CN1661359B - Method for measuring isothermal amplification of nucleic acid quantificationally - Google Patents
Method for measuring isothermal amplification of nucleic acid quantificationally Download PDFInfo
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Abstract
The present invention relates to an isothermal amplification quantative detection technique of nucleic acid. Said invention is characterized by that it adopts reverse transcriptase and RNA polymerase to combine the isothermal amplification with TaqMan fluorescent probe detection, and can implement accumulation of fluorescent signals for thermal amplification and detection of RNA sample in same reaction system, so that said invention can make qualitative and quantitative detection of micro nucleic acid. Said invention also provides several advantages of said detection technique, and its extensive application fields.
Description
Technical field
The invention belongs to technological field of biochemistry, be specifically related to the improvement of nucleic acid amplification detection technique.
background technology
In the detection of nucleic acids process, in most cases the nucleic acid content in sample is lower, in order to make to detect, reaches certain sensitivity and accuracy, need to the nucleic acid fragment to be checked in sample effectively be increased.Polymerase chain reaction (Polymerase Chain Reaction in 1985, abbreviation PCR) birth of technology has promoted the development of nucleic acid detection technique and even whole Protocols in Molecular Biology greatly, and day by day become the important means in nucleic acid detection technique, also become the basis of modern molecular biology technique simultaneously.Subsequently, derive again such as reverse transcriptase-polymerase chain reaction (Reverse Transcriptase-PCR, be called for short RT-PCR), a series of nucleic acid amplification detection methods such as real-time fluorescence quantitative polymerase chain reaction (Real TimeFluorescent Quantified PCR), can be respectively used to amplification and the qualitative and quantitative detection of DNA sample and RNA sample.Because the error of amplification procedure often directly has influence on the result of detection of nucleic acids, therefore, follow-up amplicon (being the amplified production in nucleic acid amplification reaction) testing process just seems particularly important.At present, most of nucleic acid detection methods are all the operator schemes of " gene amplification+amplicon detects ", and comparatively traditional " PCR+ electrophoresis " detection method also is widely used at present.Because PCR method easily causes non-specific amplification in actual applications, also easily be subject to the pollution of other exogenous nucleic acids in environment before and after experiment, therefore often cause false positive or the false negative phenomenon of experimental result, the practical application that makes like this this technology especially clinical detection is subject to significant limitation.Although real-time fluorescence quantitative PCR has had very large improvement technically, detection reaction has also reached quantification, and the equipment that it is expensive and expensive detection, hindered on a large scale and promoted the use of.
Within 1991, Compton has proposed amplification technique (the Nucleic AcidSequence Based Amplification based on nucleotide sequence, be called for short NASBA) (Nature, 350 (6313): 91-2,1991), for gene amplification, new concept has been proposed.This technology is with the reverse transcription of RNA molecule-be transcribed into the technology main line, without the temperature cycle that is similar to the PCR process, by unidirectional isothermal duplication, in the short period of time, forms and is equivalent to the nearly millionfold RNA amplicon of former RNA copy number.But amplicon often adopts the methods such as electrophoresis, probe hybridization, dot blot to complete qualitative detection.In addition; Gen-Probe company also develops amplification technique (the Transcription Mediated Amplification with the similar transcriptive intermediate of nucleic acid amplification principle; be called for short TMA) (Journal ofClinical Microbiology; 35 (3); 676-8,1997), its nucleic acid amplification method with NASBA is identical; and the amplification of TMA is to carry out qualitative detection by hybridization protection assay (HybridizationProtection Assay is called for short HPA) technology.Current Gen-Probe company utilized this technological development chlamydia trachomatis and tubercule bacillus detection kit.No matter be NASBA technology or TMA technology, all on method, adopt unidirectional isothermal duplication, improved the non-specific amplification difficult problem in the round pcr, improved speed of reaction, and reaction process keeps isothermal, without relying on the PCR thermal cycler, only with common heater or water-bath, get final product, can reduce testing cost like this.But, with regard to current development trend, NASBA and TMA there is no method and reach the detection by quantitative degree, required experiment material in while TMA method, especially the enzyme mixture in the reaction needs special processing, belongs to non-opening special agent, and testing cost is relatively high.In addition, the avian myeloblastosis virus used in the NASBA technology (AMV) reversed transcriptive enzyme must be that specific manufacturer produces, and must be-70 ℃ of storages, in use the effect phase shorter, so operational condition is comparatively harsh, technology popularization also is restricted.
At present, the nucleic acid quantification amplification detection method used in laboratory study and clinical detection mainly be take real-time quantitative PCR as main, be to add fluorophor in the PCR reaction system, utilize the whole reaction process of fluorescent signal accumulation Real-Time Monitoring, finally by typical curve, carry out quantitative analysis.Its fluoroscopic examination pattern is divided into the TaqMan fluorescent probe, TaqMan MGB fluorescent probe [connects non-fluorescence quenching and MGB group (Minor Groove Binder) at fluorescent probe molecule 3 ' end, also be called the MGB probe], SYBR Green fluorescence dye, molecular beacon (Molecular Beacon) probe and FRET (fluorescence resonance energy transfer) (Fluorescence Resonance Energy Transfer is called for short FRET) probe etc.Although real time fluorescence quantifying PCR method sensitivity and accuracy are higher, testing cost is relatively costly, and testing process also needs to rely on specific installation.So all there is limitation in this technology on working method and testing cost, is not suitable as routine techniques and promotes.In the nucleic acid detection technique field, yet there are no the report that integrates amplification and quantitative dual-use function and correlation technique with low cost.
