CN1405320A - Artificial sequence template primer collection and its use - Google Patents
Artificial sequence template primer collection and its use Download PDFInfo
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Abstract
The invention provides a method of detecting and/or quantifying nucleic acid by using man-made sequence mould. The mould eduction collecting includes few-ribotide solicitation pair peculiarly combined with sequence to be detected and initiating expanding reaction of destination nucleic acid in which at least one is the man-made sequence mould solicitation which includes: (a) differential-combining area; (b) public area, located on 5' end of man-made sequence mould solicitation and having reporting-molecular combining area and public solicitation area.
Description
Technical field
The present invention relates to the detection of nucleic acids field, more specifically, relate to a kind of artificial sequence template primer collection, and use this artificial sequence template primer collection to detect and/or the method for quantitative nucleic acid.
Background technology
Recently, detect rapidly and accurately and/or quantitative pathogenic agent (as virus, bacterium, fungi) in order to satisfy, and the specific nucleic acid sequence in the normal and undesired gene, have a large amount of technology of having developed.These technology detect and quantitative food, environmental sample, breeding stock and other types material in wide purposes is arranged aspect the microorganism, under these occasions, need to monitor certain and infer microorganism and whether exist.Other application comprise aspects such as being used for medical jurisprudence, molecular pathology, anthropology, archeology and biology.
A kind of common way that realizes this generic task is a nucleic acid hybridization.This method forms the ability of duplex structure based on two nucleic acid chains under conditions suitable, thereby wherein these two nucleic acid chains contain complementation or basic complementary sequence combination specifically.In order to detect and/or quantitative specific nucleotide sequence (being called " target sequence "), need the oligonucleotide (" probe ") of preparation mark, this probe contains the sequence with target complement sequence.In order to detect delicately and/or quantitatively micro-genetic material, many more proven technique have been developed, they are usually directed to amplifying target nucleic acid in specimen (DNA or RNA) and detect subsequently, comprising polymerase chain reaction (PCR), ligase chain reaction (LCR), chain replace amplification (SDA), transcriptive intermediate amplification (transcription mediatedamplication, TMA) and self-sustained synthetic reaction (3SR).
Although all these technology all are to detect and differentiate the favourable instrument of micro-target nucleic acid in the sample, they have various problem, and these problems have limited their applicabilitys when being used for routine operation under the clinical experiment room environmental.One of the most difficult problem is that amplifying target nucleic acid in every kind of test is different so that detect with the condition of quantitative analysis subsequently.In other words, be not beneficial to the unified condition of testing standardization.
In addition,, detect exactly and/or the pathogenic agent that quantitatively has different genotype or a sudden change (as causing drug-fast sudden change) is well-known challenge always based on for the detection of nucleic acids and/or quantivative approach of target sequence amplification for present.
Branched DNA (bDNA) method is a kind of emerging amplification of signal technology, and it can provide high duplication and accuracy.Yet the sensitivity of this method itself is limited, and is difficult to extensively adopt for Routine Test Lab institute.
Therefore, this area press for exploitation easy-to-use, sensitive, accurately detect and/or quantitative sample in the analytical technology of pathogenic agent and genetic expression.
Summary of the invention
Purpose of the present invention just provides and a kind ofly can be used for detecting and/or quantitatively easy-to-use, sensitive, the analytical technology accurately of pathogenic agent and genetic expression in the sample.New technology of the present invention can overcome many limitations of the prior art.
In a first aspect of the present invention, a kind of nucleic acid detection method is provided, it comprises step:
(1) in containing the nucleic acid amplification reaction system of reporter molecules, carry out nucleic acid amplification reaction with the artificial sequence template primer collection testing sample, wherein said primer collection comprise specificity be incorporated into detected nucleotide sequence and cause the primer of purpose nucleic acid amplification reaction right, and at least one primer is an artificial sequence template primer, and this artificial sequence template primer comprises:
(a) specific combination district, this specific combination district is positioned at artificial sequence template primer 3 ' end, is used for combining with detected nucleotide sequence specificity;
(b) public domain, this public domain is positioned at 5 of artificial sequence template primer ' hold and has reporter molecules land and public guiding region, but the reporter molecules specificity is incorporated into the complementary strand of this report molecule land or this report molecule land, and the state difference of described reporter molecules under following two kinds of situations, thereby produce detectable signal: (i) be incorporated into the complementary strand of reporter molecules land or this report molecule land and (ii) reporter molecules be cut off or be substituted;
(2) detectable signal that produced of examining report molecule.
In a preference, the quantity of described reporter molecules is more than or equal to the quantity of artificial sequence template primer.
In another preference, use two or more different artificial sequence template primer collection, these artificial sequence template primer collections are incorporated into the different zones of identical target sequence or its combination of the different target sequences of the target sequence of different detected bodies, same detected body, same detected body respectively specifically, and the reporter molecules land of these artificial sequence template primer collections combines with identical or different reporter molecules generation specificity respectively.
In a second aspect of the present invention, a kind of artificial sequence template primer collection is provided, described primer collection comprise specificity be incorporated into detected sequence and cause the Oligonucleolide primers of purpose nucleic acid amplification reaction right, wherein at least one primer is an artificial sequence template primer, and this artificial sequence template primer comprises:
(a) specific combination district, this specific combination district is positioned at artificial sequence template primer 3 ' end, is used for combining with detected nucleotide sequence specificity;
(b) public domain, this public domain is positioned at 5 of artificial sequence template primer ' hold and has reporter molecules land and public guiding region, but the reporter molecules specificity is incorporated into the complementary strand of this report molecule land or this report molecule land, and the state difference of described reporter molecules under following two kinds of situations, thereby produce detectable signal: (i) be incorporated into the complementary strand of reporter molecules land or this report molecule land and (ii) reporter molecules be cut off or be substituted.
In a preference, also there is transcribed spacer in artificial sequence template primer between specific combination district and reporter molecules land, and/or has transcribed spacer between public guiding region and reporter molecules land, and described transcribed spacer is 1-20bp, preferably is 5-10bp.
In another preference, described artificial sequence template primer collection also comprises the amplification template primer that is used to increase detected nucleic acid-templated quantity.
In another preference, the length of the public guiding region of described artificial sequence template primer is 0bp.
In a third aspect of the present invention, a kind of kit for detecting nucleic acid is provided, it contains artificial sequence template primer collection of the present invention.
Description of drawings
Fig. 1 has shown two kinds of structural representations of artificial sequence template primer of the present invention.
Fig. 2 has shown the various array configurations of artificial sequence template primer collection of the present invention.
Fig. 3 has shown that signal produces several synoptic diagram in the artificial sequence template detection of the present invention.
Fig. 4 is the synoptic diagram of a kind of nucleic acid detection method of the present invention, and use therein artificial sequence template primer collection contains an artificial sequence template primer, its reporter molecules in conjunction with the reporter molecules land of artificial sequence template.
Fig. 5 is the synoptic diagram of a kind of nucleic acid detection method of the present invention, use therein artificial sequence template primer collection contains an artificial sequence template primer, a conventional primer, a public primer and an amplification template primer, its reporter molecules in conjunction with the reporter molecules land of artificial sequence template.
