CN105018466A - Normal-temperature and constant-temperature nucleic acid amplification method using fluorescence probe - Google Patents
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Abstract
The invention relates to a normal-temperature and constant-temperature nucleic acid amplification method using a fluorescence probe. The normal-temperature and constant-temperature nucleic acid amplification method includes: under constant temperature, single-strand binding protein partially opens the double-strand parental strand into a single strand; recombinant enzyme is combined with primer to form complex, the complex is combined to the parental strand under the action of auxiliary protein, and the fluorescence probe is combined with a complementation area; DNA polymerase is combined to the 3' tail end of the primer to perform sub-strand extension; exonuclease identifies the tetrahydrofuran locus on the fluorescence probe under a double-strand state, fluorescent groups and quenching groups are separated after enzyme digestion to release fluorescence; after the fluorescence probe is cut off, the 3'-OH end originally sealed by probe 3' tail end modification is exposed, and DNA polymerase can continuously extend to form a sub-strand; qualitative and semi-quantitative detection can be performed on a to-be-detected sample by detecting the shape of an amplification curve and the strength of a fluorescence signal. The nucleic acid amplification method has the advantages that normal-temperature and constant-temperature detection can be performed, and qualitative and semi-quantitative detection can be performed on nucleic acid to-be-detected objects.
Description
Technical field
The invention belongs to biology field, relate to a kind of fluorescence probe method normal temperature isothermal nucleic acid amplification method, under normal temperature condition, carry out detecting fast, in real time and accurately to target set nucleic acid thing, can be applicable to the numerous areas that biological detection is relevant, such as clinical medicine, disease prevention and control or food safety etc.
Background technology
In medical science, particularly disease prevention and control field, tradition Progress of Nucleic Acid Amplification Technologies, the rapid molecular of polymerase chain reaction (PCR) to disease-producing pathogens (containing bacterium, virus and parasite) detects, also be experienced by one very long and after the process of constantly technical renovation, just obtain the accreditation of height in medical science molecular diagnosis field.Agarose gel electrophoresis detection mode after the amplification at initial stage, because of open detection, easily causes Aerosol Pollution, causes false positive frequently to occur, then namely replace by detection of fluorescent dyes mode.Although by DNA double chain combination dyestuff, such as SYBRGreen, use, the tube type closed achieving amplification system detects, thus greatly reduces the probability that false positive produces.But because of the impact of primer dimer, the specificity of detection is still under suspicion.Given this, in reaction system, introduce an oligonucleotide chain, the fluorescent probe namely with double-tagging (fluorophor and quenching group), thus target set nucleic acid thing can be detected with the common specificity of pair of primers.Therefore, fluorescence probe method (fluorescent PCR), because of its advantage such as the high specific detected and susceptibility, progressively instead of dye method, is widely used in clinical molecular diagnosis detection field.In view of the difference of target set nucleic acid thing sequence to be checked, optional fluorescent probe is also different: TaqMan double-tagging hydrolysis probes, molecular beacon probe (loop-stem structure) and novel TaqMan-MGB, wherein with TaqMan probe application at most, also the widest.Although current fluorescent PCR is applied to numerous areas, but to the dependence of precision detecting instrument (PCR instrument), make its field of employment be confined to centralab more, as need in the area of scarcity of resources, and when inconvenient long-term long-distance transport detection sample, when carrying out the Site Detection of sample, fluorescent PCR is just difficult to satisfy the demands.