Summary of the invention
The purpose of this invention is to provide a kind of nucleic acid isothermal amplification quantitative detecting method (QuantitativeDetection with Isothermal Amplification, be called for short QDIA), it is that the qualitative and quantitative detection of the isothermal duplication of nucleic acid and amplicon is combined, and makes nucleic acid samples complete amplification and detect in a reaction system.This technology has advantages of easy, quick, sensitive, can overcome the deficiencies such as in prior art, cost is high, operation is wasted time and energy, specificity is low.
Nucleic acid isothermal amplification quantitative detecting method of the present invention comprises sample preparation step and nucleic acid isothermal amplification detection by quantitative step.The sample preparation step adopted, be to obtain the RNA sample from sample to be checked, and no matter sample comes self-organization, cell or microbial pathogen, is all finally to obtain its RNA sample; Tissue, cell or microbial pathogen sample can take the RNA extraction technique to obtain the RNA sample; If the DNA sample, can obtain the RNA sample by responsive transcription.The invention is characterized in, the isothermal duplication detection by quantitative of RNA sample, to adopt reversed transcriptive enzyme and RNA polymerase that isothermal duplication and TaqMan fluorescent probe are detected to combination, in to RNA sample isothermal duplication, realize detecting the accumulation with fluorescent signal, and whole process completes in same reaction system.Described reversed transcriptive enzyme is the MMLV reversed transcriptive enzyme.The concrete operations of described isothermal duplication detection by quantitative are: RNA sample to be checked is inserted to reaction detection liquid, 5-40 minute under 58-75 ℃ of condition, to open the secondary structure of RNA, proceed to subsequently reaction 1-2 hour under 35-45 ℃ of condition, detection reaction product reading in fluorescence detector again, by the typical curve of standard fluorescent sample signal, derive the amount of detected RNA sample, complete the detection by quantitative process of RNA sample; Its reaction detection liquid is comprised of primer, TaqMan fluorescent probe, reaction buffer and enzyme mixation.
The final concentration of each component of reaction buffer of the present invention is: 25-60mM Tris-HCl damping fluid (pH8.0-9.0), 30-90mM KCl, 10-20mM MgCl
2, 0.5-5mM deoxynucleoside triphosphate (dNTPs), 1-10mM ribonucleoside triphosphote (NTPs), 4-20mM dithiothreitol (DTT) (DTT), 10-20% (v/v) dimethyl sulfoxide (DMSO) (DMSO); Described primer final concentration is: with primer (being called for short primer 1) and second each 0.1-1 μ M of chain cDNA synthetic primer (abbreviation primer 2) of RNA enzyme Promoter Recognition sequence; The amount of each component of described enzyme mixation in 25 μ L reaction solutions is: the 4-75U reversed transcriptive enzyme, the 8-3000U RNA polymerase, 0.05-0.3U ribonuclease H (RNase H), 10-50U RNA enzyme inhibitors (RNasin Inhibitor) and 0.05-5 μ g bSA (BSA); Described TaqMan fluorescent probe final concentration is 50-200nM.Described TaqMan fluorescent probe can be selected TaqMan probe or TaqMan MGB probe, and the fluorophor on its probe can be determined according to fluorescence detection device or actual demand.In addition, add the TaqMan fluorescent probe in reaction system can be for the sequences Design of RNA template, can also be according to the sequences Design of cRNA amplicon.Certainly, for specificity and the sensitivity that improves detection reaction, also can the probe of above-mentioned two kinds of Different Alkali basic sequences all be added to the detection reaction system simultaneously, only need when probe design, the sequence location of two kinds of probes be staggered and get final product, prevent their complementary and formation dimers in same system.If two kinds of probes use simultaneously, its 5 ' end fluorescent mark group also can differ from one another where necessary, in order to mutually distinguish when detecting.For the reversed transcriptive enzyme that adds in reaction, different and add different unit of activity (meaning with U) depending on its kind and the level of response that needs.For another kind of key enzyme--RNA polymerase in the RNA amplification, its add-on also can be determined on a case-by-case basis: in general, if the RNA sample size is less, the RNA molecular length is shorter, to the cRNA combined coefficient, require also not high, can use the less RNA polymerase of unit of activity, each reaction adds the polysaccharase of the following RNA of 100U to get final product; If the RNA sample size is larger, the RNA molecular length is longer, and the cRNA combined coefficient is had relatively high expectations, and can use the RNA polymerase that unit of activity is larger, even in a reaction system, can use the RNA polymerase of the high unit of activity of high density, as 1000-3000U RNA polymerase/reaction.
Feature of the present invention also is, the concrete operations of described isothermal duplication detection by quantitative can also be: RNA sample to be checked is inserted to reaction detection liquid, 5-20 minute under 60-70 ℃ of condition, to open the secondary structure of RNA, proceed to subsequently under 38-42 ℃ of condition reaction 1-2 hour, then in fluorescence detector detection reaction product reading, by the typical curve of standard fluorescent sample signal, derive the amount of detected RNA sample, complete the detection by quantitative process of RNA sample.