Embodiment
As used herein, following word/term has following meanings, unless otherwise indicated.
" nucleic acid ": the oligonucleotide analogs of polynucleotide analogue, RNA or the DNA of Yeast Nucleic Acid (RNA), thymus nucleic acid (DNA), RNA or DNA.
" template ": can be by the total length of the nucleic acid molecule of nucleic acid polymerization enzymatic amplification or partial sequence.Template can be RNA or DNA or its analogue, and can be strand, two strands or partially double stranded.
" artificial sequence template (AT) primer ": artificial sequence template primer is the synthetic oligonucleotide sequence.Referring to Figure 1A, 3 of artificial sequence template primer ' end has the specific combination district, and this specific combination district is complementary to target sequence and extends in amplified reaction as primer.In addition, artificial sequence template primer also contains public domain, and this public domain is positioned at 5 of artificial sequence template primer ' hold and has reporter molecules land (or its complementary sequence) and public guiding region.But the reporter molecules specificity is incorporated into the complementary strand of this report molecule land or this report molecule land.Artificial sequence template primer can be synthetic with the whole bag of tricks well known by persons skilled in the art.
A kind of length of public guiding region of artificial sequence template primer of special shape is 0bp, shown in Figure 1B.
As used herein, " reporter molecules land " refer to artificial sequence template primer on reporter molecules generation bonded zone.Certainly, reporter molecules can directly be incorporated into this report molecule land on the artificial sequence template primer, also can be incorporated into the complementary strand (for simplicity, the zone the same with the reporter molecules sequence on the artificial sequence template primer still is called as " reporter molecules land ") of this land.
In addition, also there is transcribed spacer in a kind of preferred artificial sequence template primer between specific combination district and reporter molecules land, and/or between public guiding region and reporter molecules land, having transcribed spacer, described transcribed spacer is 1-20bp (preferably being 3-10bp, more preferably is 5-10bp).The main purpose of adding transcribed spacer is: (1) regulates the Tm of artificial sequence template primer, makes the Tm of each primer in the artificial sequence template primer collection approaching or identical; (2) prevent the formation of some secondary structure (as hairpin structure, dimer etc.); And/or (3) prevent sterically hindered.Yet should be understood that all needs transcribed spacer under not all situation, do not need transcribed spacer sometimes.Depend on sequence to be detected, those skilled in the art can determine whether artificial sequence template primer needs transcribed spacer, and the length of transcribed spacer and composition.
" reporter molecules " is a kind of molecule that is used to produce detectable signal.The state difference of described reporter molecules under following two kinds of situations, thus produce detectable signal: (i) be incorporated into reporter molecules land or its complementary sequence zone and (ii) reporter molecules be cut off or be substituted.Suitable reporter molecules example is based on the probe of FRET (fluorescence resonance energy transfer) (FRET) principle and contains the oligonucleotide chain of rare earth element.
FRET (fluorescence resonance energy transfer) (FRET): be the interaction that utilizes between different fluorescent substance excitation wavelengths, wavelength of transmitted light, a kind of signal detection principle that some special wavelength light is detected.The probe that produces signal based on this principle is called as FRET (fluorescence resonance energy transfer) type probe.Specially suitable FRET (fluorescence resonance energy transfer) type probe comprises (but being not limited to): Taqman probe, molecular beacon (molecular beacon) (Fig. 3 D), the two probes (Fig. 2 J) of FRET and peptide nucleic acid(PNA) (peptide nucleic acid) signal probe (abbreviating the PNA signal probe as).
" Taqman probe " be a kind ofly have reporter group and 3 at its 5 ' end ' end has the oligonucleotide of quencher group, described quencher group can suppress reporter group and produce detectable signal (for example fluorescence).The Taqman probe design is a prior art, and can obtain (Heid CA, Stevens J, Livak KJ, Williams PM for information about from open approach; Real Time Quantitaive PCR, Genome Res.1996 Oct.; 6 (10): 986-94).
Fig. 3 has shown that signal produces several synoptic diagram in the artificial sequence template detection of the present invention.Fig. 3 A and Fig. 3 B are that the Taqman probe produces detectable signal because of cutting, and Fig. 3 C is that the two probes of FRET are cut because of enzyme or replaced and produce detectable signal.Fig. 3 D is that molecular beacon produces detectable signal because of hybridization.
" artificial sequence template " refers in PCR process of the present invention, the nucleotide sequence that has mixed artificial sequence template primer that amplifies, or its anti sense nucleotide sequence.These artificial sequence templates had both had the nucleotide sequence corresponding to target sequence, had again corresponding to the artificial sequence template primer public domain nucleotide sequence of (comprising reporter molecules land, public guiding region and transcribed spacer).In pcr amplification circulation subsequently, these artificial sequence templates work as template.
The method disclosed in the present is classified as the amplification of signal method in principle.Its key has been to use the template (this be called " artificial sequence template ") of the suitable nucleotide sequence that contains artificial sequence as amplification of signal, and therefore corresponding method is called as " foranalysis of nucleic acids of artificial sequence template amplification " (AT analysis).AT analyzes can sensitive, accurate and detection of stdn ground and/or quantifying target nucleic acid molecule.
In the present invention, for testing sample without limits, as long as wherein contain genetic material.Representational testing sample comprises (but being not limited to): DNA sample, RNA sample and the cDNA sample that obtains through reverse transcription from RNA, and the polynucleotide of other forms of modified.
In artificial sequence template primer of the present invention, the length in described specific combination district is not particularly limited, its length is 6-35bp usually, preferably is 15-25bp.For also being not particularly limited of described public domain, its length is 8-100bp usually, preferably is 20-60bp, more preferably is about 30-50bp.
The Nucleotide that contains in the artificial sequence template primer is selected from A, T, C, G usually.Yet, in described artificial sequence template primer, contain some other Nucleotide producing special detection effect, as increasing specificity, mixing fluorescence molecule or increase and combining of template etc.Suitable example but be not limited to is selected from down the Nucleotide of group: isoG, isoC, 2 '-O-methyl-G, 2 '-O-methyl-C and combination thereof.
In reaction system of the present invention, the quantitative relation of reporter molecules and artificial sequence template primer is not particularly limited.Yet preferably, the quantity of described reporter molecules should be more than or equal to the quantity of artificial sequence template primer, and reporter molecules and the ratio of artificial sequence template primer are greater than 1 usually: 1-10: 1, and more preferably be 1.5: 5: 1.Like this, in reaction system, artificial sequence template primer just all is in and reporter molecules bonded state basically.
When reporter molecules was the Taqman probe, the Tm of Taqman probe and artificial sequence template primer reporter molecules land should be higher than the Tm of artificial sequence template primer specific combination district and template.Usually, exceed 2-15 ℃, preferably exceed 5-12 ℃.
