CN105274096A

CN105274096A - Bridge type fluorescent probe with bridge type sequence zone doping into mismatched bases and application and method

Info

Publication number: CN105274096A
Application number: CN201510561642.3A
Authority: CN
Inventors: 黄庆; 府伟灵; 黄君富; 夏涵; 赵娜
Original assignee: First Affiliated Hospital of TMMU
Current assignee: Third Military Medical University TMMU; First Affiliated Hospital of TMMU
Priority date: 2015-09-07
Filing date: 2015-09-07
Publication date: 2016-01-27

Abstract

The present invention discloses a bridge type fluorescent probe with a bridge type sequence zone doping into mismatched bases and application and a method, the bridge type fluorescent probe 5'-end to 3'-end nucleic acid sequence successively comprises a recognition sequence zone complementary to a nucleic acid sequence of a to-be-tested mutant gene, the bridge type sequence zone, and an anchor sequence zone, wherein the anchor sequence zone is complementary to the outer side sequence zone of 3'-end of the to-be-tested mutant gene, the 3'-end of the anchor sequence is connected with an extension blocking group ; the bridge type sequence zone is a nucleotide derivative sequence containing at least one mismatched bases, an interactiveness fluorescent marking system is marked between the recognition sequence zone and the bridge type sequence zone, the bridge type fluorescent probe is simple in design, can specifically recognizes the gene, is high in sensitivity, and can be used to detect single base mutation and identify highly homologous species and subtypes.

Description

The bridge-type fluorescent probe of base mismatch and application thereof and method are mixed in bridge-type sequence area

Technical field

The invention belongs to field of nucleic acid detection, be specifically related to the bridge-type fluorescent probe that base mismatch is mixed in bridge-type sequence area, also relate to application and the method for this probe of bridge-type fluorescence.

Background technology

Single nucleotide variation comprises the transgenation (as: causing the oncogene mutation of malignant tumour) under common single nucleotide polymorphism (SNP) and morbid state.

Existing detection technique method is numerous, is mainly the fluorescent probe detection technique for single nucleotide variation, as hydrolysis probes (hydrolysisprobes; Also be TaqMan probe), hybridization probe (hybridizationprobes), molecular beacon (molecularbeacons) etc. are the most common, are also the detection techniques commonly used the most.But above-mentioned probe detects unsatisfactory to single nucleotide variation.Above-mentioned probe design difficulty on the one hand for single nucleotide variation is large, for same list nucleotide variation, usually needs to design several above probe, so that finishing screen chooses the best probe of a specificity; On the other hand due to hybridization kinetics reason, the recognition capability of probe to single nucleotide variation is limited, specificity is undesirable, due to the probe melting temperature (Tm) (meltingtemperature that different genotype is corresponding, Tm) very close, and the overall Tm value of probe is higher, the specificity that the minor differences that above-mentioned probe depends on Tm value is hybridized to control probe template; Therefore, template (or nucleic acid molecule) non-specific hybridization that the fluorescent probe for single nucleotide variation often can not make a variation with other, produces false positive results.For the mononucleotide acid polymorphism (singlenucleotidepolymorphism in single nucleotide variation, SNP), due to wild-type or saltant type abundance all higher (usually at least reaching 50% ratio), therefore, non-specificly there is not too many interference to detected result in what existing probe techniques detected SNP.But for the transgenation of disease-related, as caused the oncogene mutation of malignant tumour, the abundance of mutator gene is usually lower.Along with the development of individualized treatment, high requirement is proposed to the sensitivity that oncogene mutation detects, require at least to detect the mutator gene that sudden change abundance only has about 1%, and, require higher to the detection sensitivity of blood of cancer patients dissociative DNA.The false positive results produced owing to there is non-specific hybridization, existing probe technique is not also suitable for the detection needs of low abundance mutator gene.Therefore, be badly in need of the probe that a kind of sensitivity is higher, can be used in detecting the mutator gene that sudden change abundance only has about 1%.

Summary of the invention

In view of this, low in order to solve the sensitivity of existing probe in detecting low abundance mutator gene, easily there is false-positive problem, first the present invention is provided in the bridge-type fluorescent probe that base mismatch is mixed in bridge-type sequence area, bridge-type fluorescent probe simplicity of design of the present invention, to single nucleotide variation, there is high specific recognition capability, non-specific hybridization can be suppressed completely, the detection of low abundance list nucleotide variation can be realized with high specificity, meanwhile, this probe technique is also particularly suitable for the detection of the nucleic acid target molecule with high homology.

For achieving the above object, the invention provides following technical scheme:

The bridge-type fluorescent probe (Fig. 1) of base mismatch is mixed in a kind of bridge-type sequence area, described bridge-type fluorescent probe 5'-end holds nucleotide sequence to comprise the recognition sequence district with mutant gene place to be detected nucleic acid array complementation successively to 3'-, bridge-type sequence area and with mutant gene 3'-to be detected hold outside complementary anchor series district, the 3'-end of described anchor series is connected with extension blocking group, and its function stops the extension of bridge-type fluorescent probe in nucleic acid amplification system; Described bridge-type sequence area is the nucleotide derivative sequence containing at least one base mismatch, is marked with the fluoroscopic marker system of interaction between described recognition sequence district and bridge-type sequence area.

In the present invention, recognition sequence section length is usually at 2 ~ 16 Nucleotide (nucleotide, nt) between, be preferably 2 ~ 12, be more preferably 6 ~ 12nt, GC content is without particular requirement, be preferably 10 ~ 80%, be more preferably 40 ~ 80%, and usually do not consider its melting temperature (Tm) (meltingtemperature, Tm), but because its sequence is extremely short, its Tm value is usually extremely low, is usually less than 30 DEG C, and the complementary base preferred version of target molecule variation corresponding to base is positioned in the middle of recognition sequence district.The length in anchor series district without particular requirement, preferred length be 16 ~ 50nt, GC content without specific requirement, preferably 25 ~ 60%, Tm values are far above recognition sequence district, are usually greater than 65 DEG C, even can up to 85 DEG C.Therefore, recognition sequence district and anchor series district are low Tm value and high Tm value region respectively.

In the present invention, recognition sequence district and anchor series district are all complementary with target nucleic acid sequence to be detected, between have a spacing distance, be called bridge-type sequence area, bridge-type sequence area is the nucleotide derivative sequence containing at least one base mismatch, the sequence length of bridge-type sequence area is without specific requirement, but interval identical every the base number of target sequence with anchor series with recognition sequence district, preferred version is 2 ~ 16 Nucleotide, and further preferred version is 5 Nucleotide, nucleotide derivative can be Hypoxanthine deoxyriboside (deoxyinosine), inosine (inosine), 7-denitrification-2 '-Hypoxanthine deoxyriboside (7-deaza-2 '-deoxyinosine), 2-azepine-2 '-Hypoxanthine deoxyriboside (2-aza-2 '-deoxyinosine), 2 '-methoxyl group inosine (2 '-OMeinosine), 2 '-F inosine (2 '-Finosine), deoxidation 3-nitro-pyrrole (deoxy3-nitropyrrole), 3-nitro-pyrrole (3-nitropyrrole), 2 '-methoxyl group 3-nitro-pyrrole (2 '-OMe3-nitropyrrole), 2 '-F3-nitro-pyrrole (2 '-F3-nitropyrrole), 1-(2 '-deoxidation-β-D-RIBOSE)-3-nitro-pyrrole (1-(2 '-deoxybeta-D-ribofuranosyl)-3-nitropyrrole), deoxidation 5-nitro-pyrrole (deoxy5-nitroindole), 5-nitroindoline (5-nitroindole), 2 '-methoxyl group 5-nitroindoline (2 '-OMe5-nitroindole), 2 '-F5-nitroindoline (2 '-F5-nitroindole), deoxidation 4-nitrobenzimidazole (deoxy4-nitrobenzimidazole), 4-nitrobenzimidazole (4-nitrobenzimidazole), deoxidation 4-aminobenzimidazole (deoxy4-aminobenzimidazole), 4-aminobenzimidazole (4-aminobenzimidazole), deoxidation nebularine (deoxynebularine), 2 '-F nebularine (2 '-Fnebularine), 2 '-F4-nitrobenzimidazole (2 '-F4-nitrobenzimidazole), peptide nucleic acid(PNA)-5-nitroindoline (PNA-5-nitroindole), peptide nucleic acid(PNA)-nebularine (PNA-nebularine), peptide nucleic acid(PNA)-inosine (PNA-inosine), peptide nucleic acid(PNA)-4-nitrobenzimidazole (PNA-4-nitrobenzimidazole), peptide nucleic acid(PNA)-3-nitro-pyrrole (PNA-3-nitropyrrole), morpholinyl-5 nitroindoline (morpholino-5-nitroindole), morpholinyl-nebularine (morpholino-nebularine), morpholinyl-inosine (morpholino-inosine), morpholinyl-4-nitrobenzimidazole (morpholino-4-nitrobenzimidazole), morpholinyl-3-nitro-pyrrole (morpholino-3-nitropyrrole), phosphoramidate-5-nitroindoline (phosphoramidate-5-nitroindole), phosphoramidate-nebularine (phosphoramidate-nebularine), phosphoramidate-inosine (phosphoramidate-inosine), phosphoramidate-4-nitrobenzimidazole (phosphoramidate-4-nitrobenzimidazole), phosphoramidate-3-nitro-pyrrole (phosphoramidate-3-nitropyrrole), 2 '-0-methoxy ethyl inosine (2 '-0-methoxyethylinosine), 2 '-0-methoxy ethyl nebularine (2 ' 0-methoxyethylnebularine), 2 '-0-methoxy ethyl 5-nitroindoline (2 '-0-methoxyethyl5-nitroindole), 2 '-0-methoxy ethyl 4-nitro-benzoglyoxaline (2 '-0-methoxyethyl4-nitro-benzimidazole) and 2 '-0-methoxy ethyl 3-nitro-pyrrole (2 '-0-methoxyethyl3-nitropyrrole) one or more, preferably, bridge-type sequence area is the poly Hypoxanthine deoxyriboside (polydeoxyinosine, poly (I)) containing at least one base mismatch, the base of mixing, mispairing preferred version one is GA, CT, TT mispairing, and mispairing preferred version two is CC mispairing, mispairing preferred version three AA and GG mispairing, mispairing preferred version four CA and GT mispairing.In above-mentioned four kinds of mispairing preferred versions, mismatch binding capacity of water is mispairing preferred version one to four successively.Such as, when the complementary target sequence that bridge-type sequence area is corresponding is 5'-GTCGC-3', 3'-IITII-5' can be designed, wherein, letter I represents Hypoxanthine deoxyriboside, now, T is corresponding with the C of template, the two possibility that cannot form heteroduplex because of mispairing is the highest in above-mentioned four kinds of mispairing preferred versions, therefore, bridge-type sequence area still can when recognition sequence district and anchor series district and target molecule complementation, form " bubble " shape structure, just as the bridge of between recognition sequence district and anchor series district, this is also the reason place that probe of the present invention is named as bridge-type fluorescent probe.The object introducing base mismatch is, when the target sequence corresponding to recognition sequence district is when not being rich in nucleic acid containing G, C, A of T base, cannot there be the fluoroscopic marker system (that is: cannot simultaneously mark fluorescent reporter group and quenching group) of interaction in recognition sequence district by independent marking, can in Poly (I) sequence of bridge-type sequence area, mix the base T of the complementary target sequence base C mispairing corresponding with it, thus the fluoroscopic marker system of interaction can be marked on T, as fluorescent reporter group or quencher; The sensitivity of probe can be improved further simultaneously.

