value.
Term " target nucleic acid ", " target sequence " or " target nucleic acid sequence " refers to the oligonucleotide region expanding, detecting.Target sequence is generally of the sequence with primer or probes complementary, or in the sequence being used between two primer sequences expanded.In this patent, target nucleic acid typically refers to the wherein chain in DNA double chain.When describing primer or probe and complementary target or being identical, it is identical or complementary with the other chain in the DNA double chain of target nucleic acid.
Term " template " or " template nucleic acid molecule " refer to by polymerase (such as archaeal dna polymerase) from its synthesis complementary nucleic acid chain nucleic acid chains.Such as, Primers complementary, identification and combination in PCR reacts, then from the nucleic acid chains of its synthesis complementary nucleic acid chain.
Term " labelling " refers to and can be used for providing detectable signal, and can be attached to any atom or the molecule of nucleic acid or protein, or this signal adheres to nucleic acid or protein.Labelling can be the signal that can be detected by fluorescence, radioactivity, colorimetry, gravimetry, X-ray diffraction or absorption, magnetic, enzymatic activity etc..
Term " FRET " (FRET (fluorescence resonance energy transfer) orType Resonance energy transfer) it is commonly referred to as the interaction that the dynamic distance between the electronic state of two dye molecules relies on, wherein energy is transferred to acceptor dye molecule from donor dye molecule and is launched without the photon from donor molecule.Generally, FRET requirement, (a) donor and acceptor molecule must close proximity to (general, for instance), the absorption spectrum of (b) receptor must be overlapping with the fluorescence emission spectrum of donor, and (c) donor and acceptor transition dipole orientation must be substantially parallel.
In major part FRET application, donor and acceptor dye or reporter molecule and quencher are different, and FRET can be detected by the quencher of the appearance of receptor sensitized fluorescence or donor fluorescent in this case.In some cases, donor and receptor or reporter molecule and quencher are identical, and FRET can be detected by the fluorescence depolarization produced.
Term " reporter group " or " reporter molecules " are generally referred to as the detectable radiation of generation, for instance fluorescence or luminous radiation, the part of transmitting, this transmitting can be transferred to sufficiently close together suitable FRET quencher.Generally, such molecule is dyestuff.Statement " reporter molecules " can be interchangeably used with " reporter molecules " or " reporter marker " or " FRET mark part ".
Term " quencher " or " quenching molecules " are commonly referred to as minimizing and/or can reduce detectable radiation, for instance but be not limited to fluorescence or luminous radiation, the part of transmitting.Statement " quenching molecules " can be interchangeably used with " quenching group ".Generally, quencher refers to and can reduce photoemissive any part.In some aspects, quencher reduces the detectable radiation at least 50% that source is launched, and selectively at least 80%, and selectively and most preferably at least 90%.In general, quenching molecules causes that the fluorescent emission from reporter molecule reduces, and thus reporter molecule/quenching molecules forms FRET pair, and this quencher is referred to as " FRET quencher " and this report molecule is " FRET reporter molecule ".Term " quencher " is not limited to FRET quencher.In some embodiments, quencher refers to and can reduce photoemissive molecule.As used herein, " quencher " includes any kind of quencher, including dynamically (Energy transfer etc.) and static (ground-state complex).Still alternatively, quencher can be the form except light, for instance heat, and scatter and disappear the energy absorbed from fluorescent dye.In some embodiments, some quencher can launch the wavelength or signal type distinguished with FRET reporter molecule, and can re-emit the energy absorbed from FRET reporter molecule with the wavelength of this quencher feature or signal type.From this respect, quencher can also be " labelling ".
As it is used herein, term " corresponding to " refer to when a nucleic acid and compare with it nucleic acid sequence alignment time, the nucleotide in the first nucleotide sequence is directed at the given nucleotide in reference nucleic acid sequence.
As it is used herein, " polymerase chain reaction " or " PCR " refers to a kind of in vitro method for expanding specific polynucleotide template sequence.PCR reaction includes the temperature cycles of a series of repetition, and generally carries out in the volume of 10-100 μ l.Reactant mixture comprises dNTPs (each in four kinds of Deoxydization nucleotide dATP, dCTP, dGTP and dTTP), primer, buffer, archaeal dna polymerase such as heat-staple archaeal dna polymerase and polynucleotide template.PCR reaction can be made up of " circulation " of 5-100 degeneration of such as polynucleotide molecule and synthesis.