Technical field of nucleic acid isothermal amplification emerging nearly ten years, not only provides more choices for external nucleic acid amplification, and makes scene carry out detection of nucleic acids fast to become possibility gradually.Due to the difference of principle of work, a part of nucleic acid isothermal amplification technology does not directly use probe in reaction system, such as loop-mediated isothermal amplification technique (LAMP), single primer isothermal amplification technique (SPIA); Another part is then based on different principle, probe is utilized to realize the rapid detection of nucleic acid: the amplification (NASBA) 1) depending on nucleotide sequence, utilize the primer of two particular design, molecular beacon probe and three kinds of enzymes under constant temperature (41 degree) condition, the detection of RNA target set nucleic acid thing in 1.5-2 hour, can be completed; 2) rolling circle amplification (RCA), utilize two or many primers, padlock probe and DNA ligases and polysaccharase (there is strand-displacement activity), obtained the steps such as cyclisation strand, probe template hybridization and connection by denature template after, carry out 10 minutes or Overnight incubation at 60 or 30 degree again, after carry out product detection; 3) isothermal amplification technique (HDA) of helicase is relied on, utilize pair of primers, TaqMan double-tagging hydrolysis probes, helicase, DNA single chain binding protein and archaeal dna polymerase, the real-time detection of nucleic acid can be completed in 37 degree or 65 degree in 30 minutes to 2 hours; 4) chain substitutes amplification technique (SDA), utilize two pairs of primers, hair fastener loop-stem structure probe (double-tagging), restriction endonuclease and archaeal dna polymerase (there is strand-displacement activity), by " 95 degree of sex change, 37 degree of renaturation, 37 degree of incubations 60 minutes, last 95 degree of termination reactions " a series of process, complete the detection of nucleic acid product; 5) nicking restriction endonuclease nucleic acid constant-temperature amplification technology (NEMA), utilize pair of primers (wherein a primer 5 ' holds mark Biotin), probe (3 ' end flag F ITC), polysaccharase and a nicking restriction endonuclease, again in conjunction with improved immunochromatography development process, jointly 54 degree of incubations 30 minutes to 1 hour, after last 95 degree of sex change, complete detection.
Therefore, need to invent a kind of amplification technique to overcome the various defects of nucleic acid isothermal amplification technology and fluorescent PCR, reduce fluorescent PCR to the dependence of precision instrument.
Summary of the invention
The object of this invention is to provide a kind of fluorescence probe method normal temperature isothermal nucleic acid amplification method, overcome current fluorescent PCR comparatively large to the dependence of precision instrument, and nucleic acid isothermal amplification technology also cannot realize complete isothermal and the quantitatively accurate not problem of PCR primer.
The present invention is achieved through the following technical solutions: a kind of fluorescence probe method normal temperature isothermal nucleic acid amplification method, and the method comprises the following steps:
(1), under constant temperature, the fundamental chain of double-strand local is opened into strand by single strand binding protein;
(2) recombinase and primer are combined into complex body, be attached under the effect of accessory protein on fundamental chain, fluorescent probe is also combined with complementary region, described fluorescent probe mid labels has tetrahydrofuran (THF), tetrahydrofuran (THF) both sides are respectively fluorophor and quenching group, between fluorophor and quenching group, spacing is 2-6nt, and fluorescent probe 3 ' end mark prevents the modification group of polymerase extension or amplification;
(3) archaeal dna polymerase is attached to 3 ' end of primer, carries out the life of prolonging of subchain;
(4) the tetrahydrofuran (THF) site on the fluorescent probe under exonuclease identification double-stranded state, enzyme makes fluorophor be separated with quenching group after cutting, release fluorescence;
(5) fluorescent probe cut-off after, expose originally by probe 3' end modified the 3'-OH that closes hold, archaeal dna polymerase can continue to extend to form subchain;
(6) by detecting the shape of amplification curve, with the power of fluorescent signal, qualitative and half-quantitative detection can be carried out to sample to be tested.
Further, described fluorescent probe length is 46-52nt.
Further, described fluorescent probe 3 ' end mark prevent the modification group of polymerase extension or amplification be in C3-spacer, phosphate group, vitamin H, vitamin H-TEG or amido any one.
Further, described fluorophor is any one in FAM, HEX, VIC, JOE, Texas Red, Cy3, Cy5 or TAMRA.
Further, described quenching group is BHQ1 or BHQ2.