In the present invention interaction property fluoroscopic marker system can in recognition sequence district simultaneously mark fluorescent reporter group and quenching group (Fig. 1), or, at recognition sequence district mark fluorescent reporter group, bridge-type sequence area mark quenching group mark (Fig. 2), or, at recognition sequence district mark quenching group, bridge-type sequence area mark fluorescent reporter group.

In the present invention, when recognition sequence district while when mark fluorescent reporter group and quenching group, fluorescent reporter group is positioned at the 5'-end in this district, quenching group is positioned at the 3'-end (Fig. 1) in this district, or quenching group is positioned at the 5'-end in this district, fluorescent reporter group is positioned at the 3'-end in this district, also or fluorescent reporter group be positioned at the 5'-end in this district, quenching group is positioned at the centre in this district, or quenching group is positioned at the 5'-end in this district, fluorescent reporter group is positioned at the centre in this district, or, fluorescent reporter group is positioned at the 3'-end in this district, quenching group is positioned at the centre in this district, or, quenching group is positioned at the 3'-end in this district, fluorescent reporter group is positioned at the centre in this district.

In the present invention, when interaction fluoroscopic marker system is fluorescent reporter group and the quenching group being marked on recognition sequence district and bridge-type sequence area respectively (Fig. 2), fluorescent reporter group or quenching group can be marked on the 5'-end in recognition sequence district, centre or 3'-end, and corresponding fluorescent quenching group and reporter group can be marked on the 5'-end of bridge-type sequence area, centre or 3'-end.

In the present invention, single nucleotide variation of indication is single base deletion, sudden change, insertion or SNP, the high homology sequence of indication refers to that region to be detected nucleic acid target molecule specific sequence differs the different nucleic acid target molecules of one or more bases, as the infectious pathogen nucleic acid target molecule of the different subtype that same belongs to.

Fluorescent reporter group of the present invention is without specific requirement, preferred version comprises Fluoresceincarboxylic acid (6-FAM), chlordene fluorescein (HEX), Tetrachlorofluorescein (TET), JOE, VIC, fluorescein isothiocyanate (FITC), indoles dicarboxyl cyanines (Cy3, Cy5), TAMRA and ROX, and other fluorescence molecule or luminophore; Fluorescent quenching group without specific requirement, preferred version fluorescence class quencher (as: TAMRA, ROX) and non-fluorescence class quencher (as: DABCYL, BHQ1, BHQ2).

Further, described fluorescent reporter molecule and quencher molecule are fluorescent substance, all can utilize known any material in the technical field of the invention, comprise: Cy2 ^tM(506), YO-PRO ^tM-1 (509), YOYO ^tM-1 (509), Calcein (517), FITC (518), FluorX ^tM(519), Alexa ^tM(520), Rhodamine110 (520), OregonGreen ^tM500 (522), OregonGreen ^tM488 (524), RiboGreen ^tM(525), RhodamineGreen ^tM(527), Rhodamine123 (529), MagnesiumGreen ^tM(531), CalciumGreen ^tM(533), TO-PRO ^tM-1 (533), TOTO1 (533), JOE (548), BODIPY530/550 (550), Dil (565), BODIPYTMR (568), BODIPY558/568 (568), BODIPY564/570 (570), Cy3TM (570), Alexa ^tM546 (570), TRITC (572), MagnesiumOrange ^tM(575), PhycoerythrinR & B (575), RhodaminePhalloidin (575), CalciumOrange ^tM(576), PyroninY (580), RhodamineB (580), TAMRA (582), RhodamineRedTM (590), Cy3.5TM (596), ROX (608), CalciumCrimson ^tM(615), AlexaTM594 (615), TexasRed (615), NileRed (628), YO-PROTM-3 (631), YOYOTM-3 (631), R-phycocyanin (642), C-Phycocyanin (648), TO-PRO ^tM-3 (660), TOTO3 (660), DiDDilC (5) (665), Cy5 ^tM(670), Thiadicarbocyanine (671), Cy5.5 (694), HEX (556), TET (536), BiosearchBlue (447), CALFluorGold540 (544), CALFluorOrange560 (559), CALFluorRed590 (591), CALFluorRed610 (610), CALFluorRed635 (637), FAM (520), Fluorescein (520), Fluorescein-C3 (520), Pulsar650 (566), Quasar570 (667), Quasar670 (705) and Quasar705 (610).The numeral of bracket is the maximum emission wavelength represented with nanometer unit.

Further, described quencher molecule utilizes the known black quencher molecule of non-fluorescence can carrying out cancellation to the fluorescence of wide range of wavelength or specific wavelength in the technical field of the invention, comprises black hole quencher, (BHQ:blackholequencher; Comprise BHQ1, BHQ2, BHQ3), 4-[4-(dimethylamino) benzeneazo] phenylformic acid (DABCYL:4-[4-(Dimethylamino) phenylazo] benzoicacid).

Extension blocker group of the present invention is without specific requirement, preferred version modifies phosphate group, amino group in the 3'-terminal bases 3'-hydroxyl position in anchor series district, or the last bit base use reversion base, also or the last bit base be dideoxy nucleotide (dideoxynucleotide).

The recognition sequence district of bridge-type fluorescent probe of the present invention and/or anchor series district can mix the derivatized nucleotide improving its mismatch binding ability further, preferred version comprises lock nucleic acid (lockednucleicacids, and peptide nucleic acid(PNA) (peptidenucleicacids, PNA) LNA).

Present invention also offers the application of bridge-type fluorescent probe in the reagent of the single nucleotide variation of preparation detection.Its Cleaning Principle is as shown in Figure 3 and Figure 4: anchor series district does not possess sequence selective, the Complementary hybridization region of it and nucleic acid target molecule is positioned at the 3'-flank region of variation base to be detected, due to the Tm value that it is higher, this sequence can in wider temperature range with nucleic acid target molecule Complementary hybridization, recognition sequence district is extremely short, Tm value is lower, only after anchor series and nucleic acid target molecule Complementary hybridization, could hybridization thermokinetics effect under with its nucleic acid target molecule Complementary hybridization, now, recognition sequence district and anchor series district all with nucleic acid target molecule Complementary hybridization, with this understanding, because the Tm value of bridge-type sequence area is extremely low, under the hybridization temperature condition residing for bridge-type fluorescent probe, bridge-type sequence area and nucleic acid target molecule cannot form Complementary hybridization double-strand, thus formed one " balloon-shaped structure " between two the Complementary hybridization duplex structures formed in recognition sequence district and anchor series district and target nucleic acid sequence.When there is the single base mismatch that single nucleotide variation causes in the nucleic acid target molecule Complementary hybridization region that recognition sequence district is corresponding, single base mismatch can cause recognition sequence district cannot form Complementary hybridization double-strand with nucleic acid target molecule, with this understanding, in whole bridge-type fluorescent probe structure, anchor series district and target nucleic acid molecule is only had to form Complementary hybridization double-strand, and recognition sequence district and bridge-type sequence area all cannot form Complementary hybridization double-strand with nucleic acid target molecule, be therefore in free single-chain state.In pcr amplification circulation, in the first case described above, namely recognition sequence district and nucleic acid target molecule form Complementary hybridization double-strand, in the primer extension process of bridge-type fluorescent probe 5'-flank region, when the extension products of primer extends to the 5'-end of bridge-type fluorescent probe, under the effect of 5' → 3' exonuclease of archaeal dna polymerase, the Nucleotide in recognition sequence district is hydrolyzed into single core thuja acid successively according to 5' → 3' direction, with this understanding, when interaction fluoroscopic marker system is marked on recognition sequence district (Fig. 3), the fluorescent energy be marked between the fluorescent reporter group in recognition sequence district and quenching group is total to transferance (FRET) and is eliminated, fluorescent reporter group release fluorescence, when interaction fluoroscopic marker system is marked on recognition sequence district and bridge-type sequence area respectively (Fig. 4), fluorescent energy between the quenching group (or: fluorescent reporter group) being marked on the fluorescent reporter group (or: fluorescent quenching group) in recognition sequence district and bridge-type sequence area altogether transferance (FRET) is eliminated, fluorescent reporter group release fluorescence.After archaeal dna polymerase has been hydrolyzed recognition sequence district, primer can extend to bridge-type sequence area further, under this condition, bridge-type sequence area becomes free strand, be hydrolyzed by the tie point intactly in itself and anchor series district under the effect of 5' → 3' exonuclease of archaeal dna polymerase, subsequently, anchor series district is further hydrolyzed to single core thuja acid.In pcr amplification circulation, in above-mentioned the second situation, namely the recognition sequence district caused due to nucleic acid target molecule list nucleotide variation and the single base mismatch of nucleic acid target molecule, and when this single base mismatch causes recognition sequence district and nucleic acid target molecule cannot form Complementary hybridization double-strand, recognition sequence district and bridge-type sequence area are all in free single-chain state, only anchor series district and target nucleic acid molecule form Complementary hybridization double-strand, with this understanding, the 5'-end of anchor series directly can be extended to smoothly at the primer of bridge-type fluorescent probe 5'-flank region, subsequently, under the effect of 5' → 3' exonuclease of archaeal dna polymerase, at the joint base place in bridge-type sequence area and anchor series district, a complete fragment is cleared up in recognition sequence district and bridge-type sequence area, subsequently, the Nucleotide in anchor series district is hydrolyzed into single core thuja acid successively according to 5' → 3' direction, with this understanding, due to the integrity of recognition sequence district and bridge-type sequence area, the fluoroscopic marker system that no matter interacts is that separate marking is in recognition sequence district, still recognition sequence district and bridge-type sequence area is marked on respectively, still FRET effect is had between fluorescent reporter group and quenching group, therefore, there is not fluorescence (Fig. 3 in probe, Fig. 4).Therefore only have after recognition sequence district and nucleic acid target molecule form Complementary hybridization double-strand, recognition sequence district just can be hydrolyzed in 5' → 3' exonuclease effect of archaeal dna polymerase, and discharge the fluorescent signal that bridge-type fluorescent probe marks.But when recognition sequence district and nucleic acid target molecule cannot form Complementary hybridization double-strand, recognition sequence district and bridge-type sequence area can keep completeness, and bridge-type fluorescent probe does not discharge fluorescence.Because the Tm value in recognition sequence district is extremely low, when itself and nucleic acid target molecule only exist any one single base mismatch, recognition sequence district all can not form Complementary hybridization double-strand with nucleic acid target molecule, thus makes bridge-type fluorescent probe discharge fluorescent signal.Visible, the special construction of bridge-type fluorescent probe, and the ability of recognition sequence district identification form nucleotide variation enables bridge-type fluorescent probe effectively identification form nucleotide variation with the mode of release fluorescent signal.

