CN1186456C - General purpose template nucleic acid detection method and kit - Google Patents
General purpose template nucleic acid detection method and kit Download PDFInfo
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Abstract
The present invention provides a method for detecting a nucleic acid and/or determining the quantity of the nucleic acid by using a universal template. A primer pair of the universal template of the present invention comprises two primers which are bound specifically to the template and are used for initiating a polymerase chain reaction, wherein at least one primer is a universal template primer which comprises (a) a specific binding area and (b) a universal area; the universal area is provided with a molecular binding reporting area. The present invention also provides a corresponding detection method for the nucleic acid by using the universal template. Pathogen and gene expression in a sample can be detected and/or the quantity of the pathogen and the gene expression in the sample can be determined easily, sensitively and accurately by the present invention.
Description
The present invention relates to the detection of nucleic acids field, more specifically, relate to a kind of by using the method for common template detection and/or quantitative nucleic acid.
Recently, in satisfied detection rapidly and accurately and/or quantitative infectious agent (as virus, bacterium, fungi), 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), strand displacement amplification (SDA), transcriptive intermediate amplification (transcriptionmediated amplication, 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 controlled condition of testing standardization.
The another kind of FAQs of target sequence amplification method is the pollution of amplicon at present.
In addition,, detect impartially 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.
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 PCR reaction system of reporter molecules, react testing sample being carried out PCR with the common template primer, wherein said primer is to comprising specificity and be incorporated into template and cause two primers of PCR reaction, and at least one primer is the common template primer, and this common template primer comprises:
(a) specific combination district, this specific combination district is positioned at common template primer 3 ' end, is used for combining with the coding specificity;
(b) general district, this general district is positioned at 5 of common template primer ' hold and has the reporter molecules land, but the reporter molecules specificity is incorporated into 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 reporter molecules land, (ii) reporter molecules is cut off
And the quantity of described reporter molecules is more than or equal to the quantity of common template primer;
The detectable signal that the examining report molecule is produced.
In an example of the present invention, use two or more different common template primer right, these common template primers are to different zones or its combination of the identical target sequence that is incorporated into the different target sequences of the target sequence of different pathogens, same pathogenic agent, same pathogenic agent respectively specifically, and the right reporter molecules land of these common template primers combines with identical or different reporter molecules generation specificity respectively.
In a second aspect of the present invention, provide a kind of common template primer right, described primer is to comprising specificity and be incorporated into template and cause two primers of PCR, and wherein at least one primer is the common template primer, and this common template primer comprises:
(a) specific combination district, this specific combination district is positioned at common template primer 3 ' end, is used for combining with the coding specificity;
(b) general district, this general district is positioned at 5 of common template primer ' hold and has the reporter molecules land, but the reporter molecules specificity is incorporated into 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 reporter molecules land and (ii) reporter molecules be cut off.
In an example of the present invention, described reporter molecules comprises the Taqman probe.
In a third aspect of the present invention, a kind of kit for detecting nucleic acid is provided, it is right that it contains common template primer of the present invention.
Fig. 1 has shown the structural representation of common template primer of the present invention.
Fig. 2 has shown the various array configurations that common template primer of the present invention is right.
Fig. 3 is the synoptic diagram of a kind of nucleic acid detection method of the present invention, and use therein common template primer is to containing a common template primer.
Fig. 4 is the synoptic diagram of a kind of nucleic acid detection method of the present invention, and use therein common template primer is to containing two common template primers.
As used herein, following word/term has following meanings, unless otherwise indicated.
" nucleic acid ": the polynucleotides of ribonucleic acid (RNA), DNA (DNA), RNA or DNA The oligonucleotide analogs of analog, RNA or DNA.
" target ": treat the directly or indirectly analyte of detection, main target is the inhereditary materials such as nucleic acid.
" template ": can be by the total length of the nucleic acid molecules of nucleic acid polymerization enzymatic amplification or partial sequence. Template antigen Be RNA or DNA or its analog, and can be strand, two strands or partially double stranded.
" common template (UT) primer ": the common template primer is the oligonucleotide sequence that synthesizes. Its 3 ' end has the spy Different land, this specific bond district is complementary to target sequence and extends in amplified reaction as primer. In addition, logical Also contain general district (being also referred to as common template district or UT district) with templa-primer, this general district is positioned at common template 5 of primer ' hold and have the reporter molecules land. But the reporter molecules specific binding is in the combination of this report molecule The district. The common template primer can synthesize (Fig. 1) with the whole bag of tricks well known by persons skilled in the art.
" reporter molecules " is a kind of molecule for generation of detectable signal. Described reporter molecules is following two State difference in kind of the situation, thus detectable signal produced: (i) be incorporated into reporter molecules land and (ii) Reporter molecules is cut off. Suitable reporter molecules example comprises (but being not limited to): the Taqman probe, contain rare The oligonucleotide chain of earth metal etc.