Further, the aminoacid sequence of described recombinase is as shown in SEQ ID No.1, the aminoacid sequence of accessory protein is as shown in SEQ ID No.2, the aminoacid sequence of single strand binding protein is as shown in SEQ ID No.3, the aminoacid sequence of archaeal dna polymerase is as shown in SEQ ID No.4, and the aminoacid sequence of exonuclease is as shown in SEQ ID No.5.
Further, described constant temperature is 25-42 degree.
In more detail, in the present invention:
Pair of primers is two oligonucleotide strands, the upstream and downstream of specific binding and target area, 1) similar PCR pair of primers, but length is longer, 30-35 Nucleotide (nt); 2) should be noted during sequence selection and avoid: nonspecific binding site, multiple tumor-necrosis factor glycoproteins, palindromic sequence, formation internal secondary structure, form dimer between primer; 3) PCR is different from, the major consideration when annealing temperature (Tm) of primer is no longer design; 4) recommend for target area, design multipair primer and be optimized screening, to obtain best expanding effect; 5) amplified production is single band, without non-specific amplification.
Fluorescent probe (Fig. 1) is an oligonucleotide strand, in specific binding and target area, 1) length is 46-52nt; 2) totally 4 decorating sites: middle part distance 5 ' holds about 30nt position mark one dSpacer, i.e. tetrahydrofuran (THF) (THF), as endonuclease recognition site; Both sides, THF site mark a fluorophor and a quenching group respectively, and two group spacing are 2-6nt; 3 ' end mark prevents the modification group of polymerase extension or amplification, such as C3-spacer, phosphate group, vitamin H, vitamin H-TEG or amido; 3) should be noted during sequence selection and avoid: form dimer with primer, there is unspecific binding sites, multiple tumor-necrosis factor glycoproteins, form internal secondary structure; 4) the Tm value of probe is not the major consideration of design; 5) fluorophor optional FAM, HEX, VIC, JOE, TexasRed, Cy3, Cy5 or TAMRA, selects corresponding sense channel according to the excitation wavelength of each fluorophor with emission wavelength during detection; 6) optional BHQ1 or BHQ2 of quenching group, reduces the background fluorescence noise detected as far as possible.
Recombinase, single strand binding protein, archaeal dna polymerase, accessory protein and exonuclease, be through genetic engineering modified recombined engineering enzyme, comprise: 1) all derive from the activated protein having specific function in natural bacterium, by genetic engineering modified; 2) in vitro through chemical reagent, such as IPTG, or after the induction of temperature, the target protein of great expression can be obtained in the Bacillus coli cells of fermentation culture; 3) after cytoclasis, all through twice protein purification, such as ni-sepharose purification and heparin column purified, thus obtain highly purified single albumen; 4) after purifying, albumen is tested through enzymatic characterization, thus obtains the proteolytic enzyme with high unit of activity; 5) recombinase, is responsible for forming initial action mixture respectively with primer, can chooses from the natural bacterium such as intestinal bacteria (E.coli) recombinase RecA or T4 phage recombinase uvsX; 6) single strand binding protein, is responsible in conjunction with DNA single chain, and stablizes the Bubble Region formed when unwinding, and can choose from the natural bacterium such as the gp32 albumen of E.coli or T4; 7) archaeal dna polymerase, possesses polymerization activity and strand-displacement activity simultaneously, can choose from the natural bacterium such as the DNA polymerase i Klenow large fragment of E.coli, Bst polysaccharase, Phi-29 polysaccharase or Bacillus subtillis PolI (Bsu); 8) accessory protein, is responsible for assisting, promoting and stablize the combination of initial action mixture and DNA interchain, such as, can selects the uvsY albumen of T4; 9) exonuclease, be responsible for the DNA double chain with otch identifying that probe and subchain are formed, act on incision enzyme and cut probe, the signal of release fluorophor so that corresponding instrument detects, such as, can select exonuclease exoIII or exoIV of E.coli.The sequence of various albumen refers to sequence table.