Detect in the application of single nucleotide variation at bridge-type fluorescent probe of the present invention, real time fluorescence quantifying PCR method real time nucleic acid detection target molecule can be adopted, with this understanding, when the bridge-type fluorescent probe of the different single nucleotide variation nucleic acid target molecule of target marked different types of fluorescent reporter group respectively, just by the nucleic acid target molecule of the different single nucleotide variation of the species detection of fluorescent signal in single reaction tubes, the qualitative detection of different nucleic acid target molecule can be realized.Meanwhile, because the intensity of fluorescent signal is relevant with the starting point concentration of nucleic acid target molecule, therefore, can according to the intensity of fluorescent signal, or cycle threshold (cyclethreshold, the CT of amplification system; The cycle number that the fluorescent signal referring in each reaction tubes experiences when reaching the threshold value of setting) judge the starting point concentration of different nucleic acid target molecule to realize the detection by quantitative of different nucleic acid target molecule.

In the present invention, when utilizing bridge-type fluorescent probe to detect single nucleotide variation, when the single nucleotide variation detecting same nucleic acid target molecule divides the period of the day from 11 p.m. to 1 a.m, when detecting the different genotype of same nucleic acid target molecule in other words conj.or perhaps, target often plants genotypic bridge-type fluorescent probe can the same chain (that is: target normal chain or minus strand) of equal target nucleic acid target molecules simultaneously, now, bridge-type fluorescent probe is called as competitive bridge-type fluorescent probe (Fig. 3 and Fig. 4) of homonymy, or target often plants the different chains (that is: difference target normal chain and minus strand) that genotypic bridge-type fluorescent probe can distinguish target nucleic acid target molecule, now, bridge-type fluorescent probe is called as offside noncompetitive bridge-type fluorescent probe (Fig. 5).Under these conditions, bridge-type fluorescent probe corresponding to nucleic acid target molecule different genotype is marked with different fluorescent reporter gene respectively, therefore, in same reaction tubes, the quantitative and qualitative analysis detection of same nucleic acid target molecule different genotype can be realized by the kind of fluorescent signal and intensity (or: CT value).

Present invention also offers the application of described bridge-type fluorescent probe in the reagent of preparation detection high homology nucleic acid target molecule, such as, in various infectious pathogen kind or hypotype, and the application in the detection of its drug resistant gene.By recognition sequence district for different genera or hypotype, and the specific nucleic acid sequence of its drug resistant gene, detect pathogenic agent kind or hypotype and drug resistant gene, its Cleaning Principle is identical with detecting single nucleotide variation.With this understanding, when only having the specific nucleic acid sequence complete complementary when recognition sequence district and nucleic acid target molecule, bridge-type fluorescent probe just discharges fluorescent signal.When the bridge-type fluorescent probe of different genera or hypotype or different drug resistant gene marks upper different sorts fluorescence respectively, in same reaction tubes, just can be realized the quantitative and qualitative analysis detection of above-mentioned nucleic acid target molecule by the kind of fluorescent signal and intensity (or: CT value).

The bridge-type fluorescent probe that the present invention also provides described bridge-type sequence area to mix base mismatch is preparing the application in digital pcr detection probes, be specially and adopt micro-fluidic or droplet method, be dispersed to by reaction mixture and comprise in several microreactors of droplet or micro-reacting hole, often kind of target molecule number in each microreactor is less than or equals 1.

Present invention also offers the test kit of the bridge-type fluorescent probe mixing base mismatch containing described bridge-type sequence area, can also containing the common agents of real-time fluorescence quantitative PCR in test kit, as the pair of primers in target sequence district, bridge-type fluorescent probe land as described in increasing, pcr amplification need reacted constituent (including, but are not limited to: there is the hot resistant DNA polymerase of 5' → 3' exonuclease activity, dNTP, suitable ion component and buffer system), nucleic acid target molecule is placed in same reaction tubes.

During detection by bridge-type fluorescent probe, primer, pcr amplification are needed reacted constituent (including, but are not limited to: have the hot resistant DNA polymerase of 5' → 3' exonuclease activity, dNTP, suitable ion component and buffer system), nucleic acid target molecule is placed in same reaction tubes; Adopt real-time fluorescence quantitative PCR, through multiple cyclic amplification nucleic acid target molecules such as the annealing of high-temperature denatured, thermophilic and extension, and circulate or circulation end point determination fluorescent signal kind and intensity thereof (or: CT value) at each; The quantitative and qualitative analysis realizing nucleic acid target molecule according to the kind of fluorescent signal, intensity (or: CT value) detects.

Therefore mentioned reagent box can also adopt real time fluorescence quantifying PCR method to be used for detecting single nucleotide variation or high homology nucleic acid target molecule.

Beneficial effect of the present invention is: the present invention mixes open bridge-type sequence area the bridge-type fluorescent probe of base mismatch, simplicity of design, can high specific identification form nucleotide variation by mixing base mismatch in bridge-type district, and detection sensitivity is high, 1% or more highly sensitive can be reached, not only can avoid common single nucleotide variation (as: SNP, transgenation) detect in false positive, and be applicable to low abundance detection in Gene Mutation (as: oncogene mutation in tumor tissues or peripheral blood), also may be used for the higher infectious pathogen kind of homology or hypotype and drug resistant gene thereof to identify.Therefore, the present invention has widened the range of application that fluorescent quantitation detects, significant for medical diagnosis on disease especially.

Accompanying drawing explanation

In order to make object of the present invention, technical scheme and beneficial effect clearly, the invention provides following accompanying drawing:

Fig. 1 is the typical structure exemplary plot of bridge-type fluorescent probe.

Fig. 2 is the bridge-type fluorescent probe topology example figure of bridge-type sequence area mark function group.

Fig. 3 is that homonymy competitive bridge-type fluorescent probe single tube detects single nucleotide variation principle schematic.

Fig. 4 is the bridge-type fluorescent probe identification genotype principle exemplary plot of bridge-type sequence area mark function group.

Fig. 5 is the relative position (A: relative position 1 of offside noncompetitive bridge-type fluorescent probe and primer and target molecule; B: relative position 2).

Fig. 6 is the nucleotide sequence at BRAF gene the 600th bit codon place.

Fig. 7 is that homonymy competitive bridge-type fluorescent probe single tube detects the detection system specificity of BRAF gene V600E transgenation, linearity range and amplification efficiency (A: wild-type Quality Control plasmid amplification curve; B: saltant type Quality Control plasmid amplification curve; C: wild-type Quality Control plasmid control curve; D: saltant type Quality Control plasmid amplification curve).

Fig. 8 is abrupt climatic change sensitivity (A-C:1 × 10 that homonymy competitive bridge-type fluorescent probe single tube detects BRAF gene V600E transgenation ⁶copy number suddenlys change 0%, 50%, 1% respectively; D-F:1 × 10 ⁴copy number suddenlys change 0%, 50%, 1% respectively).

Fig. 9 is KRAS the 12nd and the 13rd bit codon place nucleotide sequence.

Figure 10 is digitizing PCR detected result.

Figure 11 is ruminating animal gene order comparison result.

Embodiment

Below in conjunction with accompanying drawing, the preferred embodiments of the present invention are described in detail.The experimental technique of unreceipted actual conditions in embodiment, usually conveniently condition, the such as condition described in Molecular Cloning: A Laboratory guide (third edition, the work such as J. Pehanorm Brooker), or according to the condition that manufacturer advises.

Embodiment 1, homonymy competitive bridge-type fluorescent probe single tube detects BRAF gene V600E transgenation

1, the design one of the competitive probe of the homonymy of target BRAF gene V600E transgenation and amplimer thereof

Design competitive bridge-type fluorescent probe (table 1) of homonymy according to the nucleic acid sequence identity (Fig. 6) at BRAF gene V600E place, comprise the wild-type bridge-type fluorescent probe one (SW-01) of target BRAF gene wild-type V600E and the saltant type bridge-type fluorescent probe one (SW-03) of target BRAF gene mutation type V600E.Structure and the sequence signature of above-mentioned bridge-type fluorescent probe are as follows: all positive-sense strands of target BRAF gene; The mutating alkali yl identified is arranged in the mid-way (corresponding sequence is the base that table 1 adds thick underline and indicates) in recognition sequence district, 3'-is end modified phosphate group (that is: extending blocking group); Wild-type (SW-01) and the separate target nucleic acid subsequence corresponding to saltant type (SW-03) bridge-type fluorescent probe are SW-02 and SW-04 respectively, and it is corresponding with the Poly (I) of bridge-type fluorescent probe that the two overstriking adds oblique base; The 5'-end in the recognition sequence district of wild-type (SW-01) and saltant type (SW-03) bridge-type fluorescent probe marks VIC and FAM fluorescent reporter group respectively, 3'-end T base place mark BHQ1 fluorescent quenching group; The position of probe sequence is positioned at the shade sequence area (Fig. 6) of BRAF gene sequence.