In addition, in artificial sequence template primer collection of the present invention, also can comprise one or more " amplification template primers ".As used herein, " amplification template primer " refers to be used to increase the primer of detected nucleotide sequence (template) quantity.The amplification template primer is incorporated into the upstream or the downstream of sequence to be detected, can improve the quantity of template effectively in amplified reaction, thereby the sensitivity of detection is provided.Should be understood that " amplification template primer " is conventional primer, just its effect is the quantity that improves template to be detected.
In the present invention, as shown in Figure 2, artificial sequence template primer collection has multiple array configuration, comprising (but being not limited to):
(1) downstream is an artificial sequence template primer, and the upstream is conventional primer, and reporter molecules combines (Fig. 2 A) with artificial sequence template primer, or the upstream and downstream primer location exchanges (Fig. 2 B);
(2) primer collection of Fig. 2 A and the combination (Fig. 2 C) that the amplification template primer forms, the primer collection of Fig. 2 B and the combination (Fig. 2 D) that the amplification template primer forms.Be that upstream and downstream is that conventional primer is right, the centre is an artificial sequence template primer, and reporter molecules combines with artificial sequence template primer.
Article (3) two, artificial sequence template primer and two combinations (Fig. 2 E) that the amplification template primer constitutes.Be that upstream and downstream is that conventional primer is right, the centre is the relative artificial sequence template primer of a pair of direction, and reporter molecules combines with artificial sequence template primer;
(4) upstream and downstream is that conventional primer is right, and middle two artificial sequence template primer directions are opposite, and reporter molecules combines (Fig. 2 F) with artificial sequence template primer;
(5) downstream is an artificial sequence template primer, and the upstream is conventional primer, and reporter molecules combines (Fig. 2 G) with the complementary sequence of artificial sequence template primer, or the upstream and downstream primer location exchanges (not shown);
(6) primer collection of Fig. 2 G and the combination (Fig. 2 H) that the amplification template primer forms, be that upstream and downstream is that conventional primer is right, the centre is an artificial sequence template primer, and reporter molecules combines with the artificial sequence template primer complementary sequence, or the artificial sequence template primer direction transformation;
(7) upstream is conventional primer, and the downstream is an artificial sequence template primer, and reporter molecules is marked at (Fig. 2 I) on artificial sequence template primer and the reporter molecules respectively, or artificial sequence template primer direction transformation (not shown); The perhaps primer collection of Fig. 2 I and combination (Fig. 2 J) that the amplification template primer forms;
(8) combination of Fig. 2 E+ Fig. 2 G, promptly upstream and downstream is that conventional primer is right, and the centre is a pair of oppositely relative artificial sequence template primer, and reporter molecules combines (not shown) with the artificial sequence template primer complementary sequence
(9) combination of Fig. 2 F+ Fig. 2 G, promptly upstream and downstream is that conventional primer is right, and middle two artificial sequence template primer directions are opposite, and reporter molecules combines (not shown) with the artificial sequence template primer complementary sequence;
(10) combination of Fig. 2 E+ Fig. 2 I, promptly upstream and downstream is that conventional primer is right, and the centre is a pair of opposite relative artificial sequence template primer, and reporter molecules is marked at (not shown) on artificial sequence template primer and the reporter molecules respectively;
(11) combination of Fig. 2 F+ Fig. 2 I, promptly upstream and downstream is that conventional primer is right, and the centre is two artificial sequence template primers that direction is opposite, and reporter molecules is marked at (not shown) on artificial sequence template primer and the reporter molecules respectively.
In the methods of the invention, on same detected nucleotide sequence, can place one or more artificial sequence template primer collections simultaneously.Can detect the different positions on the detected nucleotide sequence so simultaneously.
In the present invention, for for artificial sequence template primer bonded reporter molecules, different artificial sequence template primers can be in conjunction with identical reporter molecules, also can be in conjunction with different reporter molecules (for example have send out different the reporter group of fluorescence and the reporter molecules of corresponding quencher group, the reporter molecules that sequence is identical or different).
The length of the artificial sequence template that amplifies for artificial sequence template primer collection is not particularly limited.Apart the distance expression on template of the 3 ' end in specific combination district of two primers of pressing artificial sequence template primer collection is generally 1bp-10kb, preferably is 1-2kb, more preferably is 1-500b, is 1-100bp best.Especially be when spacing during, can design at for example primer collection of pathogenic agent high conserved region, thereby reduce false negative rate less than 100bp.In addition, spacing is short more, can bring into play the general character of artificial sequence template more.
Further specify the present invention below in conjunction with accompanying drawing.
The primer collection (combination shown in Fig. 2 A) that amplification pattern (), use contain artificial sequence template primer and conventional primer carries out the nucleic acid amplification analysis
Now referring to Fig. 4.In this example, use an artificial sequence template primer collection, this primer collection comprises an artificial sequence template primer 1 and a conventional primer 2.In addition, comprise also in the reaction system that public primer 3 and reporter molecules 4 constitute.In this example, sample to be detected is RNA or DNA.
Step 1: primer collection and testing sample are placed reaction system.
Step 2: under appropriate condition, anneal (or hybridization), promptly the specific combination district of 3 of artificial sequence template primer 1 ' end is incorporated into target RNA or dna sequence dna.
Step 3: at reverse transcriptase (for the RNA target sequence) or archaeal dna polymerase (for the DNA target sequence), 3 of artificial sequence template primer ' end extends to target sequence 5 ' end, forms RNA/DNA or DNA/DNA two strands.
Step 4: the sex change under appropriate condition of the double-strandednucleic acid of formation forms strand target sequence and new synthetic dna sequence dna (being artificial sequence template).Perhaps use RNase hydrolysis RNA chain, form new synthetic dna sequence dna (being artificial sequence template).
In case of necessity, under appropriate condition, repeat above-mentioned steps 3-4.
Step 5: conventional primer 2 is incorporated into the complementary region of new synthetic dna sequence dna.
Step 6: in the presence of archaeal dna polymerase, 3 of conventional primer 2 ' end extends to artificial sequence template sequence 5 ' end, double-stranded DNA (new synthetic dna sequence dna also is an artificial sequence template in this two strands).When 3 of conventional primer 2 ' end extended to the reporter molecules place that is incorporated into the reporter molecules land, following 3 kinds of situations can take place:
(1) extension stops;
(2) continue to extend, reporter molecules is replaced from artificial sequence template;
(3) continue to extend, and cut off or destroy reporter molecules.
Under first and second kinds of situations, reporter molecules is still complete, therefore can not produce detectable signal.And under the third situation, because the quencher group on reporter molecules (as the Taqman probe) 5 ' end is by 5 ' nuclease institute hydrolysis of archaeal dna polymerase (as the Taq polysaccharase), therefore at the reporter group at Taqman probe 3 ' end place just no longer by quencher, thereby cause producing detectable signal (as fluorescent signal).
Step 7: in next circulation, during annealing after sex change, three kinds of situations can take place:
7a: artificial sequence template primer 1 is incorporated into the new synthetic dna sequence dna that contains conventional primer 2 sequence.
7b: public primer 3 is incorporated into the new synthetic dna sequence dna that contains conventional primer 2 sequence.