Other reactive component, it is the chemical composition except above-mentioned five kinds of core enzymes, play in reaction system and energy is provided, amplification material, Optimum pH and salt concn, and provide the effects such as protection for freezing dry process subsequently, comprise 2-3% polyoxyethylene glycol (PEG), 20-30mMTris alkali, 90-110mM potassium acetate (KAc), 4-6mM dithiothreitol (DTT) (DTT), 2-3mM Triphosaden (ATP), 45-55mM disodium creatine phosphate (PCr, Creatine phosphate disodiumsalt), 90-110ng/ μ l creatine phosphokinase (CK, Creatine kinase), 200-250uM deoxyribonucleoside triphosphate (dNTP), 5.5-6.5% trehalose (Trehalose), 6-10% N.F,USP MANNITOL (Mannitol), the suitableeest working concentration of often kind of chemical composition need detect after target set nucleic acid thing does optimization Test again according to difference to be determined.
Freezing dry process, be carry out a kind of flow process of drying and other treatment to the reactive component of preparation, object is the usage period extending reaction system, and ensures not affect reactive behavior, 1 after transport) to comprise trunk dry with dry two steps again; The dry reaction conditions of trunk is-30 ~-40 degree, 3-10 hour; Drying conditions is 10-20 degree again, 1-2 hour; 2) best response procedures need be optimized according to the amount of reagent of actual drying again and determines; 3) the reaction mixture state after lyophilize is white, dry, powdery substance, after distilled water is resuspended, can dissolve completely, without any solid-state residual.
From the reaction mixture after lyophilize to final detection, also need to add some reactive components, primer, probe, template and reaction mixture are carried out end reaction, corresponding detecting instrument realizes fluoroscopic examination, comprising: 1) PEG, 350-450nM primer of 5-6%, 100-130nM fluorescent probe, certain density template, distilled water and 12-15mM magnesium acetate; 2) magnesium acetate finally adds, and plays startup reactivity; 3) for lower concentration or low copy sample, advise when reaction proceeds to 3-4 minute, carry out once the manual or dynamic operation of putting upside down or shaking mixing of machinery, greatly can increase recall rate and the detection accuracy of low concentration sample; 4) detecting instrument can have multiple choices: large-scale quantitative real time PCR Instrument, can meet high throughput testing demand; Twista or the ESE Tube Scanner of miniature portable, can meet field quick detection needs; Multifunction fluorescent microplate reader, also can meet part detection demand.
Adopt the positively effect of technique scheme:
(1) normal temperature and Constant Temperature Detection: temperature of reaction is normal temperature (25-42 degree), and DNA sample optimal reactive temperature is 35-39 degree, and RNA sample optimal reactive temperature is 40-42 degree; And whole testing process is all carried out under a steady temperature, operate without the need to any heating and cooling;
(2) specific detection is realized based on pair of primers and a fluorescent probe: the accuracy of detection is ensured by primer and fluorescent probe; Primer target is in the upstream and downstream of detection zone, and fluorescent probe then target, in the middle part of detection zone, therefore ensure that the accuracy of detection from three special districts.In addition, the signal release of the fluorophor that fluorescent probe marks is one-to-one relationship with the synthesis of subchain, therefore can go out concentration and the content of sample according to the change interpretation of testing process fluorescent signal;
(3) detect fast in real time: the release of fluorescent signal and the synthesis of subchain and quantity are positive corresponding relation, and detecting instrument can the change procedure of monitor signals in real time, thus can the amplified reaction process of Real-Time Monitoring target set nucleic acid thing.In addition, owing to carrying out under normal temperature constant temperature, and carry out collaborative enzymatic reaction primarily of multiple enzyme, therefore speed of response is exceedingly fast, and positive sample can obviously observe S type amplification curve in 20-40 minute;
(4) easy and simple to handle and result is easily understood: whole operating process comprises: sample of nucleic acid extracts, amplified reaction and detecting in real time, and two steps can complete.Result is understood and intuitively can be carried out " Yin/Yang " differentiation from the shape of amplification curve, and contained target nucleic acid amount of substance can by the exponential amplification starting point interpretation of amplification curve in sample, in the sample of such as, after 5 minutes start index amplification, contained target nucleic acid amount is necessarily higher than the sample of just start index amplification after 12 minutes.