According to wild-type (SW-01) and saltant type (SW-03) bridge-type fluorescent probe in BRAF gene sequence position, the pcr amplification forward primer SW-10 of its region of design amplification and reverse primer SW-12, its position is positioned at the straight underlined sequences district (Fig. 6) of BRAF gene sequence.

2, the design two of the competitive probe of the homonymy of target BRAF gene V600E transgenation and amplimer thereof

Design competitive bridge-type fluorescent probe (table 1) of homonymy according to the nucleic acid sequence identity (Fig. 6) at BRAF gene V600E place, comprise the wild-type bridge-type fluorescent probe two (SW-05) of target BRAF gene wild-type V600E and the saltant type bridge-type fluorescent probe two (SW-07) of target BRAF gene mutation type V600E.Structure and the sequence signature of above-mentioned bridge-type fluorescent probe are as follows: all antisense strands of target BRAF gene; The transgenation base identified is arranged in the mid-way (corresponding sequence is table 1 italic underscore base) in recognition sequence district, 3'-is end modified phosphate group (that is: extending blocking group); Wild-type (SW-05) and the separate target nucleic acid subsequence corresponding to saltant type (SW-07) bridge-type fluorescent probe are SW-06 and SW-08 respectively, and it is corresponding with the Poly (I) of bridge-type fluorescent probe that the two overstriking adds oblique base; The 5'-end in the recognition sequence district of wild-type (SW-05) and saltant type (SW-07) bridge-type fluorescent probe marks VIC and FAM fluorescent reporter group respectively, the T base place mark BHQ1 fluorescent quenching group in the middle of sequence; The position of probe sequence is positioned at the italic overstriking sequence area (Fig. 6) of BRAF gene sequence.

According to wild-type (SW-05) and saltant type (SW-07) bridge-type fluorescent probe in BRAF gene sequence position, the pcr amplification forward primer SW-09 of its region of design amplification and reverse primer SW-11, its position is positioned at the wave underline sequence area (Fig. 6) of BRAF gene sequence.

3, the design three of the competitive probe of the homonymy of target BRAF gene V600E transgenation and amplimer thereof

Design the bridge-type fluorescent probe of BRAF gene V600E positive-sense strand according to the nucleic acid sequence identity (Fig. 6) at BRAF gene V600E place, comprise wild-type bridge-type fluorescent probe three (SW-13), saltant type bridge-type fluorescent probe three (SW-14).The structure of above-mentioned bridge-type fluorescent probe is as follows with sequence signature: the positive-sense strand (corresponding sequence is the sequence adding thick underline sign in table 1) of wild-type bridge-type fluorescent probe three (SW-13) and the equal target BRAF gene of saltant type bridge-type fluorescent probe three (SW-14); The transgenation base identified is positioned at the mid-way in recognition sequence district, 3'-is end modified phosphate group (that is: extending blocking group); The wild-type (SW-13) of target positive-sense strand and the separate target nucleic acid subsequence corresponding to saltant type (SW-14) bridge-type fluorescent probe three are SW-02 and SW-04 respectively, and it is corresponding with the bridge-type sequence area of bridge-type fluorescent probe that the two overstriking adds oblique base; The 5'-end in the recognition sequence district of wild-type (SW-13) and saltant type (SW-14) bridge-type fluorescent probe marks VIC and FAM fluorescent reporter group respectively, bridge-type sequence area is mixed with the T base with the mispairing of nucleic acid target molecule complementary strand in its Poly (I), wherein, wild-type bridge-type fluorescent probe (SW-13) is CT mispairing, saltant type bridge-type fluorescent probe (SW-14) is TT mispairing, further, all at the base T place mark fluorescent quenching group BHQ1 that this mixes.

According to above-mentioned bridge-type fluorescent probe in BRAF gene sequence position, the pcr amplification forward primer SW-10 of its region of design amplification and reverse primer SW-12.

Table 1, bridge-type fluorescent probe and amplimer thereof

4, the structure of real-time fluorescence quantitative PCR reaction system one

PCR reaction system amounts to 20 μ l, and this system comprises following component: 1 × PremixExTaqmastermix (PerfectRealTime; TaKaRa company), 0.9 μM of forward primer SW-10,0.9 μM of reverse primer SW-12,0.25 μM of wild-type bridge-type fluorescent probe one (SW-01), 0.25 μM of saltant type bridge-type fluorescent probe one (SW-03), serial final concentration (1 × 10 ⁸to1 × 10 ¹copy number) wild-type Quality Control (wild-typequalitycontrol, WT-QC plasmid) and saltant type Quality Control plasmid (mutationqualitycontrol, MT-QC plasmid), or 100ng colorectal cancer BRAFV600E wild-type cell strain SW480 genomic dna and 100ng colorectal cancer BRAFV600E mutant cell strain A375 genomic dna, or the tumor tissues DNA of 10 ~ 100ng Colon and rectum patient.Real-time fluorescence PCR reaction conditions is: 95 DEG C of denaturations 30 seconds; 95 DEG C of sex change 30 seconds, 60 DEG C of renaturation with extend 1 minute (and simultaneously gathering fluorescence), amount to 40 circulations.Equipment used is CFX96 real-time fluorescence quantitative PCR instrument (Bole company, the U.S.), and fluorescence signal acquisition and analysis software are CFX96 software kit.

5, the structure of real-time fluorescence quantitative PCR reaction system two

PCR reaction system amounts to 20 μ l, and this system comprises following component: 1 × PremixExTaqmastermix (PerfectRealTime; TaKaRa company), 0.9 μM of forward primer SW-09,0.9 μM of reverse primer SW-11,0.25 μM of wild-type bridge-type fluorescent probe two (SW-05), 0.25 μM of saltant type bridge-type fluorescent probe two (SW-07), serial final concentration (1 × 10 ⁸to1 × 10 ¹copy number) wild-type Quality Control (wild-typequalitycontrol, WT-QC plasmid) and saltant type Quality Control plasmid (mutationqualitycontrol, MT-QC plasmid), or 100ng colorectal cancer BRAFV600E wild-type cell strain SW480 genomic dna and 100ng colorectal cancer BRAFV600E mutant cell strain A375 genomic dna, or the tumor tissues DNA of 10 ~ 100ng Colon and rectum patient.Real-time fluorescence PCR reaction conditions is: 95 DEG C of denaturations 30 seconds; 95 DEG C of sex change 30 seconds, 60 DEG C of renaturation with extend 1 minute (and simultaneously gathering fluorescence), amount to 40 circulations.Equipment used is CFX96 real-time fluorescence quantitative PCR instrument (Bole company, the U.S.), and fluorescence signal acquisition and analysis software are CFX96 software kit.

6, the structure of real-time fluorescence quantitative PCR reaction system three

PCR reaction system amounts to 20 μ l, and this system comprises following component: 1 × PremixExTaqmastermix (PerfectRealTime; TaKaRa company), 0.9 μM of forward primer SW-10,0.9 μM of reverse primer SW-12,0.25 μM of wild-type bridge-type fluorescent probe three (SW-13), 0.25 μM of saltant type bridge-type fluorescent probe three (SW-14), serial final concentration (1 × 10 ⁸to1 × 10 ¹copy number) wild-type Quality Control (wild-typequalitycontrol, WT-QC plasmid) and saltant type Quality Control plasmid (mutationqualitycontrol, MT-QC plasmid), or 100ng colorectal cancer BRAFV600E wild-type cell strain SW480 genomic dna and 100ng colorectal cancer BRAFV600E mutant cell strain A375 genomic dna, or the tumor tissues DNA of 10 ~ 100ng Colon and rectum patient.Real-time fluorescence PCR reaction conditions is: 95 DEG C of denaturations 30 seconds; 95 DEG C of sex change 30 seconds, 60 DEG C of renaturation with extend 1 minute (and simultaneously gathering fluorescence), amount to 40 circulations.Equipment used is CFX96 real-time fluorescence quantitative PCR instrument (Bole company, the U.S.), and fluorescence signal acquisition and analysis software are CFX96 software kit.

7, results and analysis

Probe design situation analysis: the bridge-type fluorescent probe designed by step 1 and 2 is one of the typical structure of bridge-type fluorescent probe of the present invention (Fig. 1), bridge-type fluorescent probe designed by step 3 is the another kind of typical structure (Fig. 2) of bridge-type fluorescent probe of the present invention, its fluorescent quenching group lays respectively at recognition sequence district (Fig. 1) and bridge-type sequence area (Fig. 2), and the quenching group of bridge-type sequence area is marked on base mismatch T.Bridge-type fluorescent probe in reaction system three described in reaction system two described in reaction system one, step 5 described in step 4 and step 6 and the relative position of primer and nucleic acid target molecule are as shown in Figure 3, three's essential difference is the positive-sense strand of wild-type in reaction system described in step 3 and 6 and the equal target nucleic acid target molecule of saltant type probe, and the antisense strand of wild-type in reaction system described in step 5 and the equal target nucleic acid target molecule of saltant type probe, all belong to homonymy competitive bridge-type fluorescent probe classification.