Method provided by the invention overcomes the various defects of the detection method of prior art, obtain bigger signal kinetics and better accuracy, and also have the advantage that have only to consider universal primer, the particularly recognition sequence of inner primer, enormously simplify primer and probe design, reduce difficulty.And, the method for the present invention passes through primer and probe double verification, it is possible to be greatly enhanced the verity checking result.
Accompanying drawing explanation
The nucleotide sequence of one of accompanying drawing 1 amplified reaction template
The nucleotide sequence of the two of accompanying drawing 2 amplified reaction template
The nucleotide sequence of the three of accompanying drawing 3 amplified reaction template
Accompanying drawing 4 fluorescent PCR test result.
Detailed description of the invention
It is further illustrated by the examples that follow the present invention, but these embodiments do not limit the present invention in any way.
Embodiment 1
. amplified reaction template
The method of the present invention is used for expanding three kinds of saltant type templates of following synthesis, it is respectively designated as saltant type 1801, saltant type 2042, saltant type 2361, it comprises the nucleotide sequence (as shown in Figure 3) shown in the nucleotide sequence (as shown in Figure 1) as shown in SEQIDNO.1, the nucleotide sequence (as shown in Figure 2) shown in SEQIDNO.2 and SEQIDNO.3 respectively, and is cloned in the pET30a plasmid that is purchased.
Described three kinds of saltant type templates compare with wild type, have single base mutation: wherein saltant type 1801 is at the 139th of SEQIDNO.1, the C of wild type sport A (base that in sequence shown in Fig. 1, underscore indicates)；Saltant type 2042, at the 213rd of SEQIDNO.2, is sported C (base that in sequence shown in Fig. 2, underscore indicates) by the G of wild type；Wherein saltant type 2361 is at the 308th of SEQIDNO.3, the A of wild type sport C (base that in sequence shown in Fig. 3, underscore indicates).What provide in figure is the sequence of the wherein chain in double-stranded template.Primer in following example and probe for the nucleotide sequence of template can be the sequence provided in figure, it is also possible to be the sequence of its another chain.
Primer and the synthesis with fluorescence labeling probe and labelling
Synthetic and labelling obtain following primer or probe:
General upstream outer primer: 5 '-ACGAGTCAACTAACCACTAGTAGCTGGTTAATAC-3 '
General downstream outer primer: 5 '-CCTTCTCATGCAATCCTAGTAGCTCAGATT-3 '
1801 special primer probes:
Upstream inner primer: 5 '-CCTCAGTCCGTTTCTA-3’
Downstream inner primer: 5 '-CCACATCATCCATATAACTGA-3’
Probe: 5 '-FAM-CCGTTTCTTATGGCTCAG-BHQ1-3’
2042 special primer probes:
Downstream inner primer: 5 '-TGGCTCAGTTTACTAGTG-3’
Upstream inner primer: 5 '-CCCAATACCACATCATG-3’
Probe: 5 '-HEX-CCACATCATGGATATAACT-BHQ1-3’
2361 special primer probes:
Downstream inner primer: 5 '-ATGTTTCCCTCTTGTTGCTGTA-3’
Upstream inner primer: 5 '-CCAACGTTTGGTTTTATTAGGG-3’
Probe: 5 '-CY5-TATTAGGAGTCAAATGTAT-BHQ2-3’
The 2nd base of its middle and upper reaches inner primer 3 ' end (band list underscore) designs for base mismatch.In the present embodiment, in the primer of corresponding three sudden changes to be measured and probe groups, first base of 3 ' end of each upstream inner primer and the base complementrity or identical of saltant type template to be detected, not complementary with the base of wild-type template or differ.Second base of 3 ' end of each upstream inner primer is not complementary with the base of corresponding saltant type template and wild-type template or differs, and is the base of the mispairing design of ARMS primer.
General upstream outer primer and corresponding upstream inner primer, or the Sequence that sequence is its overlap with double underline in the downstream inner primer of general downstream outer primer and correspondence.
In often organizing probe, upstream inner primer and downstream inner primer, the sequence that the 5 ' of described probe are held is identical with the target sequence identification fragment of corresponding upstream inner primer, it is to say, the site of the corresponding described sudden change of described probe is identical with the 1st and the 2nd nucleotide that described upstream inner primer 3 ' is held with the sequence of the 5 ' of this site adjacent nucleotides holding side.Upstream inner primer and downstream inner primer can saltant type template to include the fragment of mutating alkali yl to be measured be that template obtains amplified production, but wild-type template amplified production can not be obtained.The nucleotide sequence of described probe and upstream inner primer and downstream inner primer include, with saltant type template, the fragment complementation that amplified production that the fragment of mutating alkali yl to be measured obtains for template is corresponding, can specific recognition and in conjunction with the amplified production of saltant type template, but not can recognise that and in conjunction with wild-type template amplified production (if any).