The final concentration of described each component of reaction buffer also can be: 30-50mM Tris-HCl (pH8.0 9.0), 42-84mM KCl, 10-15mM MgCl
2, 1-4mM dNTPs, 2-8mM NTPs, 5-10mM dithiothreitol (DTT) (DTT), 10-15% (v/v) dimethyl sulfoxide (DMSO) (DMSO); The amount of each component of described enzyme mixation in 25 μ L reaction solutions can be: 40-60U Moloney murine leukemia virus (MMLV) reversed transcriptive enzyme, 10-2000U T7 RNA polymerase or 10-2000U SP6RNA polysaccharase or 10-50U T3 RNA polymerase, 0.1-0.2U ribonuclease H (RNase H), 15-45U RNA enzyme inhibitors (RNasin Inhibitor) and 0.1-3 μ g bSA (BSA); Described TaqMan fluorescent probe final concentration is 80-150nM.
The present invention is a kind of new nucleic acid amplification detection method, that the nucleic acid isothermal amplification is detected organically and is combined with the TaqMan fluorescent probe, form isothermal duplication and detection by quantitative in the efficient detection method of one, can carry out to micro-nucleic acid samples the detection of qualitative, quantitative.With respect to the pcr amplification technology, the present invention no longer needs the reciprocation cycle of lifting temperature, and whole augmentation detection process keeps constant temperature; Reduced like this non-specific amplification probability caused in " sex change-annealing-extension " temperature cycle; Amplification procedure, due to constant temperature, has saved thermal cycler simultaneously, and testing cost also decreases.Though testing process of the present invention has adopted with the TaqMan fluorescent probe of PCR and has detected similar principle, but concrete grammar is existing, improve, in this reaction, the accumulation of fluorescent signal is that the reverse transcription process participated in by reversed transcriptive enzyme completes, and be isothermal reaction, thereby accelerated speed of reaction and guaranteed the specificity of amplification system.In the present invention, the detection of fluorescent signal only needs common fluorescence detector, without relying on the real-time quantitative PCR amplification instrument, when reducing testing cost, has simplified operation steps.The invention has the advantages that:
(1) easy reaction system.By reversed transcriptive enzyme, isothermal amplification is reacted and couples together with detection by quantitative, " enzyme is dual-purpose ", isothermal duplication link and detection by quantitative link are carried out alternately, in the nucleic acid isothermal amplification, realize the accumulation of fluorescent signal in TaqMan fluorescent probe detection by quantitative, whole reaction completes in an individual system.
(2) amplification system of isothermal efficiency.The present invention is based on reverse transcription-transcribe the again nucleic acid isothermal amplification technique of principle, under the condition that corresponding RNA or DNA profiling, reversed transcriptive enzyme, RNA polymerase and reverse transcriptase primer are provided, form a DNA profiling according to reverse transcription reaction of a RNA template, then the RNA amplicon that DNA profiling can generate 100-1000 copy by responsive transcription calculates, under steady temperature, reaction 1-2 hour, can obtain Initial R NA sample size 10
6-10
18cRNA amplicon doubly, for round pcr, without the Thermal Cycling that repeats " sex change-annealing-extension ".One reduces the cost of detecting instrument, and two have improved the efficiency of amplified reaction greatly, has reduced again unnecessary non-specific amplification product pollution in reaction simultaneously.
(3) detection by quantitative.The present invention adopts and carry out the technology of fluorescent probe detection by quantitative in reverse transcription reaction, by the fluorescence intensity reading of real-time quantitative, and the linear relationship between RNA sample size and cRNA amplicon quantity, reach the purpose to the testing sample detection by quantitative.The same with TaqMan fluorescent probe detection technique, retained the characteristic that oligonucleotide fluorescent probe and primer pair target thing carry out high specific PCR and real-time quantitative, but the present invention to have superiority from detection time also the time testing cost than existing TaqMan fluorescent probe technique.
(4) detection speed is fast.Nucleic acid detection technique of the present invention, will increase and detect two reaction process and merge mutually, and completing in same reaction system, and whole process does not have the heating and cooling circulation, and have than DNA polyreaction rna transcription reaction system faster, save time; The TaqMan fluorescent probe is combined with reverse transcription reaction simultaneously, realizes detection by quantitative, more convenient, quick, sensitive to the detection of cRNA amplicon.
(5) detection sensitivity is high.Adopt the technology of the present invention, the RNA sample can realize 10 in 1-2 hour
6-10
18amplification amount doubly, for micro-RNA sample, can improve the sensitivity of detection greatly.I is carried out detection by quantitative to the RNA molecule of 10ng.
(6) test set is simple.The relatively existing nucleic acid qualitative and quantitative detection technology of the present invention relatively, is used common water bath with thermostatic control and fluorescence detector just can meet, and is convenient to application, is easy to promote.
The present invention is the isothermal amplification technique from the RNA sample, utilize that RNA reverse transcription-transcribe circulation is easy, isothermal, quick, sensitive characteristics, greatly improved the detection efficiency of nucleic acid, and improved targetedly temperature reciprocation cycle in the normal PCR technology, relied on specific installation, the defect such as experimental result is unstable.The more important thing is, introduced the fluorescent probe detection technique in the reverse transcription system, make under easy experiment condition, nucleic acid samples is carried out to detection by quantitative and become possibility.This will make the present invention become in common molecular biology experiment and Clinical Laboratory nucleic acid detection method conventional, highly sensitive, low-cost, less energy-consumption.The present invention is applicable to the qualitative and quantitative detection of various RNA, DNA sample, can be widely used in the link relevant to detection of nucleic acids in the fields such as experimental study, clinical detection, food safety detection, drug screening, detection of biological field agents.Because the present invention is lower to the dependency of plant and instrument, therefore on the spot, detect in application and can have great advantage.