Accompanying drawing explanation
Fig. 1 is fluorescent probe structural representation;
In figure, red F represents fluorophor, and grey Q represents quenching group, THF is the tetrahydrofuran (THF) site at middle part, 3 '-block is 3 ' terminal modifying groups, and 2-6nt is the distance between fluorescence and quenching group, and+30nt is the length of probe left arm, 46-52nt is the total length of probe, exoIII is a kind of exonuclease, specific recognition THF site, carries out enzyme and cuts, be separated fluorescence and quenching group, release fluorescence;
Fig. 2 is the amplification rear electrophoresis figure of soybean reference gene EF1b;
In figure, swimming lane M is DNA Marker, and swimming lane 1 and 2 is two to different primer pairs respectively, and swimming lane 3 is negative controls, and swimming lane 4 is filtered out positive primer pair amplifies segments;
Fig. 3 is to the sensitivity test that EF1b carries out on Twista instrument;
In figure, L9 ~ L3 is that copy number is from 10 respectively
9to 10
3nucleic acid-templated amplification curve when carrying out fluoroscopic examination, NTC is the amplification curve of negative control;
Fig. 4 is the sensitivity test carried out EF1b in BMG Omega Multifunction fluorescent microplate reader;
In figure, L9 ~ L3 is that copy number is from 10 respectively
9to 10
3nucleic acid-templated amplification curve when carrying out fluoroscopic examination, NTC is the amplification curve of negative control;
Fig. 5 is to the sensitivity test that IS6110 carries out on Twista instrument;
In figure, L9 ~ L3 is that copy number is from 10 respectively
9to 10
3nucleic acid-templated amplification curve when carrying out fluoroscopic examination, NTC is the amplification curve of negative control.
Embodiment
Below in conjunction with specific embodiment, technical scheme of the present invention is described further, but should not be construed as limitation of the present invention:
The biological material source used in the present invention:
1. the recombinant plasmid pMD18T-EF1b containing soybean reference gene EF1b, entrusts Shanghai Sheng Gong biotechnology limited-liability company to synthesize.
2. the plasmid pUC57-6110 of the insertion sequence IS6110 gene containing mycobacterium tuberculosis composite flora (MTB) entrusts Shanghai Sheng Gong biotechnology limited-liability company to synthesize.
3. amplification primers and fluorescent probe, all entrusts Shanghai Sheng Gong biotechnology limited-liability company to synthesize.
Embodiment 1
The present embodiment is for illustration of the normal temperature constant temperature fluorescence rapid reaction carried out on Twista instrument and Multifunction fluorescent microplate reader BMG Omeg.
1. report according to NCBI GenBank and pertinent literature, reference gene EF 1b according to soybean carries out design of primers, from many primer pairs, optimal screening goes out the primer pair (SEQ ID No.6/7) of a pair optimum by experiment, and the segment after amplification is 235bp (SEQ ID No.8) (Fig. 2).Subsequently this segment is cloned in plasmid pMD18-T, obtains required recombinant plasmid pMD18T-EF1b, as template during fluoroscopic examination.
EF1b-F：5’-TCACACACACACATATAGAAAGAGAGAGAC-3’(SEQ ID No.6)
EF1b-R：5’-CTGCTCCAGTTAGTTCATATAAGAGATAGG-3’(SEQ ID No.7)
SEQ ID No.8：
5’-TCACACACACACATATAGAAAGAGAGAGACAATGGTTGTCAGCATCGTCCATTTCTTTTAGACCAAGATTTTAATGCAGTCAACTCTGCAGTCATGATCCCGCAGCTCAAGTTTCTTTAATTTATATTTCATTTATGAAATTTGAAAAACAAAATAGCCAGTAATGATTCTACTTTCACACACTGATTGCAGGCAATCTCCACTTCCTATCTCTTATATGAACTAACTGGAGCAG-3’
2., based on fluorescent probe principle of design, devise a fluorescent probe EF1b-Exo (SEQID No.9) according to sequence SEQ ID No.8.