3.PCR condition
ABI7500FAST quantitative real time PCR Instrument is used to carry out PCT and fluoroscopic examination.
The archaeal dna polymerase used is: Taq DNA polymerase.
Same reaction system adds:
PCR reactant mixture:
PCR cycle is arranged:
95 DEG C, 2 minutes, 1 circulation；
95 DEG C, 10 seconds, then 56 DEG C, 30 seconds, 10 circulations；
95 DEG C, 10 seconds, then 64 DEG C, 30 seconds, 30 circulations.
Fluorescent labeling according to instrument and use detects automatically.Data are gathered during being wherein set in each 64 DEG C of steps.
Experimental result is shown in Fig. 4.
Demonstrating 4 curves in Fig. 4, it is shown in PCR process the fluorescence signal collected, and the template being respectively adopted in the PCR reaction of 4 curves from top to bottom is: 1801 saltant type template plasmid, 2361 saltant type template plasmid, 2042 saltant type template plasmid and water.As it can be seen, the signal adopting water as the reaction of template is essentially identical with baseline, it does not have significant difference.
In the present embodiment, reporter molecule and quencher are on same probe, and under free state, the signal of the described reporter group of this probe is quenched group cancellation.When upstream inner primer may identify which, when combining and expand mutant sample, namely the corresponding base complementrity of first nucleotide and saltant type template is held in the 3 ' of upstream inner primer, when second nucleotide is not complementary with template, at upstream outer primer with in the prolongation reaction that the amplified production that limited both sides by upstream outer primer and downstream outer primer is template, there is the probe that 5 ' → 3 ' exo-acting polymerase hydrolyzables are combined with this amplified production, namely polymerase can recognize that and cut away the fluorophor being marked on probe, extremely strong fluorescence signal thus detected.And on the other hand, when template does not have corresponding mutant bases, upstream inner primer or downstream inner primer cannot in conjunction with and expand, therefore occur without the reaction that probe is hydrolyzed, fluorescence signal thus can not be detected.
Experiment proves that the design of the present invention very effective can detect base mutation in a reaction system, and with the amplified production of inner primer specific recognition template and probe specificity identification inner primer, greatly reduces the probability that false positive occurs.
Method provided by the invention overcomes many defects of the detection method of prior art, obtain bigger signal kinetics and better accuracy, and also have the advantage that have only to consider universal primer, the particularly recognition sequence of inner primer, enormously simplify primer and probe design, reduce difficulty.And, the method for the present invention passes through primer and probe double verification, it is possible to be greatly enhanced the verity checking result.
Other embodiments are apparent to those skilled in the art.Will be apparent from described in detail above and and be only used as illustration set by clarification.The spirit and scope of the present invention are not limited to above-described embodiment, but are encompassed.
Claims (17)
1., for detecting a method for the targeting base in two or more target nucleic acids site to be measured in sample, it comprises the following steps:
A () provides upstream outer primer, downstream outer primer and two groups or more upstream inner primer, downstream inner primer and probe in the cyclic amplification reaction mixture containing sample and polymerase,
Wherein, often organize in upstream inner primer, downstream inner primer and probe:
3 ' ends of described upstream inner primer are upstream inner primer target sequence identification fragment, described target sequence identification fragment can 3 ' end ends of the hybridization of specificity and corresponding target nucleic acid and described target sequence identification fragment to should the site to be measured of target nucleic acid, the base of 3 ' end ends of target sequence identification fragment is complementary with the site described to be measured targeting base of corresponding target nucleic acid, additionally, 5 ' ends of described upstream inner primer are upstream outer primer overlapping fragments, and the sequence of described outer primer overlapping fragments is not complementary with the sequence of corresponding target nucleic acid；
3 ' ends of described downstream inner primer are for downstream primer target sequence identification fragment, and the complementary of its nucleotide sequence and corresponding target nucleic acid, the sequence that 5 ' ends are downstream outer primer overlapping fragments, its sequence and target nucleic acid of described downstream primer is not complementary；
Two groups or more the sequence of upstream outer primer overlapping fragments of upstream inner primer group each with in the inner primer of downstream wherein said is identical, and the sequence of the downstream outer primer overlapping fragments of each group is identical；
3 ' and 5 ' ends of wherein said probe marked reporter group and quenching group respectively, the signal of described reporter group can be quenched group cancellation, described probe can specificity and the amplified production of described upstream inner primer and downstream inner primer comprises corresponding described site to be measured fragment hybridization, and in described probe, the length of the fragment in its 5 ' site described to be measured held to its corresponding target nucleic acid is identical with the length of the target sequence identification fragment of described upstream inner primer or lacks one or more nucleotide
And,
3 ' ends of wherein said upstream outer primer are forward primer overlapping fragments, and the described upstream outer primer overlapping fragments that it is held with the 5 ' of described upstream inner primer is identical, and the 5 ' fragments held of described upstream outer primer are not complementary with target nucleic acid；
3 ' ends of described downstream outer primer are downstream primer overlapping fragments, and the described downstream outer primer overlapping fragments that it is held with the 5 ' of described downstream inner primer is identical, and 5 ' ends of described downstream outer primer are not complementary with target nucleic acid；
B () implements multiple cyclic amplification step, described cyclic amplification step includes hybridization step and extended nucleic acid step；The signal of measurement report group, thus detects the site to be measured targeting base of target nucleic acid.