7c: conventional primer 2 is in conjunction with the artificial sequence template (being similar to step 5, not shown) that contains artificial sequence primer 1 sequence.
Step 8: divide three kinds of situations:
8a: artificial sequence template primer 1 extends to 5 of the DNA chain that contains conventional primer 2 sequence ' end, forms the dna double chain.In the next round circulation, these dna double chains are exactly new artificial sequence template.
8b: public primer 3 extends to 5 of the DNA chain that contains conventional primer 2 sequence ' end, forms the dna double chain.In the next round circulation, these dna double chains are exactly new artificial sequence template.
8c:, when 3 of conventional primer 2 ' end extends to the reporter molecules place that is incorporated into the reporter molecules land, following 3 kinds of situations can take place with identical in the step 6:
(1) extension stops;
(2) continue to extend, reporter molecules is replaced from artificial sequence template;
(3) continue to extend, and cut off or destroy reporter molecules.
Under first and second kinds of situations, reporter molecules is still complete, therefore can not produce detectable signal.And under the third situation, because the quencher group on reporter molecules (as the Taqman probe) 5 ' end is by 5 ' nuclease institute hydrolysis of archaeal dna polymerase (as the Taq polysaccharase), therefore at the reporter group at Taqman probe 3 ' end place just no longer by quencher, thereby cause producing detectable signal (as fluorescent signal).
Step 9: repeating step 7 and 8.
Through after some circulations, identical with the PCR principle, also be that exponentially level (or being similar to exponential relationship) increases because of reporter molecules is cut off the detectable signal that produces.At detectable signal is under the situation of fluorescence, and these signals can be used the real-time fluorescence reading apparatus, and as Roche ' s LightCycler, perhaps ABI GeneAmp 5700 or GeneAmp 7700 etc. carry out The real time measure; Also can use static fluorescence reading apparatus, after nucleic acid amplification reaction finishes, carry out fluorometric assay.
Artificial sequence template primer collection shown in amplification pattern (two), the use Fig. 2 C carries out the nucleic acid amplification analysis
Now referring to Fig. 5.In this example, use an artificial sequence template primer 1, conventional primer 2 and amplification template primer 5 are formed a primer collection.Also comprise public primer 3 and reporter molecules 4 in the reaction system.In this example, sample to be detected is DNA or RNA.
Step 1: primer collection and testing sample are placed reaction system.
Step 2: under appropriate condition, anneal (or hybridization), two kinds of situations take place this moment:
2a: the specific combination district of 3 of artificial sequence template primer 1 ' end is incorporated into target RNA or dna sequence dna.
2b: amplification template primer 5 combines with target RNA or dna sequence dna.
Step 3:
3a: corresponding to above-mentioned first kind of situation, under reverse transcriptase (for the RNA target sequence) or archaeal dna polymerase (for the DNA target sequence) effect, 3 of artificial sequence template primer 1 ' end extends to target sequence 5 ' end, forms RNA/DNA or DNA/DNA two strands.
3b: corresponding to above-mentioned second kind of situation, under reverse transcriptase (for the RNA target sequence) or archaeal dna polymerase (for the DNA target sequence) effect, 3 of amplification template primer 5 ' end extends to target sequence 5 ' end, forms RNA/DNA or DNA/DNA two strands.
Step 4: the sex change under appropriate condition of the double-strandednucleic acid of formation forms strand target sequence and new synthetic dna sequence dna (being artificial sequence template).Perhaps use RNase hydrolysis RNA chain, form new synthetic dna sequence dna and (under first kind of situation, form artificial sequence template; Under second kind of situation, provide and conventional primer 2 bonded nucleic acid sequence).
In case of necessity, repeating step 2,3 and 4 (choosing wantonly) under appropriate condition.
Step 5: in next circulation, during annealing after sex change, four kinds of situations can take place:
5a: conventional primer 2 is incorporated into the DNA chain that new synthetic contains artificial sequence template primer 1.
5b: conventional primer 2 is incorporated into the DNA chain that new synthetic contains amplification template primer 5.
5c: the specific combination district of 3 of artificial sequence template primer 1 ' end is incorporated into target RNA or dna sequence dna (with step 2a, not shown).
5d: amplification template primer 5 combines (with step 2b, not shown) with target RNA or dna sequence dna.
Step 6:
6a: corresponding to 5a, in the presence of archaeal dna polymerase, 3 of conventional primer 2 ' end extends to artificial sequence template sequence 5 ' end, double-stranded DNA (new synthetic dna sequence dna also is an artificial sequence template in this two strands).When 3 of artificial sequence template primer 2 ' end extended to the reporter molecules place that is incorporated into the reporter molecules land, following 3 kinds of situations can take place:
(1) extension stops;
(2) continue to extend, reporter molecules is replaced from artificial sequence template;
(3) continue to extend, and cut off or destroy reporter molecules.
Under first and second kinds of situations, reporter molecules is still complete, therefore can not produce detectable signal.And under the third situation, because the quencher group on reporter molecules (as the Taqman probe) 5 ' end is by 5 ' nuclease institute hydrolysis of archaeal dna polymerase (as the Taq polysaccharase), therefore at the reporter group at Taqman probe 3 ' end place just no longer by quencher, thereby cause producing detectable signal (as fluorescent signal).
6b: corresponding to 5b, in the presence of archaeal dna polymerase, 3 of conventional primer 2 ' end extends to the nascent strand 5 that contains amplification template primer 5 sequences ' end, forms the dna double chain.In the next round circulation, these dna double chains are exactly new artificial sequence template.
6c and 6d:, form new artificial sequence template (not shown) respectively with step 3a and 3b corresponding to 5c and 5d.
Step 7: in next circulation, during annealing after sex change, four kinds of situations can take place:
7a: conventional primer 2 is incorporated into the DNA chain (same 5a, not shown) that new synthetic contains artificial sequence template primer 1.
7b: conventional primer 2 is incorporated into the DNA chain (same 5b, not shown) that new synthetic contains amplification template primer 5.
7c: the specific combination district of 3 of artificial sequence template primer 1 ' end is incorporated into target RNA or dna sequence dna (being similar to step 2a).
7d: amplification template primer 5 combines (with step 2b, not shown) with target RNA or dna sequence dna;
7e: public primer 3 is incorporated into the new synthetic dna sequence dna that contains conventional primer 2 sequence.
Step 8: in extension process next time, following several situation may take place:
8a: in the presence of archaeal dna polymerase, 3 of conventional primer 2 ' end extends to artificial sequence template sequence 5 ' end, double-stranded DNA (new synthetic dna sequence dna also is an artificial sequence template in this two strands).When 3 of artificial sequence template primer 2 ' end extended to the reporter molecules place that is incorporated into the reporter molecules land, following 3 kinds of situations can take place:
(1) extension stops;
(2) continue to extend, reporter molecules is replaced from artificial sequence template;
(3) continue to extend, and cut off or destroy reporter molecules.