Cleaning Principle: in same reaction tubes, add the bridge-type fluorescent probe of target BRAF gene V600E wild-type (as: SW-01) and saltant type (as: SW-03), wild-type and saltant type probe mark VIC and FAM fluorescent reporter group respectively simultaneously.When only there is wild-type template in reaction system, wild-type probe (as: SW-01; P1 probe in Fig. 3) recognition sequence district can form Complementary hybridization double-strand with wild-type template, and under the effect of primer extension and archaeal dna polymerase, be hydrolyzed into single core thuja acid, release fluorescent signal, and saltant type probe (as: SW-03; P2 in Fig. 3) recognition sequence district and wild-type template can not form Complementary hybridization double-strand, under the acting in conjunction of primer and archaeal dna polymerase, recognition sequence district and bridge-type sequence area are hydrolyzed into a complete fragment, do not discharge fluorescent signal.Similar to the above case, when only there is saltant type template in reaction system, only have saltant type probe (as: SW-03; P2 in Fig. 3) discharge fluorescent signal, wild-type probe (as: SW-01; P1 probe in Fig. 3) do not discharge fluorescent signal.When there is wild-type and mutant nucleic acid target molecule in reaction system simultaneously, wild-type probe (as: SW-01; P1 probe in Fig. 3) and saltant type probe (as: SW-03; P2 in Fig. 3) all can discharge fluorescent signal.Because wild-type is different with the fluorescent reporter group of saltant type probe mark, therefore, can according to the genotype of the kind qualitative analysis nucleic acid target molecule of fluorescent signal, simultaneously, the CT value obtained due to intensity or the real-time fluorescence quantitative PCR of fluorescent signal and the initial amount of nucleic acid target molecule linear, therefore, can according to the different genotype of the intensity of different fluorescent signal or CT value detection by quantitative nucleic acid target molecule.Visible, and the fluorescent signal kind that can discharge in amplification process according to the bridge-type fluorescent probe of different genotype or intensity (or: CT value) realize the genotypic qualitative and quantitative analysis of nucleic acid target molecule to be detected respectively.In addition, relative to recognition sequence district mark quenching group (as: SW-01, SW-03) recognition sequence district (Fig. 1 compared with the hybridization efficiency of target molecule can likely be affected, Fig. 3), bridge-type sequence area is by (as: SW-13 after base mismatch introducing quenching group, SW-14), do not affect hybridization efficiency (Fig. 2 of recognition sequence district and target molecule, Fig. 4), therefore, its detection sensitivity can higher than the former, and main manifestations is when there is the target molecule of same concentrations in reaction system, the CT value of the fluorescent probe of bridge-type shown in Fig. 2 can lower than the fluorescent probe of bridge-type shown in Fig. 1, and, the detection in Gene Mutation sensitivity of the fluorescent probe of bridge-type shown in Fig. 2 can higher than the fluorescent probe of bridge-type shown in Fig. 1.

The detected result of reaction system one described in step 4 and analysis

Specificity and linearity range: when only there is serial final concentration (1 × 10 in reaction system ⁸to1 × 10 ¹copy number) wild-type Quality Control plasmid and saltant type Quality Control plasmid condition under, all only there is the fluorescent signal that its genotype is corresponding, without any non-specific amplification, but, no matter be wild-type or mutant plasmids, all can only lowest detection to 1 × 10 ⁴copy number.In addition, when reaction system target molecule used is SW480 or A375 cell strain genomic dna, SW480 cell strain used is BRAF gene V600E wild-type, and A375 cell strain is BRAF gene V600E saltant type, and target molecule usage quantity is 100ng, be equivalent to have 3 × 10 in reaction system ⁴the BRAF gene of copy number, can meet lowest detection line that this reaction system adopts Quality Control plasmid to obtain (that is: 1 × 10 ⁴copy number).Detected result shows, when only having cell line SW480 genomic dna in reaction system, only has wild-type bridge-type fluorescent probe one (SW-01) to discharge fluorescent signal in reaction system one described in step 4; When only having cell strain A375 genomic dna in reaction system, saltant type bridge-type fluorescent probe one (SW-03) in reaction system one described in step 4, is only had to discharge fluorescent signal; When there is cell line SW480 and A375 genomic dna in reaction system simultaneously, the wild-type in reaction system described in step 4 and saltant type bridge-type fluorescent probe all discharge fluorescent signal.

Clinical verification: adopt described reaction system to have detected the mutation rate of 100 routine colorectal cancer patients tumor tissues BRAF gene V600E altogether, detect that 4 routine patients there occurs V600E sudden change altogether, this mutation rate (4%, 4/100), lower than the BRAF gene V600E mutation rate of bibliographical information, its reason may be relevant with linearity range with the lower detection sensitivity of this system probe used.

The detected result of reaction system two described in step 5 and analysis

Specificity and linearity range: when only there is serial final concentration (1 × 10 in reaction system ⁸to1 × 10 ¹copy number) wild-type Quality Control plasmid and saltant type Quality Control plasmid condition under, all only there is the fluorescent signal that its genotype is corresponding, without any non-specific amplification, but, no matter be wild-type or mutant plasmids, all can only lowest detection to 1 × 10 ⁴copy number.When reaction system target molecule used is SW480 or A375 cell strain genomic dna, and target molecule usage quantity meets lowest detection line (that is: 100ng) needed for reaction system, when only having cell line SW480 genomic dna in reaction system, wild-type bridge-type fluorescent probe two (SW-05) in reaction system one described in step 5, is only had to discharge fluorescent signal; When only having cell strain A375 genomic dna in reaction system, saltant type bridge-type fluorescent probe two (SW-07) in reaction system two described in step 5, is only had to discharge fluorescent signal; When there is cell line SW480 and A375 genomic dna in reaction system simultaneously, the wild-type in reaction system described in step 5 and saltant type bridge-type fluorescent probe all discharge fluorescent signal.

The detected result of reaction system three described in step 6 and analysis

Be marked on recognition sequence district with step 4 with the quenching group of the bridge-type fluorescent probe in reaction system described in step 5 to compare, the quenching group of the bridge-type fluorescent probe that reaction system described in step 6 uses is marked on bridge-type sequence area, the quenching group being marked on this district's base mismatch does not affect the hybridization efficiency of recognition sequence district and target molecule, simultaneously, the bridge architecture of blister can be formed better between target molecule and the bridge-type sequence area of bridge-type fluorescent probe, do not affecting on the basis of hybrid specificities, significantly improve hybridization efficiency, thus, relative to reaction system described in step 4 and step 5, significantly improve the detection sensitivity of bridge-type fluorescent probe, make it have wider linear detection range, in the actual detection application of clinical samples, there is more obvious practical application advantage.

Specificity and linearity range: when only there is serial final concentration (1 × 10 in reaction system ⁸to1 × 10 ¹copy number) wild-type Quality Control plasmid (in Fig. 7 A) and saltant type Quality Control plasmid (in Fig. 7 B) condition under, all only there is the fluorescent signal that its genotype is corresponding, without any non-specific amplification.The above results demonstrates the present invention and completely eliminates the false positive results that the non-specific hybridization existing for existing similar technique causes.Simultaneously, the detected result of above-mentioned serial final concentration Quality Control plasmid shows, the present invention has good linearity range, at least can reach the linearity range of 8 orders of magnitude, and, in this linearity range, the amplification efficiency (in Fig. 7 C) of wild-type and mutant-type genotype is 96.59% (R respectively ²=0.999) and 99.11% (R ²=0.998) amplification efficiency (in Fig. 7 D) of the wild-type, in heterozygous template and mutant-type genotype is 95.07% (R respectively ²=0.998) and 98.27% (R ²=0.999).This result further illustrates wild-type and saltant type has equivalence amplification in the methods of the invention.When employing SW480 and A375 cell strain genomic dna in reaction system, this reaction system all can identify the genotype of cell strain special, delicately.

Sensitivity: when wild-type Quality Control plasmid (A-C:1 × 10 in Fig. 8 of certain concentration ⁶copy number; D-F:1 × 10 in Fig. 8 ⁴copy number) and series concentration (1 × 10 ⁶copy number ~ 1 × 10 ¹copy number) saltant type Quality Control plasmid when jointly existing, the detection sensitivity of the method for the invention to saltant type reaches 1%.

Clinical verification: adopt described reaction system to have detected the mutation rate of 100 routine colorectal cancer patients tumor tissues BRAF gene V600E altogether; detect that 12 routine patients there occurs V600E sudden change altogether; this mutation rate (12%, 12/100), consistent with bibliographical information; and; all detected results are all through nucleic acid sequencing validation, and the two result is completely the same, and; in testing process, the Quality Control plasmid gene type result of Parallel testing is entirely true, illustrates that detected result accurately and reliably.

Visible, the specificity of reacting described in the detection specificity of reaction system described in step 6 and step 4 and step 5 is suitable, but the abrupt climatic change of its detection sensitivity, linearity range and clinical samples goes out rate all higher than reaction system described in step 4 and step 5, absolutely prove that bridge-type sequence area can obviously promote the methodology parameters such as detection sensitivity of the present invention and linearity range after introducing base mismatch, there is more obvious clinical application advantage.

Embodiment 2, offside noncompetitive bridge-type fluorescent probe single tube detect BRAF gene V600E transgenation

1, the offside noncompetitive probe of target BRAF gene V600E transgenation and the design one of amplimer thereof

Nucleic acid sequence identity (Fig. 6) according to BRAF gene V600E place designs offside noncompetitive bridge-type fluorescent probe (table 1), comprises the wild-type bridge-type fluorescent probe one (SW-01) of target BRAF gene wild-type V600E and the saltant type bridge-type fluorescent probe two (SW-07) of target BRAF gene mutation type V600E.Structure and the sequence signature of above-mentioned bridge-type fluorescent probe are as follows: the positive-sense strand of difference target BRAF gene and antisense strand; The mutating alkali yl identified is positioned at the mid-way (corresponding sequence is table 1 overstriking underline position) in recognition sequence district, 3'-is end modified phosphate group (that is: extending blocking group); Wild-type (SW-01) and the separate target nucleic acid subsequence corresponding to saltant type (SW-07) bridge-type fluorescent probe are SW-02 and SW-08 respectively, and it is corresponding with the Poly (I) of bridge-type fluorescent probe that the two overstriking adds oblique base; The 5'-end in the recognition sequence district of wild-type (SW-01) bridge-type fluorescent probe marks VIC fluorescent reporter group respectively, 3'-end T base place mark BHQ1 fluorescent quenching group; The 5'-end mark FAM fluorescent reporter group in the recognition sequence district of saltant type (SW-07) bridge-type fluorescent probe, 3'-end T base place mark BHQ1 fluorescent quenching group; The position of probe sequence is positioned at the shade sequence area (Fig. 6) of BRAF gene sequence.

According to above-mentioned bridge-type fluorescent probe in BRAF gene sequence position, the pcr amplification forward primer SW-09 of its region of design amplification and reverse primer SW-12, its position is positioned at wave underline and the straight underlined sequences district (Fig. 6) of BRAF gene sequence.