" Taqman probe " be a kind of its 5 ' end with reporter group and 3 ' end is with the quencher group Oligonucleotides, described quencher group can suppress reporter group and produce detectable signal (for example fluorescence). The Taqman probe design is prior art, and can obtain for information about from open approach (Heid CA, Stevens J, Livak KJ, Williams PM; Real Time Quantitaive PCR, Genome Res.1996 Oct.; 6 (10): 986-94).
" common template " refer in PCR process of the present invention, and what amplify has mixed the common template primer Nucleotide sequence, or its anti sense nucleotide sequence. These common templates had both had the nucleotides sequence corresponding to target sequence Row have again the nucleotide sequence of (containing the reporter molecules land) corresponding to the general district of common template primer. With After pcr amplification circulation in, these common templates work as template.
The method disclosed in the present is classified as the amplification of signal method in principle. Its key is the nuclear that has used general adaptation Acid sequence is as the template (referred to here as " common template ") of amplification of signal, and therefore corresponding method is called as " logical Amplification Analysis with template mediation " (UT analysis). UT analyzes can be sensitive, accurately and standardization ground detects and/ Or quantifying target nucleic acid molecules.
In the present invention, for testing sample without limits, as long as wherein contain inhereditary material. Representative Testing sample comprise (but being not limited to): DNA sample, RNA sample and obtain through reverse transcription from RNA The cDNA sample, and the polynucleotide of other forms of modified.
In common template primer of the present invention, the length in described specific bond district is not particularly limited, logical Normal its length is 8-30bp, preferably is 15-25bp. For also being not particularly limited of described general district, usually Its length is 15-100bp, preferably is 20-40bp, more preferably is about 25-35bp.
The nucleotides that contains in the common template primer is selected from A, T, C, G usually. Yet, described logical With containing some other nucleotides in the templa-primer with the combination of increase with template, for example be selected from lower group nucleosides Acid: isoG, isoC, 2 '-O-methyl-G, 2 '-O-methyl-C and combination thereof.
In reaction system of the present invention, the not special limit of the quantitative relation of reporter molecules and common template primer System. Yet preferably, the quantity of described reporter molecules is more than or equal to the quantity of common template primer, usually The ratio of reporter molecules and common template primer is greater than 1: 1-10: 1, and more preferably be 1.5: 1-5: 1. Like this, In reaction system, the common template primer just all is in the state of being combined with reporter molecules basically.
When reporter molecules is the Taqman probe, Taqman probe and common template primer reporter molecules land Tm, should be higher than the Tm of common template primer specific land and template. Usually, exceed 2-15 ℃, Exceed 5-12 ℃ goodly.
In the present invention, as shown in Figure 2, the common template primer is to there being 3 kinds of array configurations: (1) upstream is the common template primer, and the downstream is conventional primer; (2) upstream is the common template primer, and the downstream is the common template primer; (3) upstream is conventional primer, and the downstream is the common template primer.
In addition, for common template primer bonded reporter molecules, different common templates 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).
Be not particularly limited for the length of common template primer the common template that amplifies.Apart the distance expression on template of the 3 ' end in specific combination district of pressing two right primers of common template primer is generally 1bp-10kb, preferably is 1-2kb, more preferably is 1-500b, is 1-100bp best.Especially be that it is right to design at the primer of for example pathogenic agent high conserved region, thereby reduces false negative rate when spacing during less than 100bp.In addition, spacing is short more, can bring into play the versatility of common template more.
Further specify the present invention below in conjunction with accompanying drawing.
One, use the primer that contains common template primer and conventional primer to analyze to carrying out UT
Now referring to Fig. 3.In this example, use a pair of common template primer right, this primer is to being made of a common template primer 1 and a conventional primer 2.In this example, sample to be detected is RNA or DNA.
Step 1: primer to placing reaction system with testing sample, then, under appropriate condition, is annealed (or hybridization), and promptly the specific combination district of 3 of common template primer 1 ' end is incorporated into target RNA or dna sequence dna.
Step 2: at reverse transcriptase (for the RNA target sequence) or archaeal dna polymerase (for the DNA target sequence), 3 of common template primer ' end extends to target sequence 5 ' end, forms RNA/DNA or DNA/DNA two strands.
Step 3: the sex change under appropriate condition of the double-strandednucleic acid of formation forms strand target sequence and new synthetic dna sequence dna (being common template).Perhaps use RNase hydrolysis RNA chain, form new synthetic dna sequence dna (being common template).
Step 4 (optional): in case of necessity, repeating step 1,2 and 3 under appropriate condition.