Accompanying drawing and explanation thereof
(1) sample preparation in figure, obtain detecting required RNA sample.
(2) set up the QDIA reaction system, be combined with the RNA template annealing with primer 1 and the TaqMan fluorescent probe of the sub-recognition sequence of rna polymerase promoter, this completes at 58-75 ℃.
(3) synthetic cDNA the first chain under the reversed transcriptive enzyme effect, TaqMan fluorescent probe 5 ' end fluorophor is cut under reversed transcriptive enzyme 5 '-3 ' 5 prime excision enzyme activity effect simultaneously, is free in liquid-phase reaction system emitting fluorescence; The synthetic continuation of its cDNA the first chain.
(4) process the RNA single strand in the RNA-DNA heterozygote with RNase H.
(5) primer 2 and cDNA the first chain hybridization combination.
(6) synthetic cDNA the second chain under the reversed transcriptive enzyme effect.
(7) under the RNA polymerase effect, the cDNA two strands of take with the sub-recognition sequence of rna polymerase promoter is template, and isothermal duplication obtains a large amount of cRNA.
(8) primer 2 and cRNA strand hybridization combination, start the QDIA reaction cycle.
(9) synthetic cDNA the first chain under the reversed transcriptive enzyme effect.
(10) process the RNA single strand in the RNA-DNA heterozygote with RNase H.
(11) primer 1 and TaqMan fluorescent probe and cDNA strand hybridization combination.
(12) TaqMan fluorescent probe 5 ' end fluorophor is cut under reversed transcriptive enzyme 5 '-3 ' 5 prime excision enzyme activity effect, is free in liquid-phase reaction system emitting fluorescence; Continue synthetic cDNA the second chain simultaneously.
(13) read the fluorescence intensity in system by fluorescence detector.
(14) under the RNA polymerase effect, with the cDNA with the sub-recognition sequence of rna polymerase promoter.
Below in conjunction with accompanying drawing, with regard to the technology of the present invention mechanism, elaborate.
Two reactions steps have been the present invention includes, i.e. sample preparation, isothermal duplication detection by quantitative.
(1) sample preparation (as shown in (1) in figure).This step is to isolate nucleic acid molecule from sample to be checked, finally obtains the RNA sample.The present invention, by the detection to the RNA target molecule, proposes the new approaches of technique of gene detection, and the detection of RNA amplicon is also best embodied to technical superiority of the present invention.Discharge the RNA molecule from sample, can take the RNA extraction technique, modal is to extract (United States Patent (USP): No.5,346,994) by Trizol reagent single stage method, and this method is relatively efficient, quick, repeatability is high.In addition, also can take enzymatic lysis method and heating method.If need to extract mRNA, can directly adopt the FastTrack mRNA that American I nvitrogen company produces to extract test kit, simple and easy to do.In a word, effectively discharging the RNA molecule from sample cell gets final product.Therefore the RNA sample size obtained at this is relatively less, and RNA is easy to degraded, is not easy to direct-detection, need to just can reach the detection sensitivity needed by the amplification approach.If the DNA sample, can, by add rna polymerase promoter sequence on DNA sequence dna, in conjunction with transcription, obtain the RNA sample.Concrete method is by the sex change of DNA sample at 94-98 ℃, then go to 55-65 ℃, make to add in advance the upstream primer with rna polymerase promoter sequence of system to be combined with the downstream primer without rna polymerase promoter sequence, then under the Taq archaeal dna polymerase participates in, carry out extension, thereby obtain the DNA sample of 5 ' end with rna polymerase promoter sequence.Subsequently, under corresponding RNA polymerase exists, carry out responsive transcription, obtain specific RNA sample to be checked.(RNA polymerase of using in this process should be distinguished mutually with the RNA polymerase used in subsequent process, in order to avoid mutually pollute in system, affects detected result.As: select the T7 RNA polymerase in isothermal duplication detection by quantitative reaction system, in this RNA sample preparation process, select SP6 RNA polymerase or other suitable RNA polymerase).
(2) isothermal duplication detection by quantitative (as shown in (2) in figure-(14)).Reverse transcription link (2)-(6), RNA isothermal duplication link (7) and detection by quantitative link (8)-(14) that comprise the RNA target molecule.The reverse transcription link is that the RNA sample obtained is carried out to special reverse transcription, for next step isothermal duplication provides effective special template.At the beginning of specific gene section or gene locus, by selecting specific Oligonucleolide primers pair, determine constant gene segment C to be measured or gene locus in detecting sample.Because the present invention is according to transcribing principle, therefore, the promoter sequence that special primer (being positioned at RNA sample gene direction downstream) needs join dependency to identify in the RNA polymerase of DNA at its 5 ' end; Like this, the gene downstream direction of the double-stranded cDNA obtained by reverse transcription will be with upper promoter sequence.In addition, in view of the effect of reverse transcriptase primer in the later stage detection by quantitative, need consider the relation between required TaqMan fluorescent probe annealing temperature in the annealing temperature of primer and detection by quantitative when design of primers.