2, the offside noncompetitive probe of target BRAF gene V600E transgenation and the design two of amplimer thereof

Nucleic acid sequence identity (Fig. 6) according to BRAF gene V600E place designs offside noncompetitive bridge-type fluorescent probe (table 1), comprises the wild-type bridge-type fluorescent probe two (SW-05) of target BRAF gene wild-type V600E and the saltant type bridge-type fluorescent probe one (SW-03) of target BRAF gene mutation type V600E.Structure and the sequence signature of above-mentioned bridge-type fluorescent probe are as follows: the antisense strand of difference target BRAF gene and positive-sense strand; The transgenation base identified is arranged in the mid-way (corresponding sequence is that table 1 adds thick underline base) in recognition sequence district, 3'-is end modified phosphate group (that is: extending blocking group); Wild-type (SW-05) and the separate target nucleic acid subsequence corresponding to saltant type (SW-03) bridge-type fluorescent probe are SW-06 and SW-04 respectively, and it is corresponding with the Poly (I) of bridge-type fluorescent probe that the two overstriking adds oblique base; The 5'-end flag F AM fluorescent reporter group respectively in the recognition sequence district of wild-type (SW-05) and bridge-type fluorescent probe, 3'-end T base place mark BHQ1 fluorescent quenching group; The 5'-end in the recognition sequence district of saltant type (SW-03) bridge-type fluorescent probe marks VIC fluorescent reporter group respectively, the T base place mark BHQ1 fluorescent quenching group in the middle of sequence; The position of probe sequence is positioned at the italic overstriking sequence area (Fig. 6) of BRAF gene sequence.

3, the offside noncompetitive probe of target BRAF gene V600E transgenation and the design three of amplimer thereof

Nucleic acid sequence identity (Fig. 6) according to BRAF gene V600E place designs offside noncompetitive bridge-type fluorescent probe (table 1), comprises wild-type bridge-type fluorescent probe three (SW-13) and saltant type bridge-type fluorescent probe four (SW-15).The structure of above-mentioned bridge-type fluorescent probe is as follows with sequence signature: the positive-sense strand (corresponding sequence is the sequence adding thick underline sign in table 1) of wild-type bridge-type fluorescent probe three (SW-13) target BRAF gene, the antisense strand (in corresponding sequence table 1 italic overstriking base) of saltant type bridge-type fluorescent probe four (SW-15) target BRAF gene; The transgenation base identified is positioned at the mid-way in recognition sequence district, 3'-is end modified phosphate group (that is: extending blocking group); The separate target nucleic acid subsequence corresponding to wild-type (SW-13) bridge-type fluorescent probe three of target positive-sense strand is SW-02, the separate target nucleic acid subsequence corresponding to saltant type bridge-type fluorescent probe four (SW-15) of target antisense strand is SW-08, and it is corresponding with the bridge-type sequence area of bridge-type fluorescent probe that the two overstriking adds oblique base; The 5'-end in the recognition sequence district of wild-type (SW-13) and saltant type (SW-15) bridge-type fluorescent probe marks VIC and FAM fluorescent reporter group respectively, bridge-type sequence area is mixed with the T base with the mispairing of nucleic acid target molecule complementary strand in its Poly (I), wherein, wild-type bridge-type fluorescent probe (SW-13 and SW-15) is CT mispairing, saltant type bridge-type fluorescent probe (SW-14) is TT mispairing, further, all at the base T place mark fluorescent quenching group BHQ1 that this mixes.

4, the structure of real-time fluorescence quantitative PCR reaction system one

PCR reaction system amounts to 20 μ l, and this system comprises following component: 1 × PremixExTaqmastermix (PerfectRealTime; TaKaRa company), 0.9 μM of forward primer SW-09,0.9 μM of reverse primer SW-12,0.25 μM of wild-type bridge-type fluorescent probe one (SW-01), 0.25 μM of saltant type bridge-type fluorescent probe two (SW-07), serial final concentration (1 × 10 ⁸to1 × 10 ¹copy number) wild-type Quality Control (wild-typequalitycontrol, WT-QC plasmid) and saltant type Quality Control plasmid (mutationqualitycontrol, MT-QC plasmid), or 100ng colorectal cancer BRAFV600E wild-type cell strain SW480 genomic dna and 100ng colorectal cancer BRAFV600E mutant cell strain A375 genomic dna, or the tumor tissues DNA of 10 ~ 100ng Colon and rectum patient.Real-time fluorescence PCR reaction conditions is: 95 DEG C of denaturations 30 seconds; 95 DEG C of sex change 30 seconds, 60 DEG C of renaturation with extend 1 minute (and simultaneously gathering fluorescence), amount to 40 circulations.Equipment used is CFX96 real-time fluorescence quantitative PCR instrument (Bole company, the U.S.), and fluorescence signal acquisition and analysis software are CFX96 software kit.

5, the structure of real-time fluorescence quantitative PCR reaction system two

PCR reaction system amounts to 20 μ l, and this system comprises following component: 1 × PremixExTaqmastermix (PerfectRealTime; TaKaRa company), 0.9 μM of forward primer SW-09,0.9 μM of reverse primer SW-12,0.25 μM of saltant type bridge-type fluorescent probe one (SW-03), 0.25 μM of wild-type bridge-type fluorescent probe two (SW-05), serial final concentration (1 × 10 ⁸to1 × 10 ¹copy number) wild-type Quality Control (wild-typequalitycontrol, WT-QC plasmid) and saltant type Quality Control plasmid (mutationqualitycontrol, MT-QC plasmid), or 100ng colorectal cancer BRAFV600E wild-type cell strain SW480 genomic dna and 100ng colorectal cancer BRAFV600E mutant cell strain A375 genomic dna, or the tumor tissues DNA of 10 ~ 100ng Colon and rectum patient.Real-time fluorescence PCR reaction conditions is: 95 DEG C of denaturations 30 seconds; 95 DEG C of sex change 30 seconds, 60 DEG C of renaturation with extend 1 minute (and simultaneously gathering fluorescence), amount to 40 circulations.Equipment used is CFX96 real-time fluorescence quantitative PCR instrument (Bole company, the U.S.), and fluorescence signal acquisition and analysis software are CFX96 software kit.

PCR reaction system amounts to 20 μ l, and this system comprises following component: 1 × PremixExTaqmastermix (PerfectRealTime; TaKaRa company), 0.9 μM of forward primer SW-09,0.9 μM of reverse primer SW-12,0.25 μM of saltant type bridge-type fluorescent probe one (SW-15), 0.25 μM of wild-type bridge-type fluorescent probe two (SW-13), serial final concentration (1 × 10 ⁸to1 × 10 ¹copy number) wild-type Quality Control (wild-typequalitycontrol, WT-QC plasmid) and saltant type Quality Control plasmid (mutationqualitycontrol, MT-QC plasmid), or 100ng colorectal cancer BRAFV600E wild-type cell strain SW480 genomic dna and 100ng colorectal cancer BRAFV600E mutant cell strain A375 genomic dna, or the tumor tissues DNA of 10 ~ 100ng Colon and rectum patient.Real-time fluorescence PCR reaction conditions is: 95 DEG C of denaturations 30 seconds; 95 DEG C of sex change 30 seconds, 60 DEG C of renaturation with extend 1 minute (and simultaneously gathering fluorescence), amount to 40 circulations.Equipment used is CFX96 real-time fluorescence quantitative PCR instrument (Bole company, the U.S.), and fluorescence signal acquisition and analysis software are CFX96 software kit.

7, results and analysis

Probe design situation analysis: the bridge-type fluorescent probe designed by step 1 and 2 is one of the typical structure of bridge-type fluorescent probe of the present invention (Fig. 1), bridge-type fluorescent probe designed by step 3 is the another kind of typical structure (Fig. 2) of bridge-type fluorescent probe of the present invention, its fluorescent quenching group lays respectively at recognition sequence district (Fig. 1) and bridge-type sequence area (Fig. 2), and the quenching group of bridge-type sequence area is marked on base mismatch T.Bridge-type fluorescent probe in reaction system one described in step 4 and primer and nucleic acid target molecule, and the relative position of bridge-type fluorescent probe in reaction system two described in step 5 and primer and nucleic acid target molecule is respectively as shown in A in Fig. 5, the two essential difference is wild-type in reaction system one described in step 4 and the saltant type probe positive-sense strand of target nucleic acid target molecule and antisense strand respectively, and wild-type in reaction system two described in step 5 and the saltant type probe antisense strand of target nucleic acid target molecule and positive-sense strand respectively.Bridge-type fluorescent probe in reaction system three described in step 6 and the relative position of primer and nucleic acid target molecule are as shown in B in Fig. 5.Bridge-type fluorescent probe described in step 1 to step 6 all belongs to offside noncompetitive bridge-type fluorescent probe.Bridge-type fluorescent probe in reaction system three described in step 6 derives from the design of step 3.Relative to recognition sequence district mark quenching group (as: step 1, step 2, step 4, step 5) likely can affect recognition sequence district (Fig. 1 compared with the hybridization efficiency of target molecule, A in Fig. 5), bridge-type sequence area is by (as: step 1 after base mismatch introducing quenching group, step 6), do not affect hybridization efficiency (Fig. 2 of recognition sequence district and target molecule, B in Fig. 5), therefore, its detection sensitivity can higher than the former, and main manifestations is when there is the target molecule of same concentrations in reaction system, in Fig. 5, the CT value of the fluorescent probe of bridge-type shown in B can lower than the fluorescent probe of bridge-type shown in A in Fig. 5, and, the detection in Gene Mutation sensitivity of bridge-type fluorescent probe shown in Fig. 5 B can higher than the fluorescent probe of bridge-type shown in B in Fig. 5.

Detected result: in reaction system described in step 4 to step 6, SW480 cell strain is BRAF gene V600E wild-type, A375 cell strain is BRAF gene V600E saltant type.Detected result shows, three kinds of reaction systems described in step 4 to step 6 all accurately can identify the genotype of 100ng cell line SW480 and A375, have consistent specificity.But for wild-type and the saltant type Quality Control plasmid of serial final concentration, step 4 and step 5 can only detect 1 × 10 ⁴the Quality Control plasmid of copy number, and step 6 can detect 1 × 10 ¹the Quality Control plasmid of copy number, the detection sensitivity of the latter is at least higher than the former 1000 times.The detected result of 100 routine clinical samples shows, reaction system described in step 4 to step 6 detects that the patient of 4% (4/100), 5% (5/100) and 12% (12/100) has BRAFV600E transgenation respectively.Detected result shows, the specificity of reacting described in the detection specificity of reaction system described in step 6 and step 4 and step 5 is suitable, but the abrupt climatic change of its detection sensitivity, linearity range and clinical samples goes out rate all higher than reaction system described in step 4 and step 5, absolutely prove that bridge-type sequence area can obviously promote the methodology parameters such as detection sensitivity of the present invention and linearity range after introducing base mismatch, there is more obvious clinical application advantage.