Under first and second kinds of situations, reporter molecules is still complete, therefore can not produce detectable signal.And under the third situation, because the quencher group on reporter molecules (as the Taqman probe) 5 ' end is by 5 ' nuclease institute hydrolysis of archaeal dna polymerase (as the Taq polysaccharase), therefore at the reporter group at Taqman probe 3 ' end place just no longer by quencher, thereby cause producing detectable signal (as fluorescent signal).
8b: same 6b, in the presence of archaeal dna polymerase, 3 of conventional primer 2 ' end extends to the nascent strand 5 that contains amplification template primer 5 sequences ' end, forms the dna double chain.In the next round circulation, these dna double chains are exactly new artificial sequence template.
8c: same 6c forms new artificial sequence template (being similar to step 3a).
8d: same 6d forms new artificial sequence template (being similar to 3b).
8e: public primer 3 extends to 5 of the DNA chain that contains conventional primer 2 sequence ' end, forms the dna double chain.In the next round circulation, these dna double chains are exactly new artificial sequence template.
Step 9: repeating step 7 and 8.
Through after some circulations, identical with the PCR principle, also be that exponentially level (or being similar to exponential relationship) increases because of reporter molecules is cut off the detectable signal that produces.At detectable signal is under the situation of fluorescence, and these signals can be used the real-time fluorescence reading apparatus, and as Roche ' s LightCycler, perhaps ABI GeneAmp 5700 or GeneAmp 7700 etc. carry out The real time measure; Also can use static fluorescence reading apparatus, after nucleic acid amplification reaction finishes, carry out fluorometric assay.
In the methods of the invention, reporter molecules also can combine (Fig. 2 G and Fig. 2 H) with the complementary strand of reporter molecules land on the artificial sequence template, perhaps use two artificial sequence templates and two conventional primers (combination of Fig. 2 E and 2F), its amplification process and amplification pattern () and (two) are basic identical
In the present invention, the another kind of mode that produces signal is to use the two probes of FRET, and promptly the report signal group is marked at respectively on artificial sequence template and the reporter molecules.Referring to Fig. 3 C and 3C '.At this moment, artificial sequence template primer collection comprises: an artificial sequence template primer 1 and an a pair of conventional primer 2 that is marked with FRET group such as FAM fluorescein.In reaction system, also comprise reporter molecules 4 (oligonucleotide) with another FRET group such as mark Dabcyl.
Step 1-5, step 1-5 is similar with amplification pattern ().
In step 6: under first kind of situation, in the presence of archaeal dna polymerase, 3 of conventional primer 2 ' end extends to artificial sequence template sequence 5 ' end, double-stranded DNA (new synthetic dna sequence dna also is an artificial sequence template in this two strands).When 3 of artificial sequence template primer 2 ' end extended to the reporter molecules place that is incorporated into the reporter molecules land, following 3 kinds of situations can take place:
(1) extension stops;
(2) continue to extend, reporter molecules is replaced from artificial sequence template;
(3) continue to extend, and cut off or destroy reporter molecules.
Under first situation, reporter molecules is combined in artificial sequence template primer, with the FAM molecule on the Dabcyl cancellation artificial sequence template primer of mark on it, therefore can not produce detectable signal.Under second kind of situation, though reporter molecules is complete,, make the mutual cancellation between reporter molecules and artificial sequence template primer destroyed, thereby produce detectable signal (seeing Fig. 3 C ') because nascent strand has replaced reporter molecules.Under the third situation, the quencher group of reporter molecules is by 5 ' nuclease institute hydrolysis of archaeal dna polymerase (as the Taq polysaccharase), therefore reporter molecules on the artificial sequence template primer such as FAM be just no longer by quencher, thereby cause producing detectable signal (as fluorescent signal) (Fig. 3 C).
Under second kind of situation, in the presence of archaeal dna polymerase, 3 of conventional primer 2 ' end extends to the nascent strand 5 that contains amplification template primer 5 sequences ' end, forms artificial sequence template.
Step 7-9 is similar in step 7-9 and the above-mentioned amplification pattern ().
Through after some circulations, identical with the PCR principle, also be that exponentially level (or being similar to exponential relationship) increases because of reporter molecules is cut off or replaces the detectable signal that produces.At detectable signal is under the situation of fluorescence, and these signals can be used the real-time fluorescence reading apparatus, and as Roche ' s LightCycler, perhaps ABI GeneAmp5700 or GeneAmp 7700 etc. carry out The real time measure; Also can use static fluorescence reading apparatus, after nucleic acid amplification reaction finishes, carry out fluorometric assay.
The present invention has the advantage that obviously is better than prior art, and its major advantage comprises:
(1) being easy to combined type detects
Mix this artificial sequence template district and can in new synthetic dna sequence dna, introduce the one section sequence that does not belong to target sequence.And new synthetic dna sequence dna can be used as the template of further DNA cloning.Irrelevant with the number of different target sequences, new synthetic dna sequence dna is that major part is identical-artificial sequence template region sequence, and this makes variant target sequence be transformed into the sequence with denominator.Therefore, can under identical or essentially identical condition, continue to handle or operation, be easy to realize that for example combined type such as multitube identical conditions or the multiple target sequence of a pipe detects this dna sequence dna.
(2) higher sensitivity for analysis
Contain the primer collection of two artificial sequence template primers by use, perhaps the different loci to single target sequence designs a plurality of artificial sequence template primer collections, can produce the higher levels of signal to noise ratio of single probe patterns in the prior art.
In addition, use the amplification template primer also to can further improve detection sensitivity.
(3) high accuracy
Artificial sequence template primer of the present invention and artificial sequence template nucleic acid detection method, can reduce the probability of false negative that is caused by following factors:
(a) in the secondary structure at probe (or primer) the binding site place of target sequence, these secondary structures may influence the combination of probe (or primer) effectively;
(b) in the change of probe (or primer) the binding site place of target sequence sequence, these variations can be because different hypotype or sudden changes causes.For example in HCV,, can cause HCV to undergo mutation or make a variation owing to used various medicines.As using the conventional nucleic acid detection method, regular meeting causes false negative.And find out high conservative short zone (as 100bp or shorter) in the various biologies genetic material of (comprising pathogenic agent) is very easily.Utilize the present invention, can design at these artificial sequence template primer collections short, high conserved region territory (as 40-50bp), thereby reduce false negative.According to, the inventor finds that with many detections to artificial sequence template primer collection HCV mutant strain false negative rate reduces 50%.
(c) fracture of long segment target sequence in sample processing or treating processes, and this fracture occurs in the region intermediate of the amplification of long segment, what can cause that complementary dna sequence duplicates stops.
(4) simplify or eliminate multiple detection
When needs detect multiple pathogenic agent, often need carry out independent detection at present to certain pathogenic agent.This is because the optimum reaction condition of different detection reaction has nothing in common with each other, and therefore, when carrying out nucleic acid amplification reaction in same reaction tubes, the efficient of each amplified reaction is difficult near consistent, thereby is difficult to detect simultaneously in same reaction tubes multiple pathogenic agent.
Use technology of the present invention, because the major part of artificial sequence template is identical, promptly therefore the artificial sequence template district is convenient in the reaction conditions stdn that makes the PCR that detects each pathogenic agent, thereby realizes the detection to multiple pathogenic agent in a pipe.This makes the technology of the present invention occasions such as sample survey that are specially adapted to donate blood.