Reverse transcription link (as shown in (2) in figure-(6)) comprise the cDNA two strands synthetic, TaqMan fluorescent probe participate in the accumulation with fluorescent signal.Obtain double-stranded experience cDNA the first chain and two synthesis steps of the second chain of needing of cDNA by the reverse transcription process in prior art, the first step often completes by reversed transcriptive enzyme, then in the presence of DNA polymerase i, RNase H (sometimes in long segment cDNA reverse transcription process, also using DNA ligase), completes second step.Method of the present invention is merged into a step by these two steps and is completed, and participates in the detection by quantitative of TaqMan fluorescent probe.In this process, the reversed transcriptive enzyme adopted has the DNA polymerase activity of dependenc RNA, can, under Oligonucleolide primers exists, using single stranded RNA as synthetic the first chain cDNA fragment of template.At the beginning of cDNA the first chain is synthetic, specificity T aqMan fluorescent probe just is incorporated on template ribonucleic acid together with primer 1, under the reversed transcriptive enzyme effect, synthesize cDNA the first chain on one side, on one side by 5 ' → 3 ' 5 prime excision enzyme activity of reversed transcriptive enzyme, make the fluorophor of TaqMan probe 5 ' end cut, carry out the fluorescent signal accumulation in the middle of being free on solution, this is the fluorescent signal accumulation of first round detection by quantitative.Then, after another Oligonucleolide primers and cDNA the first chain annealing, this reversed transcriptive enzyme is usingd cDNA the first chain as template, carries out primer extension, thus synthetic cDNA the second chain.Therefore, the double-stranded building-up process of whole cDNA is to complete in a reaction system, only needs a kind of enzyme catalysis of reversed transcriptive enzyme, without archaeal dna polymerase, participates in.Reversed transcriptive enzyme in this step reaction is key enzyme in reaction, and it has determined that can the RNA amplicon be increased efficiently; Simultaneously, this enzyme is not identical with the reversed transcriptive enzyme used in existing TMA and NASBA technology, and it has the dual function of the RNA sample being carried out to reverse transcription and accumulation fluorescent signal in reaction simultaneously.Detailed process is, when synthetic the first chain cDNA, take the single stranded RNA sample as template, and the primer 1 in system is the first chain cDNA reverse transcriptase primer, utilizes the DNA polymerase activity of reversed transcriptive enzyme dependenc RNA, synthetic the first chain cDNA; Next under primer 2 and ribonuclease H participation, utilize reversed transcriptive enzyme to rely on the DNA polymerase activity of DNA, synthesize the second chain cDNA.Like this, the end of the double-stranded cDNA of synthesized will be with rna polymerase promoter sequence.By the reverse transcription process from single stranded RNA to double-stranded cDNA, obtain the double-stranded cDNA with the sub-recognition sequence of rna polymerase promoter, thereby provide template for follow-up RNA isothermal duplication process.
In RNA isothermal duplication link (as shown in (7) in figure), the double-stranded cDNA with the sub-recognition sequence of rna polymerase promoter that utilization of the present invention has been synthesized is as template, in the presence of RNA polymerase, by the mode of transcribing, synthesize in a large number the RNA amplicon (complementary RNA is called for short cRNA) with the complementation of Initial R NA template.This is the nucleic acid rapid amplifying process of unidirectional a, isothermal.
Detection by quantitative link (as shown in (8) in figure-(14)).After the present invention obtains a large amount of cRNA amplicons by isothermal duplication, under the reversed transcriptive enzyme in system and primer 1 and primer 2 participate in, the cRNA amplicon can repeat again synthetic cDNA two strands.Because initial RNA sample in cRNA and detection system is complementary relationship, therefore, in the cDNA of new round building-up process, the first chain cDNA will be take the cRNA strand as template, extension by primer 2 is synthesized, it is template that the second chain cDNA will be take the first newly synthetic chain cDNA, by the extension of primer 1, synthesizes.In this case, TaqMan probe in reaction system is after hybridizing with new synthetic cDNA the first chain, understanding the reversed transcriptive enzyme that is had 5 ' → 3 ' 5 prime excision enzyme activity in the process synthetic at cDNA the second chain acts on, make the fluorophor of TaqMan probe 5 ' end cut, be free in the middle of solution, its fluorescent signal is along with the circulation of reaction increases, and then run up to can be by the fluorescence detector reading, then react the linear relationship fluorescent signal that obtain between with it through QDIA by the standard rna sample size, derive the amount of surveyed RNA sample, complete RNA sample detection by quantitative process.Otherwise if, probe and not hybridization fully of new synthetic cDNA, the fluorophor on probe can not be reversed the record enzyme yet and decompose, and does not just have fluorescent signal to read in solution yet.
Hybridize and participate in the accumulation (as shown in (2) in figure-(4) and (11)-(13)) of fluorescent signal in the process that the TaqMan fluorescent probe is mainly double-stranded cDNA at the RNA reverse transcription with the cDNA strand.Certainly, add the TaqMan fluorescent probe in reaction system can be for the sequences Design of RNA template, (2) in the drawings-(4) and (11)-(13) also produce by reversed transcriptive enzyme the accumulation that detects the use fluorescent signal with the hybridization of cDNA strand; Also can, according to the sequences Design of cRNA amplicon, in (5) in the drawings-(6) and (9)-(10), with the hybridization of cDNA strand and by reversed transcriptive enzyme, produce and detect the accumulation of using fluorescent signal.For specificity and the sensitivity that improves detection reaction, also can two kinds of probes all be added to detection system simultaneously, only need when probe design, the sequence location of two kinds of probes be staggered and get final product, prevent their complementary and formation dimers in same system.In addition, if two kinds of probes use simultaneously, its 5 ' fluorescent mark group also can differ from one another where necessary, in order to mutually distinguish when detecting.