Embodiment 3, bridge-type fluorescent probe technique detect KRAS the 12nd bit codon transgenation

The missense mutation of KRAS gene the 12nd and the 13rd bit codon (Fig. 9) is the Several Kinds of Malignancy common mutations site comprising colorectal cancer, and its catastrophe is directly related with malignant tumour molecule parting and individualized treatment effect.

1, the design of the competitive probe of the homonymy of target KRAS gene the 12nd and amplimer thereof

Design the bridge-type fluorescent probe of target KRAS gene the 12nd bit codon antisense strand according to the nucleic acid sequence identity (in Fig. 9, shade base is the 12nd and the 13rd bit codon nucleotide sequence of KRAS) at KRAS gene the 12nd and the 13rd bit codon place, comprise wild-type bridge-type fluorescent probe (SW-16), c.34G>A saltant type bridge-type fluorescent probe (SW-17), c.34G>C saltant type bridge-type fluorescent probe (SW-18), c.34G>T saltant type bridge-type fluorescent probe (SW-19).Structure and the sequence signature of above-mentioned bridge-type fluorescent probe are as follows: bridge-type fluorescent probe and KRAS gene the 12nd bit codon place nucleotide sequence antisense strand (SW-20; The single underscore base of corresponding sequence chart 8) corresponding, Serotype-dependent base is arranged in 5'-end (Fig. 8 adds thick underline base), 3'-is end modified phosphate group (that is: extending blocking group), different genotype is FAM, VIC, TexasRed and CY5 fluorescent reporter gene at 5'-end mark respectively, corresponding fluorescent quenching base group modification in the bridge-type sequence area of 2 bases longs with the base T (table 2) of target molecule mispairing.

According to above-mentioned bridge-type fluorescent probe in KRAS gene order position, the pcr amplification forward primer SW-21 of its region of design amplification and reverse primer SW-22 (table 2; The nucleotide sequence of SW-21 and SW-22 corresponds to double underline base in Fig. 8).

Table 2, KRAS gene test probe and primer

2, the structure of real-time fluorescence quantitative PCR reaction system

PCR reaction system amounts to 20 μ l, and this system comprises following component: 1 × PremixExTaqmastermix (PerfectRealTime, TaKaRa company), 0.9 μM of forward primer SW-21, 0.9 μM of reverse primer SW-22, 0.25 μM of wild-type bridge-type fluorescent probe (SW-16), 0.25 μM of saltant type bridge-type fluorescent probe (comprising: SW-17, SW-18, SW-19), the each cell strain genomic dna of 50ng, comprise cell strain HT-29 genomic dna (KRAS gene the 12nd and the 13rd bit codon wild-type cell strain), cell line A549 genomic dna (KRAS gene the 12nd bit codon is (p.G12S) homozygous mutant cell strain c.34G>A), cell strain H157 genomic dna (KRAS gene the 12nd bit codon is (p.G12R) homozygous mutant cell strain c.34G>C), cell strain SW-1573 genomic dna (KRAS gene the 12nd bit codon is (p.G12C) homozygous mutant cell strain c.34G>T).Real-time fluorescence PCR reaction conditions is: 95 DEG C of denaturations 30 seconds; 95 DEG C of sex change 30 seconds, 60 DEG C of renaturation with extend 1 minute (and simultaneously gathering fluorescence), amount to 40 circulations.Equipment used is CFX96 real-time fluorescence quantitative PCR instrument (Bole company, the U.S.), and fluorescence signal acquisition and analysis software are CFX96 software kit.

4, results and analysis

Probe design situation analysis: the bridge-type fluorescent probe designed by step 1 is typical structure described in bridge-type fluorescent probe Fig. 2 of the present invention, bridge-type fluorescent probe and primer and nucleic acid target molecule relative position as shown in Figure 4, belong to homonymy competitive bridge-type fluorescent probe classification.

Detected result: detected result shows, when only having cell strain HT-29, A549, H157 and SW-1573 genomic dna in reaction system respectively, FAM, VIC, TexasRed and CY5 fluorescent signal of probe SW-16, SW-17, SW-18 and SW-19 release corresponding to its KRAS the 12nd bit codon genotype can only be detected respectively in reaction system, illustrate that often kind of probe all can detect the genotype of its correspondence specifically, there is not the false positive results that non-specific hybridization causes.Meanwhile, when there is the genomic dna of four kinds of cell strains such as HT-29, A549, H157 and SW-1573 in reaction system simultaneously, in reaction system, four kinds of fluorescent signals such as FAM, VIC, TexasRed and CY5 can be detected simultaneously.

Embodiment 4, digitizing PCR single tube detect KRAS and BRAF gene mutation

KRAS gene the 12nd and the 13rd bit codon and BRAFV600E transgenation are the Several Kinds of Malignancy common mutations sites comprising colorectal cancer, and its catastrophe is directly related with malignant tumour molecule parting and individualized treatment effect.The present embodiment adopts digitizing PCR method, by the fluorescent reporter gene different to target same genotypic bridge-type fluorescent probe mark difference two kinds, in PCR amplification system, use the bridge-type fluorescent probe of different abundance ratio, thus break through the limitation that traditional dichromatism fluorescence channel only can detect two kinds of target molecules.In this embodiment, utilize the existing digitizing PCR test set only only having dichromatism, the single tube realizing 6 kinds of target molecules detects, and greatly improves the detection efficiency based on bridge-type fluorescent probe.

1, the design of bridge-type fluorescent probe

According to KRAS the 12nd bit codon place nucleotide sequence (Fig. 9) and BRAFV600E place nucleotide sequence (Fig. 6), design the bridge-type fluorescent probe (table 3) of target KRAS the 12nd bit codon and BRAFV600E wild-type and mutant allele respectively according to the principle of embodiment 1 ~ 3, bridge-type fluorescent probe corresponding to genotype has following characteristics respectively:

Wild-type bridge-type fluorescent probe flag F AM fluorescent reporter group (SW-16) of KRAS the 12nd bit codon;

C.34G>A, bridge-type fluorescent probe mark VIC fluorescent reporter group (SW-17) that KRAS the 12nd bit codon suddenlys change;

C.34G>C, the bridge-type fluorescent probe that KRAS the 12nd bit codon suddenlys change is flag F AM and VIC fluorescent reporter group respectively, but the nucleotide sequence of the two identical (SW-23, SW-24);

C.34G>T, the bridge-type fluorescent probe that KRAS the 12nd bit codon suddenlys change is flag F AM and VIC fluorescent reporter group respectively, but the nucleotide sequence of the two identical (SW-25, SW-26);

Bridge-type fluorescent probe flag F AM and the VIC fluorescent reporter group respectively of BRAFV600E wild-type, but the nucleotide sequence of the two identical (SW-27, SW-13);

Bridge-type fluorescent probe flag F AM and the VIC fluorescent reporter group respectively of BRAFV600E saltant type, but the nucleotide sequence of the two identical (SW-14, SW-28).

Principle according to embodiment 1 ~ 3, according to above-mentioned bridge-type fluorescent probe in KRAS and BRAF gene sequence position, pcr amplification forward primer SW-21, SW-09 of its region of design amplification and reverse primer SW-22, SW-11.

Table 3, KRAS the 12nd bit codon and BRAFV600E wild-type and mutant allele bridge-type fluorescent probe and primer

2, the structure of digitizing PCR reaction system

PCR reaction system amounts to 50 μ l, and this system comprises following component: 1 × PremixExTaqmastermix (PerfectRealTime; TaKaRa company), 0.9 μM of forward primer SW-21 and SW-09,0.9 μM of reverse primer SW-22 and SW-11, the bridge-type fluorescent probe of specific final concentration and various cell strain genomic dna.Wherein, final concentration and the correlation proportion thereof of the bridge-type fluorescent probe corresponding to various genotype are as follows:

KRAS gene wild-type bridge-type fluorescent probe SW-16 (flag F AM fluorescent reporter group): 120nM;

KRAS gene c.34G>A saltant type bridge-type fluorescent probe SW-17 (mark VIC fluorescent reporter group): 120nM;

The bridge-type fluorescent probe SW-23 (flag F AM fluorescent reporter group) of KRAS gene c.34G>C saltant type: 120nM, SW-24 (mark VIC fluorescent reporter group): 120nM;

The bridge-type fluorescent probe SW-25 (flag F AM fluorescent reporter group) of KRAS gene c.34G>T saltant type: 60nM, SW-26 (mark VIC fluorescent reporter group): 60nM;

The bridge-type fluorescent probe SW-27 (flag F AM fluorescent reporter group) of BRAF gene V600E wild-type: 120nM, SW-13 (mark VIC fluorescent reporter group): 60nM;

The bridge-type fluorescent probe SW-14 (flag F AM fluorescent reporter group) of BRAF gene V600E saltant type: 60nM, SW-28 (mark VIC fluorescent reporter group): 120nM;

KRAS gene c.35G>A saltant type bridge-type fluorescent probe SW-29 (flag F AM fluorescent reporter group): 60nM;

KRAS gene c.35G>T saltant type bridge-type fluorescent probe SW-30 (mark VIC fluorescent reporter group): 60nM;

Cell strain genomic dna is all each reaction system 50ng, respectively:

Cell strain CACO2 genomic dna (KRAS and BRAF gene are the cell strain of wild-type);

Cell line A549 genomic dna (KRAS gene the 12nd bit codon c.34G>A homozygous mutant, BRAF gene is the cell strain of wild-type);

Cell strain H157 genomic dna (KRAS gene the 12nd bit codon c.34G>C homozygous mutant, BRAF gene is the cell strain of wild-type);

Cell strain SW-1573 genomic dna (KRAS gene the 12nd bit codon c.34G>T homozygous mutant, BRAF gene is the cell strain of wild-type);

Cell strain PK-45H genomic dna (KRAS gene the 13rd bit codon c.35G>A homozygous mutant, BRAF gene is the cell strain of wild-type);

Cell line SW480 genomic dna (KRAS gene the 13rd bit codon c.35G>T homozygous mutant, BRAF gene is the cell strain of wild-type);

Cell strain A375 genomic dna (cell strain of KRAS gene wild-type, BRAF gene V600E homozygous mutant);

3, the preparation of microreactor

Adopt the RainDance droplet preparing device of commercialization RainDrop company, the 50 μ L reaction solutions 2nd step prepared are dispersed in the microreactor of 2,000,000 " oily bag drop " forms, and the quantity of the nucleic acid target molecule of the same race in the target response device in each microreactor or copy number are less than or equal one.