(5) reduce many times of analysis costs that use the fluorescent mark technology
In the prior art, for a plurality of target nucleic acid sequences to be measured, need the fluorescence labeling probe of similar number.In contrast, in the present invention, only need a kind of public reporter probe, therefore can reduce cost greatly for detecting a plurality of target sequences.
Below in conjunction with specific embodiment, further set forth the present invention.Should be understood that these embodiment only to be used to the present invention is described and be not used in and limit the scope of the invention.
Embodiment 1
The artificial sequence template primer nucleic acid amplification detects HCV virus (RNA viruses)
In this embodiment, designed the artificial sequence template primer collection that comprises an artificial sequence template primer and a conventional primer, the reaction process part as shown in Figure 4.
In the PCR scheme be: become DNA:RT (50 ℃, 20 minutes, 95 ℃, 5 minutes) from the RNA reverse transcription; PCR:50 ℃, 2 minutes, 94 ℃ 5 minutes; 94 ℃ 20 seconds; With 61 ℃ 40 seconds, totally 40 circulations.
Artificial sequence template primer is:
5-AAGGAACAGG?CGGCGACGAA?TCAACGACAG?AACGCAACCC?AACGCTACTC-3′(SEQ?IDNO：1)
Conventional primer:
5′-GTGCCCCCGC?AAGACT-3′(SEQ?ID?NO：2)
The Taqman probe sequence that uses is:
5′-TCGTCGCCGC?CTGTTCCTTA-3′(SEQ?ID?NO：3)
Use therein reporter group is 6-Fluoresceincarboxylic acid (6-carboxyfluorescein, 6-FAM) (be positioned at 5 ' end), the quencher group is 6-carboxyl-tetramethyl-rhodamine (6-carboxy-tetramethyl-rhodamine, TAMRA) (is positioned at 3 ' end).
The detecting instrument that uses during detection is ABI GeneAmp 5700, and excitation light source is a halogen lamp, and wavelength is 488nm.
Increase at the ABI5700 gene-amplificative instrament with above-mentioned primer and corresponding Taqman probe.As a result, add different extent of dilution positive and fluorescent signal occurs, and fluorescent signal does not all appear when (Ct＞40) in negative sample and other non-special control samples (sample that contains other pathogenic agent) when amplified reaction finishes in difference circulation (Ct).
Embodiment 2
Artificial sequence template augmentation detection HBV virus (dna virus)
In this embodiment, designed the artificial sequence template primer collection that comprises an artificial sequence template primer and a conventional primer, the reaction process part as shown in Figure 4.
In the PCR scheme be: PCR:50 ℃, 2 minutes, 94 ℃ 5 minutes; 94 ℃ 20 seconds; With 61 ℃ 40 seconds, totally 40 circulations.
Artificial sequence template primer is respectively:
5-AAGGAACAGG?CGGCGACGAA?TCAACGACAG?AACCAGCGAT?AGCCA?GGACA-3′(SEQ?IDNO：4)
Conventional primer:
5′-CCTCCAATCA?CTCACCAACC-3′(SEQ?ID?NO：5)
The Taqman probe sequence that uses is:
5′-TCGTCGCCGC?CTGTTCCTTA-3′(SEQ?ID?NO：3)
Use therein reporter group is 6-Fluoresceincarboxylic acid (6-carboxyfluorescein, 6-FAM) (be positioned at 5 ' end), the quencher group is 6-carboxyl-tetramethyl-rhodamine (6-carboxy-tetramethyl-rhodamine, TAMRA) (is positioned at 3 ' end).
The detecting instrument that uses during detection is ABI GeneAmp 5700, and excitation light source is a halogen lamp, and wavelength is 488nm.
Increase at the ABI5700 gene-amplificative instrament with above-mentioned primer and corresponding Taqman probe.As a result, add different extent of dilution positive and fluorescent signal occurs, and fluorescent signal does not all appear when (Ct＞40) in negative sample and other non-special control samples (sample that contains other pathogenic agent) when amplified reaction finishes in difference circulation (Ct).
Artificial sequence template augmentation detection HCV virus (RNA viruses)
In this embodiment, designed the artificial sequence template primer collection that comprises an artificial sequence template primer, a conventional primer and a public primer, reaction process as shown in Figure 4.
In the PCR scheme be: become DNA:RT (50 ℃, 20 minutes, 95 ℃, 5 minutes) from the RNA reverse transcription; PCR:50 ℃, 2 minutes, 94 ℃ 5 minutes; 94 ℃ 20 seconds; With 61 ℃ 40 seconds, totally 40 circulations.
Artificial sequence template primer is:
5-TCATCCACAT CCCACCTCAT CAGGAACAGG CGGCGACGAA?TCAACGACAGAACGCAACCC?AACGCTACTC-3′(SEQ?ID?NO：6)
Conventional primer:
5′-GTGCCCCCGC?AAGACT-3′(SEQ?ID?NO：2)
The public primer sequence that uses is:
5′-TCATCCACAT CCCACCTCAT-3′(SEQ?ID?NO：7)
The Taqman probe sequence that uses is:
5′-TCGTCGCCGC?CTGTTCCTTA-3′(SEQ?ID?NO：3)
Use therein reporter group is 6-Fluoresceincarboxylic acid (6-carboxyfluorescein, 6-FAM) (be positioned at 5 ' end), the quencher group is 6-carboxyl-tetramethyl-rhodamine (6-carboxy-tetramethyl-rhodamine, TAMRA) (is positioned at 3 ' end).
The detecting instrument that uses during detection is ABI GeneAmp 5700, and excitation light source is a halogen lamp, and wavelength is 488nm.
Increase at the ABI5700 gene-amplificative instrament with above-mentioned primer and corresponding Taqman probe.As a result, add different extent of dilution positive and fluorescent signal occurs, and fluorescent signal does not all appear when (Ct＞40) in negative sample and other non-special control samples (sample that contains other pathogenic agent) when amplified reaction finishes in difference circulation (Ct).
Embodiment 4
Artificial sequence template augmentation detection HCV virus (RNA viruses)
In this embodiment, designed the artificial sequence template primer collection that comprises an artificial sequence template primer, a pair of conventional primer and a public primer, reaction process as shown in Figure 5.
In the PCR scheme be: become DNA:RT (50 ℃, 20 minutes, 95 ℃, 5 minutes) from the RNA reverse transcription; PCR:50 ℃, 2 minutes, 94 ℃ 5 minutes; 94 ℃ 20 seconds; With 61 ℃ 40 seconds, totally 40 circulations.