Embodiment
Embodiment 1: the QDIA detection method of bacterium rRNA sample
Below introduce and carry out discriminating bacteria by the present invention, the detection of Pseudomonas aeruginosa (Pseudomonasaeruginosa) (also claiming Pseudomonas aeruginosa) of take is example.The clinical samples processing that will contain Pseudomonas aeruginosa obtains bacterial suspension, or directly from the Pseudomonas aeruginosa culture, obtains bacteria suspension; Add bacterial lysate to discharge bacteria RNA (will have a large amount of bacterium rRNA in bacterial lysate), or carry out the bacteria RNA extracting by the amount of 107 bacterial cells/mL Trizol, obtain the RNA sample.Prepare 13 μ L reaction system damping fluid [40mM Tris-HCl (pH8.5), 42mM KCl, 15mM MgCl
2, 1mM dNTPs, 2mM NTPs, 10mM DTT, 15% (v/v) DMSO].At first add 0.2 μ M Pseudomonas aeruginosa 23S rRNA primer 1 (with the sub-recognition sequence of T7 rna polymerase promoter), 0.2 μ M Pseudomonas aeruginosa 23S rRNA primer 2 in damping fluid, (this probe is for Pseudomonas aeruginosa 23S rRNA sequences Design for 100nM TaqMan fluorescent probe, at its 5 ' end mark FAM fluorophor, playing the reporter group effect, can certainly be other fluorophors; 3 ' end mark TAMRA cancellation fluorophor, play the effect of cancellation fluorophor) and 2 μ L RNA sample liquid, mix and be placed on 65 ℃ of effects 15 minutes, then reaction tubes is placed in to 40 ℃ of water-baths, add enzyme mixation (to comprise 50U MMLV reversed transcriptive enzyme, the 40UT7 RNA polymerase, 0.2U RNase H, 20U RNasin Inhibitor and 2.6 μ g BSA, supply reaction system to 25 μ L), mix reaction solution, hatch 2 hours.After the ice bath termination reaction, reactant is placed in to fluorescence detector, according to reporter group used, chooses suitable spectral filter, fluorescence intensity.If the ratio of the fluorescence intensity in reaction tubes and blank reaches certain numerical value (as more than 5 times), can determine that specificity fluorescent probe and RNA isothermal duplication product have carried out specific hybrid, have proved the existence of Pseudomonas aeruginosa.Simultaneously, the fluorescence intensity obtained through same reaction conditions according to RNA template positive control and the typical curve between the RNA normal content, can derive the initial RNA sample size of sample Pseudomonas aeruginosa, thereby complete the detection by quantitative of Pseudomonas aeruginosa.
Carrying out discriminating bacteria by bacterial detection 16S or 23SrRNA has been a kind of very common method, but prior art is that employing PCR method bacterial detection rDNA is main.The method that the present invention adopts the conservative section amplification of bacterium 23SrRNA to be combined with specific probe, the detection by quantitative bacterium.The primer pair used in detection is the primer 2 of the conservative section design of directed toward bacteria 23SrRNA and with the primer 1 of the sub-recognition sequence of rna polymerase promoter.Start with from the detection of RNA, by the inventive method, identify bacterium, especially bacterial detection from clinical sample, will have a great deal of practical meanings.Bacterium in sample is not increasing before bacterium, and quantity is very low, should not detect.RNA copy number in bacterium is larger than genomic dna, therefore RNA is detected, and in the first step, has just increased and has detected probability, simultaneously again in conjunction with isothermal duplication, the RNA amount is increased, and then with quantitative means, detects.The present invention can be applicable to equally clinical in to the rapid detection of some other pathogenic micro-organism, such as tubercule bacillus, treponema pallidum etc.In addition, in food safety detection, also can carry out detection by quantitative to common disease substances such as intestinal bacteria, Listeria, Salmonella, virus and other noncellular microorganisms.
Embodiment 2: the QDIA detection method of eukaryotic cell mRNA sample
Below introducing the mRNA amount corresponding to the human cancer specific gene by the present invention is detected.The FastTrack mRNA produced with American I nvitrogen company extracts test kit and extract the mRNA sample from human Hela cell's suspension.(it comprises 13 μ L reaction buffer [50mM Tris-HCl (pH8.3), 80mM KCl, 12mM MgCl to prepare 25 μ L reaction system mixed solutions
2, 2mM dNTPs, 4mM NTPs, 6mM DTT, 10% (v/v) DMSO], each 250nM of primer 1 and primer 2, this probe of TaqMan fluorescent probe 120nM[designs for the human cancer genes involved, at its 5 ' end mark VIC fluorophor, play the reporter group effect, can certainly be other fluorophors, 3 ' end mark NFQ-MGB group (mixture of non-fluorescence quenching and MGB group)], 3 μ L RNA sample liquid, enzyme mixation [3 μ g BSA, 40U MMLV reversed transcriptive enzyme, 2000U T7 RNA polymerase, 0.1U RNaseH, 15U RNasin Inhibitor]).Reaction system, 62 ℃ of effects 10 minutes, is then proceeded to 42 ℃ of water-baths and hatches 1 hour, then reactant is placed in to fluorescence detector, according to reporter group used, choose suitable spectral filter, fluorescence intensity.Method is same as Example 1, derives the initial RNA sample size of human Hela cell in sample, thereby completes the detection by quantitative of human cancer gene mRNA.