Step 5: under conditions suitable, 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 common template sequence 5 ' end, double-stranded DNA (new synthetic dna sequence dna also is a common 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 common 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 the rapid synthase of DNA (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, common template primer 1 is incorporated into the new synthetic dna sequence dna that contains conventional primer 2 sequence.Simultaneously, conventional primer 2 is also in conjunction with the common template that contains universal primer 1 sequence.
Step 8: common template primer 1 extends to target sequence 5 ' end, forms the dna double chain.These dna double chains are exactly new common template.Simultaneously, with identical in the step 6, 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:
(1) extension stops;
(2) continue to extend, reporter molecules is replaced from common 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 fluorescence reading apparatus, as Roche ' s LightCycler, or ABI GeneAmp 5700 measure.
Two, use the primer that contains two common template primers to analyze to carrying out UT
Now referring to Fig. 4.In this example, use a pair of common template primer right, this primer is to being made of two common template primers 1 and 2, when 3 of common template primer 2 ' end extends to the reporter molecules place that is incorporated into the reporter molecules land.In this example, sample to be detected is DNA or RNA.
Step 1: primer to placing reaction system with testing sample, then, under appropriate condition, is annealed (or hybridization), and promptly the specific combination district of 3 of common template primer 1 ' end is incorporated into target RNA or dna sequence dna.
Step 2: at reverse transcriptase (for the RNA target sequence) or archaeal dna polymerase (for the DNA target sequence), 3 of common template primer ' end extends to target sequence 5 ' end, forms RNA/DNA or DNA/DNA two strands.
Step 3: the sex change under appropriate condition of the double-strandednucleic acid of formation forms strand target sequence and new synthetic dna sequence dna (being common template).Perhaps use RNase hydrolysis RNA chain, form new synthetic dna sequence dna (being common template).
Step 4 (optional): in case of necessity, repeating step 1,2 and 3 under appropriate condition.
Step 5: under conditions suitable, the common template 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 common template primer 2 ' end extends to common template sequence 5 ' end, double-stranded DNA (new synthetic dna sequence dna also is a common template in this two strands).When 3 of common 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 common 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, common template primer 1 is incorporated into the new synthetic dna sequence dna that contains common template primer 2 sequence.Simultaneously, the common template primer 2 is also in conjunction with the common template that contains universal primer 1 sequence.
Step 8: common template primer 1 extends to target sequence 5 ' end, forms the dna double chain.With identical in the step 6, when 3 of common template primer 1 ' end extends to the reporter molecules place that is incorporated into the reporter molecules land and 3 ' end of common template primer 2 when extending to the reporter molecules place that is incorporated into the reporter molecules land, following 3 kinds of situations all can take place:
(1) extension stops;
(2) continue to extend, reporter molecules is replaced from common 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 fluorescence reading apparatus, as Roche ' s LightCycler, or ABI GeneAmp 5700 measure.
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 UT region sequence 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-UT 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
By using a pair of primer that contains two common template primers right, it is right perhaps the different loci of single target sequence to be designed a plurality of common template primers, can produce the higher levels of signal to noise ratio of single probe patterns in the prior art.
(3) high accuracy
Common template primer of the present invention and UT 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 HIV 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, it is right to design at these common template primers short, high conserved region territory (as 40-50bp), thereby reduces false negative.According to, the inventor to the detection of common template primer to the HIV mutant strain, finds that false negative rate reduces 50% with many.
(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 the PCR 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 common template is identical, promptly therefore the UT 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 UT fluorescent mark technology
In the prior art, for a plurality of target nucleic acid sequences to be measured, need the Taqman probe of similar number.In contrast, in the present invention, only need a kind of UT fluorescent 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.
UT analyzing and testing HCV virus (RNA viruses)
In this embodiment, it is right to have designed the common template primer that comprises a common template primer and a conventional primer, and reaction process as shown in Figure 3.
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 common template primer is:
5-AAGGAACAGG CGGCGACGAA TCAACGACAG AACGCAACCCAACGCTACTC-3′(SEQ?ID?NO：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).
UT analyzing and testing HBV virus (dna virus)
In this embodiment, it is right to have designed the common template primer that comprises a common template primer and a conventional primer, and reaction process as shown in Figure 3.
In the PCR scheme be: PCR:50 ℃, 2 minutes, 94 ℃ 5 minutes; 94 ℃ 20 seconds; With 61 ℃ 40 seconds, totally 40 circulations.
The common template primer is respectively:
5-AAGGAACAGG?CGGCGACGAA?TCAACGACAG?AACCAGCGAT?AGCCAGGACA-3′(SEQ?ID?NO：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).
All quote in this application as a reference at all documents that the present invention mentions, just quoted as a reference separately as each piece document.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.