Artificial sequence template primer is:
5-TCATCCACAT CCCACCTCAT CAGGAACAGG CGGCGACGAA TCAACGACAGAACGCAACCC AACGCTACTC-3′(SEQ?ID?NO：6)
Conventional primer 1:
5′-GTGCCCCCGC AAGACT-3′(SEQ?ID?NO：2)
Conventional primer 2:
5′-TGAGTGTCGTACAGC CTCCAGG-3′(SEQ?ID?NO：8)
The public primer sequence that uses is:
5′-TCATCCACAT CCCACCTCAT-3′(SEQ?ID?NO：7)
The Taqman probe sequence that uses is:
5′-TCGTCGCCGC?CTGTTCCTTA-3′(SEQ?ID?NO：3)
Use therein reporter group is 6-Fluoresceincarboxylic acid (6-carboxyfluorescein, 6-FAM) (be positioned at 5 ' end), the quencher group is 6-carboxyl-tetramethyl-rhodamine (6-carboxy-tetramethyl-rhodamine, TAMRA) (is positioned at 3 ' end).
The detecting instrument that uses during detection is ABI GeneAmp 5700, and excitation light source is a halogen lamp, and wavelength is 488nm.
Increase at the ABI5700 gene-amplificative instrament with above-mentioned primer and corresponding Taqman probe.As a result, add different extent of dilution positive and fluorescent signal occurs, and fluorescent signal does not all appear when (Ct＞40) in negative sample and other non-special control samples (sample that contains other pathogenic agent) when amplified reaction finishes in difference circulation (Ct).
Artificial sequence template augmentation detection HCV virus (RNA viruses) and HBV virus (dna virus)
In this embodiment, designed the artificial sequence template primer collection that comprises two artificial sequence template primers, two pairs of conventional primers and a res communes, reaction process as shown in Figure 5.
In the PCR scheme be: become DNA:RT (50 ℃, 20 minutes, 95 ℃, 5 minutes) from the RNA reverse transcription; PCR:50 ℃, 2 minutes, 94 ℃ 5 minutes; 94 ℃ 20 seconds; With 61 ℃ 40 seconds, totally 40 circulations.
HCV virus artificial sequence template primer is:
5-TCATCCACAT CCCACCTCAT CAGGAACAGG CGGCGACGAA TCAACGACAGAACGCAACCC AACGCTACTC-3′(SEQ?ID?NO：6)
HBV virus artificial sequence template primer is:
5′-TCATCCACAT CCCACCTCAT CAGGAACAGG CGGCGACGAA TCATCCAGTCTATGTTTCCC TCTTGTTGCT-3′(SEQ?ID?NO：9)
The conventional primer 1 of HCV virus:
5′-GTGCCCCCGC AAGACT-3′(SEQ?ID?NO：2)
The conventional primer 2 of HCV virus:
5′-TGAGTGTCGTACAGC CTCCAGG-3′(SEQ?ID?NO：8)
The conventional primer 1 of HBV virus:
5′-CCTCCAATCA CTCACCAACC-3′(SEQ?ID?NO：5)
The conventional primer 2 of HBV virus:
5′-AGTTTCCGTC CGAAGGTTT-3′(SEQ?ID?NO：10)
The public primer sequence that uses is:
5′-TCATCCACAT CCCACCTCAT-3′(SEQ?ID?NO：7)
The Taqman probe sequence that uses is:
5′-TCGTCGCCGC CTGTTCCTTA-3′(SEQ?ID?NO：3)
Use therein reporter group is 6-Fluoresceincarboxylic acid (6-carboxyfluorescein, 6-FAM) (be positioned at 5 ' end), the quencher group is 6-carboxyl-tetramethyl-rhodamine (6-carboxy-tetramethyl-rhodamine, TAMRA) (is positioned at 3 ' end).
The detecting instrument that uses during detection is ABI GeneAmp 5700, and excitation light source is a halogen lamp, and wavelength is 488nm.
Increase at the ABI5700 gene-amplificative instrament with above-mentioned primer and corresponding Taqman probe.As a result, add different extent of dilution HCV with (or) the HBV positive fluorescent signal occurs in different circulations (Ct), contains HBV simultaneously and the HCV positive does not influence detection sensitivity, its Ct value is mainly determined by high concentration virus.And fluorescent signal does not all appear when (Ct＞40) in negative sample and other non-special control samples (sample that contains other pathogenic agent) when amplified reaction finishes.
Embodiment 6
Artificial sequence template augmentation detection HCV virus (RNA viruses)
In this embodiment, designed the artificial sequence template primer collection that comprises a fluorescein-labeled artificial sequence template primer and a pair of conventional primer, the reaction process part as shown in Figure 8.
In the PCR scheme be: become DNA:RT (50 ℃, 20 minutes, 95 ℃, 5 minutes) from the RNA reverse transcription; PCR:50 ℃, 2 minutes, 94 ℃ 5 minutes; 94 ℃ 20 seconds; With 61 ℃ 40 seconds, totally 40 circulations.
The artificial sequence template primer sequence is:
5′-AAGGAACAGG CGGCGACGAA TCAACGACAG AACGCAACCC AACGCTACTC-3′(SEQ?IDNO：11)
Use therein reporter group is 6-Fluoresceincarboxylic acid (6-carboxyfluorescein, 6-FAM) (is positioned at 5 ' end)
Conventional primer:
5′-GTGCCCCCGC AAGACT-3′(SEQ?ID?NO：2)
The amplification template primer:
5′-TGAGTGTCGTACAGC CTCCAGG-3′(SEQ?ID?NO：8)
The reporter probe sequence of using is:
5′-TCGTCGCCGC CTGTTCCTTA-3′(SEQ?ID?NO：3)
Use therein quencher group is Dabcyl (i.e. four (4-methylamino phenyl azo)-phenylformic acid (be positioned at 3 ' end).
The detecting instrument that uses during detection is ABI GeneAmp 5700, and excitation light source is a halogen lamp, and wavelength is 488nm.
Increase at the ABI5700 gene-amplificative instrament with above-mentioned primer and corresponding Taqman probe.As a result, if only use conventional primer 1 and artificial sequence template primer to increase, compare with the artificial sequence template primer collection of amplification template primer with adding conventional primer simultaneously, its detection sensitivity can differ 10～100 times.Add different extent of dilution positive and fluorescent signal occurs, and fluorescent signal does not all appear when (Ct＞40) in negative sample and other non-special control samples (sample that contains other pathogenic agent) when amplified reaction finishes in difference circulation (Ct).
Should be understood that in addition those skilled in the art can make various changes or modifications the present invention after having read above-mentioned teachings of the present invention, these equivalent form of values fall within the application's appended claims institute restricted portion equally.