2. the process of claim 1 wherein that described upstream inner primer is ARMS primer, the second to four nucleotide of its 3 ' end end has the mispairing of at least one nucleotide with corresponding target nucleic acid sequence, it is preferred that described mispairing is to hold second nucleotide of end 3 '.
3. the method for claim 1 or 2, the nucleotide sequence of wherein said probe and the fragment complementation comprising corresponding described site to be measured in the amplified production of the described upstream inner primer that the target nucleic acid being targeting base with described site to be measured is template and downstream inner primer.
4. the method any one of claim 1-3,5 ' end ends of wherein said probe are identical to the sequence of the fragment in the site comprising corresponding described sudden change and the target sequence identification fragment of described upstream inner primer.
5. the method any one of claim 1-4, wherein described in sample, two or more site to be measured targeting base can be located on a target nucleic acid, or is positioned on different target nucleic acids.
6. the method any one of claim 1-5, the cyclic amplification reaction mixture wherein containing sample and polymerase described in step a) is a mixture.
7. the method any one of claim 1-6, in two groups or more upstream inner primer wherein said, downstream inner primer and probe, the reporter group of each probe mark is identical or different.
8. the method for claim 1, wherein multiple cyclic amplification steps described in step (b) include the first amplification stage and the second amplification stage, first amplification stage can obtain with target nucleic acid for template, the amplified production limited by corresponding upstream inner primer and downstream inner primer, the amplified production that can obtain in second amplification stage obtaining with the first amplification stage for template, upstream outer primer and downstream outer primer the amplified production limited.
9. the method any one of claim 1-8, wherein at the signal of described extended nucleic acid step measurement report group, thus detects the site to be measured targeting base of target nucleic acid.
10. the method any one of claim 1-9, in wherein said probe, the position of the base in corresponding target nucleic acid site to be measured is positioned in the middle of described probe.
11. the method any one of claim 1-10, wherein said polymerase has 5 ' → 3 ' exo-acting, it is furthermore preferred that described polymerase does not have 3' → 5' 5 prime excision enzyme activity.
12. the method any one of claim 1-11, it is used for detecting in sample the sudden change to be measured of two or more target nucleic acids, and it comprises the following steps:
A () provides upstream outer primer, downstream outer primer and two groups or more upstream inner primer, downstream inner primer and probe in the cyclic amplification reaction mixture containing sample and polymerase, wherein often organize one of them in the sudden change to be measured of upstream inner primer, downstream inner primer said two corresponding to probe or two or more target nucleic acid
Wherein, often organize in upstream inner primer, downstream inner primer and probe:
3 ' ends of described upstream inner primer are upstream inner primer target sequence identification fragment, described target sequence identification fragment can 3 ' end ends of the hybridization of specificity and corresponding target nucleic acid and described target sequence identification fragment to should the mutational site to be measured of target nucleic acid, the base of 3 ' end ends of described target sequence identification fragment is complementary with the mutant bases in the described mutational site of corresponding target nucleic acid, additionally, 5 ' ends of described upstream inner primer are upstream outer primer overlapping fragments, and the sequence of described outer primer overlapping fragments is not complementary with the sequence of corresponding target nucleic acid；
3 ' ends of described downstream inner primer are for downstream primer target sequence identification fragment, and the complementary of its nucleotide sequence and corresponding target nucleic acid, the sequence that 5 ' ends are downstream outer primer overlapping fragments, its sequence and target nucleic acid of described downstream primer is not complementary；
3 ' and 5 ' ends of wherein said probe marked saltant type reporter group and quenching group respectively, the signal of described reporter group can be quenched group cancellation, described probe can specificity and the amplified production of the described upstream inner primer that the target nucleic acid being mutating alkali yl with described site to be measured is template and downstream inner primer comprises corresponding described site to be measured fragment hybridization, and in described probe, the length of the fragment in its 5 ' site described to be measured held to its corresponding target nucleic acid is identical with the length of the target sequence identification fragment of described upstream inner primer or lacks one or more nucleotide；
B () implements multiple cyclic amplification step, described cyclic amplification step includes hybridization step and extended nucleic acid step；Measure the signal of sudden change reporter group, thus the sudden change to be measured of multiple target nucleic acids is detected.