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Claims (10)
1. a common template primer is right, it is characterized in that, described primer is incorporated into two primers of template and initiated polymerization enzyme chain reaction to comprising specificity, and wherein at least one primer is the common template primer, and this common template primer comprises:
(a) specific combination district, this specific combination district is positioned at common template primer 3 ' end, is used for combining with the coding specificity;
(b) general district, this general district is positioned at 5 of common template primer ' hold and has the reporter molecules land, but the reporter molecules specificity is incorporated into 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 reporter molecules land and (ii) reporter molecules be cut off.
2. common template primer as claimed in claim 1 is right, it is characterized in that, described reporter molecules comprises the Taqman probe.
3. common template primer as claimed in claim 1 is right, it is characterized in that, described specific combination section length is 8-30bp, and the length in described general district is 15-100bp.
4. common template primer as claimed in claim 1 is right, it is characterized in that, described specific combination section length is 15-25bp, and the length in described general district is 20-40bp.
5. common template primer as claimed in claim 1 is right, it is characterized in that, contains to be selected from the Nucleotide of group down in described common template primer: isoG, isoC, 2 '-O-methyl-G, 2 '-O-methyl-C and combination thereof.
6. a detection kit is characterized in that, it is right that it contains the described common template primer of claim 1.
7. nucleic acid detection method is characterized in that it comprises step:
(1) in containing the PCR reaction system of reporter molecules, react testing sample being carried out PCR with the common template primer, wherein said primer is to comprising specificity and be incorporated into template and cause two primers of PCR reaction, and at least one primer is the common template primer, and this common template primer comprises:
(a) specific combination district, this specific combination district is positioned at common template primer 3 ' end, is used for combining with the coding specificity;
(b) general district, this general district is positioned at 5 of common template primer ' hold and has the reporter molecules land, but the reporter molecules specificity is incorporated into 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 reporter molecules land and (ii) reporter molecules be cut off;
(2) detectable signal that produced of examining report molecule.
8. method as claimed in claim 7 is characterized in that described reporter molecules comprises the Taqman probe.
9. method as claimed in claim 7 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 7, it is characterized in that, use two or more different common template primer right, these common template primers are to different zones or its combination of the identical target sequence that is incorporated into the different target sequences of the target sequence of different pathogens, same pathogenic agent, same pathogenic agent respectively specifically, and the right reporter molecules land of these common template primers combines with identical or different reporter molecules generation specificity respectively.
Priority Applications (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNB011128151A CN1186456C (en) | 2001-04-30 | 2001-04-30 | General purpose template nucleic acid detection method and kit |
| PCT/CN2002/000113 WO2002088385A1 (en) | 2001-04-30 | 2002-02-25 | Method of nucleic acid sequence detection using general primers and the related kits |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNB011128151A CN1186456C (en) | 2001-04-30 | 2001-04-30 | General purpose template nucleic acid detection method and kit |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN1384206A CN1384206A (en) | 2002-12-11 |
| CN1186456C true CN1186456C (en) | 2005-01-26 |
Family
ID=4659568
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CNB011128151A Expired - Fee Related CN1186456C (en) | 2001-04-30 | 2001-04-30 | General purpose template nucleic acid detection method and kit |
Country Status (2)
| Country | Link |
|---|---|
| CN (1) | CN1186456C (en) |
| WO (1) | WO2002088385A1 (en) |
Families Citing this family (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP6012605B2 (en) | 2010-09-22 | 2016-10-25 | アリオス バイオファーマ インク． | Substituted nucleotide analogs |
| CA2860234A1 (en) | 2011-12-22 | 2013-06-27 | Alios Biopharma, Inc. | Substituted phosphorothioate nucleotide analogs |
| NZ631601A (en) | 2012-03-21 | 2016-06-24 | Alios Biopharma Inc | Solid forms of a thiophosphoramidate nucleotide prodrug |
| EP2827876A4 (en) | 2012-03-22 | 2015-10-28 | Alios Biopharma Inc | Pharmaceutical combinations comprising a thionucleotide analog |
| 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 |
Family Cites Families (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| IE61148B1 (en) * | 1988-03-10 | 1994-10-05 | Ici Plc | Method of detecting nucleotide sequences |
| EP0746627B1 (en) * | 1994-02-21 | 2002-08-14 | Stichting Researchfonds Pathologie | Human papilloma virus detection in a nucleic acid amplification process using general primers |
-
2001
- 2001-04-30 CN CNB011128151A patent/CN1186456C/en not_active Expired - Fee Related
-
2002
- 2002-02-25 WO PCT/CN2002/000113 patent/WO2002088385A1/en not_active Application Discontinuation
Also Published As
| Publication number | Publication date |
|---|---|
| WO2002088385A1 (en) | 2002-11-07 |
| CN1384206A (en) | 2002-12-11 |
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