Present method can be applicable to the research of system of gene expression in eukaryote amount.Detected object is eukaryotic cell mRNA, selected primer is random primer and with the Oligo-dT of the sub-recognition sequence of rna polymerase promoter, then in conjunction with the TaqMan fluorescent probe designed for specific gene, the mRNA amount that this gene pairs is answered is detected, thereby reaches the purpose that gene expression amount detects.In this example, adopt high density T7 RNA polymerase purpose to have and improve cRNA output at two: one, two improve the fluorescent probe detection efficiency.
The QDIA detection method of embodiment 3:DNA sample
When the DNA sample need to adopt the inventive method to be detected, need from testing sample, isolate DNA.The method of separating can adopt the method for heating, enzymolysis or chemical extracting.The DNA sample is mixed with upstream primer (with the sub-recognition sequence of SP6 rna polymerase promoter) and downstream primer, make the sex change of DNA double chain at 95 ℃, then make primer and DNA annealing at 65 ℃, and under participating in, the Taq archaeal dna polymerase carries out chain extension reaction, thereby obtain the DNA double chain of upstream with the sub-recognition sequence of SP6 rna polymerase promoter, form positive chain RNA under the effect of SP6 RNA polymerase, this just obtains the RNA sample of follow-up isothermal duplication.Prepare in advance 50 μ L reaction system mixed solutions, it comprises 32mM Tris-HCl (pH8.5), 50mM KCl, 10mM MgCl
2, 3mM dNTPs, 6mM NTPs, 8mM DTT, 6% (v/v) DMSO, each 0.3 μ M of primer 1 and primer 2, (this probe designs for the human cancer genes involved TaqMan fluorescent probe A 80nM, at its 5 ' end mark TET fluorophor, play the reporter group effect, 3 ' end mark DABCYL group, play the effect of cancellation fluorophor), (this probe designs for the human cancer genes involved TaqMan fluorescent probe B75nM, at its 5 ' end mark ROX fluorophor, 3 ' end mark DABCYL group), RNA sample liquid 2 μ L, enzyme mixation (1 μ g BSA, 60U MMLV reversed transcriptive enzyme, 50U T3 RNA polymerase, 0.2U RNase H, 40U RNasin Inhibitor).The reaction system that does not contain enzyme mixation is acted on to 5 minutes at 68 ℃, then proceed to 41 ℃ of water-baths and hatch 1.5 hours, then reactant is placed in to fluorescence detector, according to reporter group used, choose suitable spectral filter, fluorescence intensity.Method is same as Example 1, derives the RNA sample size for the treatment of in sample, then by the DNA sample, is transcribed in the RNA process quantitative relation between two kinds of nucleic acid, can extrapolate DNA sample size to be measured.
Present method is carried out the situation of QDIA detection for the DNA sample and is designed, with difference in example 1,2, be, before QDIA detects, the process that has a step with rna polymerase promoter sequence, DNA to be modified, then, by Transcription, the DNA sample is converted into to the RNA sample.The QDIA primer used can be selected for the nucleic acid samples source.In addition, adopt two fluorescent probes to carry out the specificity detection by quantitative, probe A is for the design of RNA template sequence simultaneously, and probe B is for cRNA amplicon sequences Design, two probes are in the fluorescent mark group difference of 5 ' end, and the fluorescent signal in detected result can be distinguished from each other.
Embodiment 4: the QDIA detection method of RNA sample in tissue
Below will introduce by the QDIA method corresponding RNA in animal tissues will be detected.In mouse tissue, in the 50-100mg/ml ratio, add TRIZOL reagent to be processed, according to TRIZOL test kit explanation, the total RNA in release cells, required RNA sample in being detected.(it comprises 13 μ L reaction buffer [55mM Tris-HCl (pH8.6), 70mM KCl, 12mM MgCl to prepare 25 μ L reaction system mixed solutions
2, 4mM dNTPs, 8mM NTPs, 5mM DTT, 15% (v/v) DMSO], each 0.6 μ M of primer 1 and primer 2, this probe of TaqMan fluorescent probe 120nM[designs for the mouse genes involved, at its 5 ' end mark VIC fluorophor, play the reporter group effect, can certainly be other fluorophors, 3 ' end mark NFQ-MGB group (mixture of non-fluorescence quenching and MGB group)], 1.5 μ L RNA sample liquid, enzyme mixation [38U avian myeloblastosis virus (AMV) reversed transcriptive enzyme, 1000U SP6 RNA polymerase, 0.15U RNase H, 28U RNasin Inhibitor, 0.5 μ g BSA]).Reaction system, 58 ℃ of effects 20 minutes, is then proceeded to 38 ℃ of water-baths and hatches 2 hours, then reactant is placed in to fluorescence detector, according to reporter group used, choose suitable spectral filter, fluorescence intensity.Method is same as Example 1, derives the initial RNA sample size of human Hela cell in sample, thereby completes the detection by quantitative of human cancer gene mRNA.