13. the method for claim 12, in the cyclic amplification reaction mixture containing sample and polymerase, wherein also provide one or more groups upstream inner primer of wild type of corresponding described target nucleic acid, downstream inner primer and probe, in the described corresponding upstream inner primer of wild type of target nucleic acid, downstream inner primer and probe:
3 ' ends of described upstream inner primer are upstream inner primer target sequence identification fragment, and described target sequence identification fragment can hold the base of ends complementary with the wild-type base in the described mutational site of corresponding target nucleic acid with the 3 ' of the hybridization of corresponding target nucleic acid and described target sequence identification fragment by specificity；
3 ' and 5 ' ends of wherein said probe marked wild-type reporter group and quenching group respectively, the signal of described reporter group can be quenched group cancellation, and described probe can specificity and the fragment hybridization comprising corresponding described site to be measured in the amplified production of the described upstream inner primer that the target nucleic acid being wild-type base with described site to be measured is template and downstream inner primer.
14. the method for claim 13, wherein said saltant type reporter group is different with the reporter group of wild-type reporter group.
15. one kind for detecting the test kit of the method for the targeting base in two or more target nucleic acids site to be measured in sample as according to any one of claim 1-14, comprising: upstream outer primer, downstream outer primer and two groups or more upstream inner primer, downstream inner primer and probe, wherein often organize one of them in the site to be measured targeting base of upstream inner primer, downstream inner primer said two corresponding to probe or two or more target nucleic acid
Wherein, often organize in upstream inner primer, downstream inner primer and probe:
3 ' ends of described upstream inner primer are upstream inner primer target sequence identification fragment, described target sequence identification fragment can 3 ' end ends of the hybridization of specificity and corresponding target nucleic acid and described target sequence identification fragment to should the site to be measured of target nucleic acid, the base of 3 ' end ends of target sequence identification fragment is complementary with the site described to be measured targeting base of corresponding target nucleic acid, additionally, 5 ' ends of described upstream inner primer are upstream outer primer overlapping fragments, and the sequence of described outer primer overlapping fragments is not complementary with the sequence of corresponding target nucleic acid；
3 ' ends of described downstream inner primer are for downstream primer target sequence identification fragment, and the complementary of its nucleotide sequence and corresponding target nucleic acid, the sequence that 5 ' ends are downstream outer primer overlapping fragments, its sequence and target nucleic acid of described downstream primer is not complementary；
Two groups or more the sequence of upstream outer primer overlapping fragments of upstream inner primer group each with in the inner primer of downstream wherein said is identical, and the sequence of the downstream outer primer overlapping fragments of each group is identical；
3 ' and 5 ' ends of wherein said probe marked reporter group and quenching group respectively, the signal of described reporter group can be quenched group cancellation, described probe can specificity and the amplified production of described upstream inner primer and downstream inner primer comprises corresponding described site to be measured fragment hybridization, and in described probe, the length of the fragment in its 5 ' site described to be measured held to its corresponding target nucleic acid is identical with the length of the target sequence identification fragment of described upstream inner primer or lacks one or more nucleotide
And,
3 ' ends of wherein said upstream outer primer are forward primer overlapping fragments, and the described upstream outer primer overlapping fragments that it is held with the 5 ' of upstream inner primer is identical, and the 5 ' fragments held of described upstream outer primer are not complementary with target nucleic acid；
3 ' ends of described downstream outer primer are downstream primer overlapping fragments, and it is identical with the downstream outer primer overlapping fragments that the 5 ' of downstream inner primer hold, and 5 ' ends of described downstream outer primer are not complementary with target nucleic acid.
16. the test kit of claim 15, wherein also including having 5 ' → 3 ' exo-acting polymerases, wherein said polymerase has 5 ' → 3 ' exo-acting, it is furthermore preferred that described polymerase does not have 3' → 5' 5 prime excision enzyme activity.
17. the test kit of claim 15 or 16, it is used for detecting nucleic acid mutation.
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