EF1b-Exo:5 '-CATGATCCCGCAGCTCAAGTTTCTTTAA
tt
ta
taTTTCATTTATGA-3 ', 46nt, 29 bit base T mark 6-FAM, and 31 bit base T mark dSpacer, and 33 bit base T mark BHQ1,3 ' end mark Biotin-TEG; (SEQ ID No.9)
3. utilize freeze drying technology, reaction main ingredient prepared by following system, after making dry powder, carry out follow-up test again:
| PEG35000 | 2.5％ |
| Trehalose | 6％ |
| N.F,USP MANNITOL | 7％ |
| Recombinase | 0.25ug/ul |
| Accessory protein | 0.10ug/ul |
| Single strand binding protein | 0.26ug/ul |
| Archaeal dna polymerase | 0.1ug/ul |
| Exonuclease | 90ng/ul |
| ATP | 2.4mM |
| dNTP | 235uM |
| Tris alkali | 26mM |
| DTT | 5mM |
| PCr | 48mM |
| CK | 100ng/ul |
| KAc | 105mM |
4. adopt Auele Specific Primer EF1b-F/R, fluorescent probe EF1b-Exo, carries out fluorescence with recombinant plasmid pMD18T-EF1b for template and detects real-time.The reaction system of amplification is:
| Lyophilized powder | 1 pipe |
| PEG35000 | 5％ |
| EF1b-F | 400nM |
| EF1b-R | 400nM |
| EF1b-Exo | 120nM |
| Template | 1μl |
| Magnesium acetate | 14mM |
| Distilled water | Mend to 50ul |
5. amplified reaction program: 39 degree of constant temperature, 30 minutes.
Detected result as shown in Figure 3, Figure 4, Fig. 3. be that the normal temperature constant temperature nucleic acid amplification that carries out on Twista instrument detects, L9-L3 is positive sample, for the recombinant plasmid pMD18T-EF1b containing goal gene, NTC are for contrasting, negative sample; L9-L3 is the positive sample of high density, after 10 times of gradient dilutions, and the positive sample that the copy number obtained is different; L9 is copy number is 10
9the sample of copy/microlitre, the like, L3 is copy number is 10
3the sample of copy/microlitre; Amplification is as expected, and the amplification curve of L9-L3 is all rendered as S type, and therefore qualitative interpretation is positive; The curve of NTC is an almost flat line, can qualitative interpretation be therefore negative; And peak position point (starting point of the start index amplification) interpretation from L9 to L3, also obviously semi-quantitatively can find out that the concentration of sample is also successively decreased successively from L9 toward L3, this also with to the gradient dilution process of positive sample L9-L3 matches.
In like manner, Fig. 4 is the detection that same test is carried out on Multifunction fluorescent microplate reader BMG Omeg, the instruction of L9-L3 and NTC is the same with Fig. 3, and this figure result illustrates that this invention described normal temperature constant temperature fluorescence nucleic acid amplification can complete test on broad variety fluorescence detection device.
Embodiment 2
The present embodiment for illustration of on Twista instrument for the fluoroscopic examination that MTB insertion sequence IS6110 gene carries out.
1. report according to NCBI GenBank and pertinent literature, the sequence (SEQ ID No.10) that have chosen one section of 523bp from the insertion sequence IS6110 of MTB carries out gene chemical synthesis, and be cloned in carrier pUC57, be prepared into recombinant plasmid pUC57-6110, as the template of fluoroscopic examination.