Present method can be applicable to the detection analysis of animal tissues's specific gene and expression amount.Detected object is the RNA in animal tissues, the QDIA primer of selecting is random primer and with the OlIgo-dT of the sub-recognition sequence of rna polymerase promoter, then in conjunction with the TaqMan fluorescent probe designed for specific gene, this gene and expression amount are detected to analysis.
Main agents related in the present invention illustrates:
MMLV (murine leukemia virus) reversed transcriptive enzyme (production of U.S. Epicentre company)
AMV (avian myeloblastosis virus) reversed transcriptive enzyme (production of U.S. Seikagaku America company)
RNase H (ribonuclease H) (production of American I nvitrogen company)
T7 RNA polymerase (production of U.S. Epicentre company)
SP6 RNA polymerase (production of U.S. Epicentre company)
T3 RNA polymerase (production of U.S. Epicentre company)
Tris-HCl damping fluid (laboratory preparation)
DTT (dithiothreitol (DTT)) (production of U.S. Epicentre company)
DMSO (dimethyl sulfoxide (DMSO)) (production of U.S. Sigma company)
BSA (bSA) (production of U.S. Sigma company)
DNTPs (deoxynucleoside triphosphate mixed solution) (production of American I nvitrogen company)
NTPs (ribonucleoside triphosphote mixed solution) (production of U.S. Epicentre company)
RNasin Inhibitor (RNA enzyme inhibitors) (production of U.S. Amersham Pharmacia company)
TaqMan fluorescent probe (precious biotechnology (Dalian) company limited)
Primer 1 (with the sub-recognition sequence of rna polymerase promoter) (precious biotechnology (Dalian) company limited is synthetic)
Primer 2 (precious biotechnology (Dalian) company limited is synthetic)
RNA standard model (laboratory is synthetic voluntarily)
TRIZOL reagent (production of American I nvitrogen company)
FastTrack mRNA extracts test kit (production of American I nvitrogen company)
Taq archaeal dna polymerase (precious biotechnology (Dalian) company limited)
Claims (3)
1. nucleic acid isothermal amplification quantitative detecting method, adopting has reverse transcription-transcribe principle, its augmentation detection to as if obtain RNA sample to be checked from sample, sample is cell, tissue or microorganism or has obtained any of DNA; It is characterized in that, the isothermal duplication detection by quantitative of RNA sample, be to adopt reversed transcriptive enzyme and RNA polymerase that isothermal duplication and TaqMan fluorescent probe are detected to combination, realize detecting the accumulation with fluorescent signal in to RNA sample isothermal duplication, it is to complete in same reaction system; Described reversed transcriptive enzyme is the MMLV reversed transcriptive enzyme; The concrete operations of described isothermal duplication detection by quantitative are: RNA sample to be detected is inserted to reaction detection liquid, and reaction 5-20 minute under 60-70 ℃ of condition, then proceed to reaction 1-2 hour under 38-42 ℃ of condition, then with fluorescence detector, detect; Wherein reaction detection liquid is comprised of primer, TaqMan fluorescent probe, reaction buffer and enzyme mixation;
Wherein, the final concentration of described each component of reaction buffer is: 25-60mM Tris-HCl, 30-90mM KCl, 10-20mM MgCl
2, 0.5-5mM deoxynucleoside triphosphate, 1-10mM ribonucleoside triphosphote, 4-20mM dithiothreitol (DTT), the dimethyl sulfoxide (DMSO) of 10-20% volume ratio; Described primer final concentration is: the primer 1 with RNA enzyme Promoter Recognition sequence is 0.1-1 μ M, with the primer 2 of the second chain CDNA synthetic primer, is 0.1-1 μ M; The amount of each component of described enzyme mixation in 25 μ L reaction solutions is: 4-75U reversed transcriptive enzyme, 8-3000U RNA polymerase, 0.05-0.3U ribonuclease H, 10-50U RNA enzyme inhibitors and 0.05-5 μ g bovin serum albumin; Described TaqMan fluorescent probe final concentration is 50-200nM.
2. detection method according to claim 1, is characterized in that, the final concentration of described each component of reaction buffer is: 30-50mM Tris-HCl, 42-84mM KCl, 10-15mM MgCl
2, the 1-4mM deoxynucleoside triphosphate, the 2-8mM ribonucleoside triphosphote, the 5-10mM dithiothreitol (DTT), 10--the dimethyl sulfoxide (DMSO) of 15% volume ratio; The amount of each component of described enzyme mixation in 25 μ L reaction solutions is: 40-60U MMLV reversed transcriptive enzyme, 10-2000U t7 rna polymerase or 10-2000U SP6RNA polysaccharase or 10-50UT3RNA polysaccharase, 0.1-0.2U ribonuclease H, 15-45URNA enzyme inhibitors and 0.1-3 μ g bovin serum albumin; Described TaqMan fluorescent probe final concentration is 80-150nM.
3. detection method according to claim 1, is characterized in that, described enzyme mixation is to add after 60-70 ℃ of processing at RNA sample and reaction buffer, then carry out the augmentation detection reaction of 38-42 ℃.
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| 曹晓红，唐英 等.艾滋病病毒的核酸检测法.渝西学院学报（自然科学版）1 4.2002,1(4),43-44. |
| 曹晓红,唐英 等.艾滋病病毒的核酸检测法.渝西学院学报(自然科学版)1 4.2002,1(4),43-44. * |
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