＜110〉,＜120〉＜130〉014338＜160〉11＜170〉PatentIn version 3.0＜210〉1＜211〉50＜212〉DNA＜213〉＜400〉1aaggaacagg cggcgacgaa tcaacgacag aacgcaaccc aacgctactc 50＜210〉2＜211〉16＜212〉DNA＜213〉＜400〉2gtgcccccgc aagact 16＜210〉3＜211〉20＜212〉DNA＜213〉＜400〉3tcgtcgccgc ctgttcctta 20＜210〉4＜211〉50＜212〉DNA＜213〉＜400〉4aaggaacagg cggcgacgaa tcaacgacag aaccagcgat agccaggaca 50＜210〉5＜211〉20＜212〉DNA＜213〉＜400〉5cctccaatca ctcaccaacc 20＜210〉6＜211〉70＜212〉DNA＜213〉＜400〉6tcatccacat cccacctcat caggaacagg cggcgacgaa tcaacgacag aacgcaaccc 60aacgctactc 70＜210〉7＜211〉20＜212〉DNA＜213〉＜400〉7tcatccacat cccacctcat 20＜210〉8＜211〉22＜212〉DNA＜213〉＜400〉8tgagtgtcgt acagcctcca gg 22＜210〉9＜211〉70＜212〉DNA＜213〉＜400〉9tcatccacat cccacctcat caggaacagg cggcgacgaa tcatccagtc tatgtttccc 60tcttgttgct 70＜210〉10＜211〉19＜212〉DNA＜213〉＜400〉10agtttccgtc cgaaggttt 19＜210〉11＜211〉50＜212〉DNA＜213〉＜400〉11aaggaacagg cggcgacgaa tcaacgacag aacgcaaccc aacgctactc 50
Claims (10)
1. an artificial sequence template primer collection is characterized in that, described primer collection comprises:
It is right that specificity is incorporated into detected nucleotide sequence and causes the Oligonucleolide primers of purpose nucleic acid amplification reaction, and wherein at least one primer is an artificial sequence template primer, and this artificial sequence template primer comprises:
(a) specific combination district, this specific combination district is positioned at artificial sequence template primer 3 ' end, is used for combining with detected nucleotide sequence specificity;
(b) public domain, this public domain is positioned at 5 of artificial sequence template primer ' hold and has reporter molecules land and public guiding region, but the reporter molecules specificity is incorporated into the complementary strand of this report molecule land or this report molecule land, and the state difference of described reporter molecules under following two kinds of situations, thereby produce detectable signal: (i) be incorporated into the complementary strand of reporter molecules land or this report molecule land and (ii) reporter molecules be cut off or be substituted.
2. artificial sequence template primer collection as claimed in claim 1 is characterized in that, described reporter molecules comprises FRET (fluorescence resonance energy transfer) type signal probe and contains the oligonucleotide chain of rare earth element.
3. artificial sequence template primer collection as claimed in claim 1 is characterized in that, described specific combination section length is 6-35bp, and the length of described public domain is 8-100bp.
4. as arbitrary described artificial sequence template primer collection in the claim 2, it is characterized in that, described specific combination section length is 15-25bp, the length of described public domain is 20-50bp, and described FRET (fluorescence resonance energy transfer) type signal probe is selected from down group: Taqman probe, the two probes of FRET, molecular beacon and PNA signal probe, and between specific combination district and reporter molecules land, there is transcribed spacer, and/or between public guiding region and reporter molecules land, having transcribed spacer, described transcribed spacer is 1-20bp.
5. artificial sequence template primer collection as claimed in claim 1 is characterized in that, contains to be selected from the Nucleotide of group down in described artificial sequence template primer: isoG, isoC, 2 '-O-methyl-G, 2 '-O-methyl-C and combination thereof.
6. artificial sequence template primer collection as claimed in claim 1 is characterized in that, also comprises the amplification template primer that is used to increase detected nucleic acid-templated quantity.
7. a detection kit is characterized in that, it contains the described artificial sequence template primer collection of claim 1.
8. one kind is detected nucleic acid detection method, it is characterized in that it comprises step:
(1) in containing the nucleic acid amplification reaction system of reporter molecules, carry out nucleic acid amplification reaction with artificial sequence template primer collection and testing sample, wherein said primer collection comprise specificity be incorporated into detected nucleotide sequence and cause the primer of purpose nucleic acid amplification reaction right, and at least one primer is an artificial sequence template primer, and this artificial sequence template primer comprises:
(a) specific combination district, this specific combination district is positioned at artificial sequence template primer 3 ' end, is used for combining with detected nucleotide sequence specificity;
(b) public domain, this public domain is positioned at 5 of artificial sequence template primer ' hold and has reporter molecules land and public guiding region, but the reporter molecules specificity is incorporated into the complementary strand of this report molecule land or this report molecule land, and the state difference of described reporter molecules under following two kinds of situations, thereby produce detectable signal: (i) be incorporated into the complementary strand of reporter molecules land or this report molecule land and (ii) reporter molecules be cut off or be substituted;
(2) detectable signal that produced of examining report molecule.
9. method as claimed in claim 8 is characterized in that, described testing sample is selected from down group: DNA sample, RNA sample and the cDNA sample that obtains through reverse transcription from RNA.
10. method as claimed in claim 8, it is characterized in that, use two or more different artificial sequence template primer collection, these artificial sequence template primer collections are incorporated into the different zones of identical target sequence or its combination of the different target sequences of the target sequence of different detected bodies, same detected body, same detected body respectively specifically, and the reporter molecules land of these artificial sequence template primer collections combines with identical or different reporter molecules generation specificity respectively.
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105734116A (en) * | 2014-12-08 | 2016-07-06 | 常州金麦格生物技术有限公司 | Detection method of multiplex nucleic acid sites |
| CN105734117A (en) * | 2014-12-08 | 2016-07-06 | 常州金麦格生物技术有限公司 | Method for detecting nucleic acid target to-be-detected sites by virtue of universal primers |
| CN105734167A (en) * | 2014-12-09 | 2016-07-06 | 常州金麦格生物技术有限公司 | Multi-target nucleic acid detection method |
| CN105734119A (en) * | 2014-12-09 | 2016-07-06 | 常州金麦格生物技术有限公司 | Method for detecting target to-be-detected position of nucleic acid through general probe |
| CN106868111A (en) * | 2017-01-13 | 2017-06-20 | 中玉金标记（北京）生物技术股份有限公司 | Using the method and kit of universal TaqMan probe detection SNP |
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2001
- 2001-08-08 CN CNB011264233A patent/CN1174104C/en not_active Expired - Fee Related
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105734116A (en) * | 2014-12-08 | 2016-07-06 | 常州金麦格生物技术有限公司 | Detection method of multiplex nucleic acid sites |
| CN105734117A (en) * | 2014-12-08 | 2016-07-06 | 常州金麦格生物技术有限公司 | Method for detecting nucleic acid target to-be-detected sites by virtue of universal primers |
| CN105734167A (en) * | 2014-12-09 | 2016-07-06 | 常州金麦格生物技术有限公司 | Multi-target nucleic acid detection method |
| CN105734119A (en) * | 2014-12-09 | 2016-07-06 | 常州金麦格生物技术有限公司 | Method for detecting target to-be-detected position of nucleic acid through general probe |
| CN106868111A (en) * | 2017-01-13 | 2017-06-20 | 中玉金标记（北京）生物技术股份有限公司 | Using the method and kit of universal TaqMan probe detection SNP |
| CN106868111B (en) * | 2017-01-13 | 2020-12-25 | 中玉金标记（北京）生物技术股份有限公司 | Method and kit for detecting SNP (Single nucleotide polymorphism) by using universal TaqMan probe |
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| CN1174104C (en) | 2004-11-03 |
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