SEQ ID No.10：
5’-CTCCGACCGACGGTTGGATGCCTGCCTCGGCGAGCCGCTCGCTGAACCGGATCGATGTGTACTGAGATCCCCTATCCGTATGGTGGATAACGTCTTTCAGGTCGAGTACGCCTTCTTGTTGGCGGGTCCAGATGGCTTGCTCGATCGCGTCGAGGACCATGGAGGTGGCCATCGTGGAAGCGACCCGCCAGCCCAGGATCCTGCGAGCGTAGGCGTCGGTGACAAAGGCCACGTAGGCGAACCCTGCCCAGGTCGACACATAGGTGAGGTCTGCTACCCACAGCCGGTTAGGTGCTGGTGGTCCGAAGCGGCGCTGGACGAGATCGGCGGGACGGGCTGTGGCCGGATCAGCGATCGTGGTCCTGCGGGCTTTGCCGCGGGTGGTCCCGGACAGGCCGAGTTTGGTCATCAGCCGTTCGACGGTGCATCTGGCCACCTCGATGCCCTCACGGTTCAGGGTTAGCCACACTTTGCGGGCACCGTAAACACCGTAGTTGGCGGCGTGGACGCGGCTGATGTGCTC-3’
2. subsequently based on primer and fluorescent probe principle of design, Auele Specific Primer and fluorescent probe is designed for sequence SEQ ID No.10, by experiment after screening, select a pair the suitableeest primer I S6110-F/R (SEQ ID No.11/12) and a fluorescent probe Pro-6110 (SEQ ID No.13).
IS6110-F：5’-GTGGATAACGTCTTTCAGGTCGAGTACGC-3’(SEQ ID No.11)
IS6110-R：5’-CGCCGATCTCGTCCAGCGCCGCTTCGGAC-3’(SEQ ID No.12)
Pro-6110:5 '-CGAACCCTGCCCAGGTCGACACATAGGTGAGGTC
tGc
taCCCACAGCCGGTTA-3 ', 52nt, 35 bit base T mark 6-FAM, and 36 bit base G mark dSpacer, and 38 bit base T mark BHQ1,3 ' end mark Biotin-TEG; (SEQ ID No.13)
3. utilize freeze drying technology, reaction main ingredient prepared by following system, after making dry powder, carry out follow-up test again:
| PEG35000 | 2.5％ |
| Trehalose | 6％ |
| N.F,USP MANNITOL | 7％ |
| Recombinase | 0.25ug/ul |
| Accessory protein | 0.10ug/ul |
| Single strand binding protein | 0.26ug/ul |
| Archaeal dna polymerase | 0.1ug/ul |
| Exonuclease | 90ng/ul |
| ATP | 2.4mM |
| dNTP | 235uM |
| Tris alkali | 26mM |
| DTT | 5mM |
| PCr | 48mM |
| CK | 100ng/ul |
| KAc | 105mM |
4. adopt Auele Specific Primer 6110-F/R, fluorescent probe Pro-6110, carries out fluorescence with recombinant plasmid pUC57-6110 for template and detects real-time.The reaction system of amplification is:
| Lyophilized powder | 1 pipe |
| PEG35000 | 5.3％ |
| 6110-F | 410nM |
| 6110-R | 410nM |
| Pro-6110 | 120nM |
| Template | 1μl |
| Magnesium acetate | 14mM |
| Distilled water | Mend to 50ul |
5. amplified reaction program: 39 degree of constant temperature, 35 minutes.
As shown in Figure 5, Fig. 5 shows test Twista instrument carrying out normal temperature constant temperature fluorescence detection of nucleic acids for the goal gene IS6110 of pathogenic bacteria MTB to detected result; Wherein L9-L3 is that concentration of specimens is followed successively by 10
9-10
3the positive sample of copy/microlitre, NTC is negative sample; Equally, the S type curve of L9-L3, can be qualitative interpretation for positive sample and provides foundation, and straight curve is qualitative interpretation NTC is that negative sample also provides foundation; In addition, what L9-L3 postponed successively plays peak time point, also illustrate that concentration of specimens is successively decreased to L3 successively by L9.
Integrated embodiment 1 and 2, nucleic acid amplification of the present invention can at normal temperature and Constant Temperature Detection, and to PCR primer accurate quantification, can overcome current fluorescent PCR comparatively large to the dependence of precision instrument, and nucleic acid isothermal amplification technology also cannot realize complete isothermal and the quantitatively accurate not problem of PCR primer.
Claims (7)
1. a fluorescence probe method normal temperature isothermal nucleic acid amplification method, is characterized in that: the method comprises the following steps:
(1), under constant temperature, the fundamental chain of double-strand local is opened into strand by single strand binding protein;
(2) recombinase and primer are combined into complex body, be attached under the effect of accessory protein on fundamental chain, fluorescent probe is also combined with complementary region, described fluorescent probe mid labels has tetrahydrofuran (THF), tetrahydrofuran (THF) both sides are respectively fluorophor and quenching group, between fluorophor and quenching group, spacing is 2-6nt, and fluorescent probe 3 ' end mark prevents the modification group of polymerase extension or amplification;
(3) archaeal dna polymerase is attached to 3 ' end of primer, carries out the life of prolonging of subchain;
(4) the tetrahydrofuran (THF) site on the fluorescent probe under exonuclease identification double-stranded state, enzyme makes fluorophor be separated with quenching group after cutting, release fluorescence;
(5) fluorescent probe cut-off after, expose originally by probe 3' end modified the 3'-OH that closes hold, archaeal dna polymerase can continue to extend to form subchain;
(6) by detecting the shape of amplification curve, with the power of fluorescent signal, qualitative and half-quantitative detection can be carried out to sample to be tested.
2. fluorescence probe method normal temperature isothermal nucleic acid amplification method according to claim 1, is characterized in that: described fluorescent probe length is 46-52nt.
3. fluorescence probe method normal temperature isothermal nucleic acid amplification method according to claim 1, is characterized in that: described fluorescent probe 3 ' end mark prevent the modification group of polymerase extension or amplification be in C3-spacer, phosphate group, vitamin H, vitamin H-TEG or amido any one.
4. fluorescence probe method normal temperature isothermal nucleic acid amplification method according to claim 1, is characterized in that: described fluorophor is any one in FAM, HEX, VIC, JOE, Texas Red, Cy3, Cy5 or TAMRA.
5. fluorescence probe method normal temperature isothermal nucleic acid amplification method according to claim 1, is characterized in that: described quenching group is BHQ1 or BHQ2.
6. fluorescence probe method normal temperature isothermal nucleic acid amplification method according to claim 1, it is characterized in that: the aminoacid sequence of described recombinase is as shown in SEQ ID No.1, the aminoacid sequence of accessory protein is as shown in SEQ ID No.2, the aminoacid sequence of single strand binding protein is as shown in SEQ ID No.3, the aminoacid sequence of archaeal dna polymerase is as shown in SEQID No.4, and the aminoacid sequence of exonuclease is as shown in SEQ ID No.5.
7. fluorescence probe method normal temperature isothermal nucleic acid amplification method according to claim 1, is characterized in that: described constant temperature is 25-42 degree.
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| CN105695600A (en) * | 2016-03-25 | 2016-06-22 | 苏州达麦迪生物医学科技有限公司 | Primer for isothermal amplification as well as design method and application thereof |
| CN106979940A (en) * | 2017-04-17 | 2017-07-25 | 浙江大学 | A kind of device and method for carrying out nucleic acid amplification detection |
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| CN107016258B (en) * | 2016-01-27 | 2020-06-05 | 应清界 | Method for fluorescence quantitative calculation based on recombinase-mediated isothermal nucleic acid amplification (RAA) method |
| CN107016258A (en) * | 2016-01-27 | 2017-08-04 | 应清界 | One kind is based on recombinase-mediated isothermal nucleic acid amplification（RAA）The method that method carries out fluorescent quantitation calculating |
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| CN117551818B (en) * | 2024-01-11 | 2024-05-10 | 天津欧德莱生物医药科技有限公司 | Target gene, primer probe combination and kit for detecting respiratory syncytial virus and application |
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