4, the amplification of target molecule and fluoroscopic examination

The microreactor place reaction tubes of " the oily bag drop " form the 3rd step prepared is placed on thermal cycler and increases, and reaction conditions is: 95 DEG C of denaturations 30 seconds; 95 DEG C of sex change 30 seconds, 60 DEG C of renaturation with extend 1 minute (and simultaneously gathering fluorescence), amount to 40 circulations.After completion of the reaction, adopt the RainDance droplet detector of commercialization RainDrop company, detect the microreactor of FAM and the VIC passage positive, and detect according to the absolute quantitation of its positive Numerical Implementation target molecule.

5, results and analysis

The Cleaning Principle of digitizing PCR: digitizing PCR (digitalPCR) allows amplification from the technology of minimum dilute sample single nucleic acid template, in other words, being a kind of nucleic acid quantification method of carrying out counting based on single-molecule PCR method, is a kind of method of absolute quantitation.The basic premise of this technology is the micro-fluidic or droplet method adopting the popular research field of analytical chemistry, be dispersed to by nucleic acid solution after Macrodilution in the microreactor comprising droplet or micro-reacting hole, the nucleic acid-templated number in each microreactor is less than or equals 1.Like this, after PCR circulation, there is the reactor of a nucleic acid templates to provide fluorescent signal, do not have the microreactor of template just there is no fluorescent signal.According to the volume of relative proportion and microreactor, the nucleic acid concentration of original solution just can be extrapolated.Be with the difference of traditional quantitative PCR, digitizing PCR, by the method for direct census, can realize the absolute quantitation of initial nucleic acid template.In addition, digitizing PCR can also be a kind of method that can identify micro-mutant in a large amount of wild-type nucleic acid template background.The microreactor nucleic acid-templated use can separated in advance due to digitizing round pcr carries out the independent amplification of single molecules level, like this, just avoid the nucleic acid-templated amplification nucleic acid-templated to low abundance genotype of high abundance genotype to suppress, therefore, greatly can improve the genotypic detector efficiency of trace, such as, emulsion droplet digitizing round pcr can detect low to 0.001% mutated genes, and nucleic acid sequencing and conventional quantitative real-time PCR are helpless to the sudden change recall rate being less than 1%, therefore, the detection sensitivity of digitizing PCR at least can improve 1000 times.

But existing digitizing PCR detection system all only has dichromatism fluorescence channel, if merely only for a kind of fluorescence molecule of certain genotypic markers, can greatly reduce its detection efficiency.In the present embodiment, end point determination is carried out to the fluorescence intensity of several microreactors, namely after pcr amplification reaction stops, its genotype is judged by the fluorescent signal kind of each microreactor and intensity again, finally, then realized the absolute quantitation of specific gene type by the quantity of the microreactor of counting specific gene type.When in microreactor for different genotype but when marked the bridge-type fluorescent probe of identical fluorescent reporter group, be less than due to the genotype quantity in each microreactor or copy number or equal one, therefore, only has the bridge-type fluorescent probe mated with this genotype to fluorescence can be discharged, and, its fluorescence intensity and it is at microreactor, concentration in other words in initial reaction system is directly proportional, when the bridge-type fluorescent probe concentration used of different genotype is different, the fluorescence signal intensity discharged by microreactor judges it is which kind of genotype.Another similar situation is, for certain the specific genotype simultaneously different fluorescent signal of applying marking but identical bridge-type fluorescent probe of nucleotide sequence, with this understanding, can the absolute strength of different fluorescence corresponding to certain genotype and relative intensity, realize the absolute quantitation of different genotype.Therefore, when the limited fluorescence channel of existing digitizing PCR detection system, by the abundance of the microreactor bridge-type fluorescent probe corresponding to change different genotype, the genotypic detection of multiple target molecule can be realized.Present embodiments provide and utilize existing dichromatism fluorescence channel digitizing PCR detection system to realize 8 kinds of genotypic single tubes detections, drastically increase sense channel and the detection efficiency of existing digitizing PCR.

In the present embodiment, by concentration and probe concentration corresponding to adjustment different genotype, as SW-16, SW-17, SW-29, SW-30, wherein, SW-16 and SW-17 is the probe of FAM mark, and the two concentration ratio in microreactor is 4:2; SW-29, SW-30 are the probe of VIC mark, and the two concentration ratio in microreactor is 4:2;

The KRAS gene c.34G>C bridge-type fluorescent probe SW-23 (flag F AM fluorescent reporter group) of saltant type and the concentration ratio of SW-24 (mark VIC fluorescent reporter group) is 4:4;

The KRAS gene c.34G>T bridge-type fluorescent probe SW-25 (flag F AM fluorescent reporter group) of saltant type and the concentration ratio of SW-26 (mark VIC fluorescent reporter group) is 2:2;

The concentration ratio of the bridge-type fluorescent probe SW-27 (flag F AM fluorescent reporter group) of BRAF gene V600E wild-type and SW-13 (mark VIC fluorescent reporter group) is 4:2;

The concentration ratio of bridge-type fluorescent probe SW-14 (flag F AM fluorescent reporter group) 60nM, SW-28 (mark VIC fluorescent reporter group) of BRAF gene V600E saltant type is 2:4;

KRAS gene c.35G>T saltant type bridge-type fluorescent probe SW-30 (mark VIC fluorescent reporter group): 60nM.

The detected result of cell mixing pnca gene group DNA: the detected result (Figure 10) of 8 kinds of cell strain genomic DNA pools such as CACO2, A549, H157, SW-1573, PK-45H, SW480 and A375 shows, relative abundance and the fluorescent mark kind of FAM and the VIC fluorescence intensity relative value of each cell strain gene strain DNA in microreactor and its bridge-type fluorescent probe used in reaction system have good dependency, can distinguish well above-mentionedly amount to 8 kinds of genotype according to the fluorescence intensity relative value of FAM and VIC.When only having a kind of cell strain genomic dna in above-mentioned 8 kinds of cell strains in detection system, all can only obtain the microreactor of the relative intensity of fluorescence value corresponding with region shown in Figure 10, and its detected result and mixing sample has good consistence.

Embodiment 5, bridge-type fluorescent probe technique differentiate the animal derived gene with high homology

The conventional anticoagulant heparin agent used derives from the heparin raw product that chitterlings extract usually clinically.Because heparin is clinical common medicine, the regulations such as American-European countries, above-mentioned heparin raw product must not derive from the small intestine of the ruminating animal comprising cattle and sheep, and major cause is the animal borne diseases such as the mad cow disease that the heparin raw product preventing ruminating animal from originating causes.Therefore, usually need to identify the source of heparin raw product, mainly identify the gene whether containing ruminating animal in heparin raw product.Because ruminating animal and pig gene have high homology, meanwhile, the gene of different ruminating animal also has high homology, and conventional method is often difficult to the Species origin differentiating ruminating animal with high specificity.The present embodiment adopts bridge-type fluorescent probe of the present invention to detect three kinds of ruminating animal genes such as milk cow, goat and sheep.

1, the design of the competitive probe of the homonymy of target ruminating animal species specificity mtDNA sequence and amplimer thereof

According between ruminating animal and with comparison result (Figure 11 of porcine mtdna nucleotide sequence; The GeneBank numbering of the Mitochondrial gene sequence of pig, milk cow, sheep, goat is NC_000845, NC_001567, NC_001941, NC_005044 respectively; Adding frame sequence in figure is ruminating animal species specificity sequence) design the competitive bridge-type fluorescent probe of homonymy, the species specificity sequence of species specificity probe is arranged in recognition sequence district (Figure 11 adds thick underline nucleotide sequence), comprise the SW-31 of target milk cow kind gene, the SW-33 of target sheep kind gene, the SW-35 of target goat kind gene, the nucleotide sequence that above-mentioned probe is corresponding is be SW-32, SW-34 and SW-36 (table 4) respectively respectively.

According to nucleotide sequence position, above-mentioned bridge-type fluorescent probe place, design of amplification primers SW-37 and SW-38 (table 4).

The competitive probe of homonymy of table 4, target ruminating animal species specificity mtDNA sequence and amplimer thereof

2, the structure of real-time fluorescence quantitative PCR reaction system

PCR reaction system amounts to 20 μ l, and this system comprises following component: 1 × PremixExTaqmastermix (PerfectRealTime; TaKaRa company), 0.9 μM of forward primer SW-37,0.9 μM of reverse primer SW-38, the SW-31 of 0.25 μM of milk cow kind gene, the SW-33 of 0.25 μM of target sheep kind gene, the SW-35 of 0.25 μM of target goat kind gene, 50ng ruminating animal detection group DNA (comprising: milk cow, sheep and goat).

3, interpretation of result

When only there is milk cow, sheep and goat genomic dna in reaction system respectively, the fluorescent signal that its bridge-type fluorescent probe discharges can only be detected respectively, when to there is above-mentioned three kinds of detection group DNA simultaneously, then three kinds of fluorescent signals can be detected simultaneously.

Embodiment 6, bridge-type fluorescent probe technique differentiate infectious pathogen kind

The present embodiment adopts bridge-type fluorescent probe of the present invention to detect Dallas's strain and Knoxville strain two kinds of different subtypes of legionella pneumophilia.

1, the competitive probe of homonymy of the different tight type of target and the design of amplimer thereof

According to Dallas's strain of legionella pneumophilia and the hypospecificity sequence of Knoxville strain, design detects the competitive bridge-type fluorescent probe of homonymy of different subtype, the species specificity sequence of species specificity probe is positioned at recognition sequence district, comprise the SW-41 of the SW-39 of target Dallas strain, the strain of target Knoxville, the nucleotide sequence that above-mentioned probe is corresponding is SW-40 and SW-42 (table 5) respectively.

According to nucleotide sequence position, above-mentioned bridge-type fluorescent probe place, design of amplification primers SW-43 and SW-44 (table 5).

The competitive probe of table 5, Dallas's strain and Knoxville strain homonymy and amplimer thereof

2, the structure of real-time fluorescence quantitative PCR reaction system

PCR reaction system amounts to 20 μ l, and this system comprises following component: 1 × PremixExTaqmastermix (PerfectRealTime; TaKaRa company), 0.9 μM of forward primer SW-43,0.9 μM of reverse primer SW-44, the SW-39 of 0.25 μM of target Dallas strain, the SW-40 of target Knoxville strain, 10ng legionella pneumophilia hypospecificity genomic dna (comprising: Dallas's strain and Knoxville strain).

3, interpretation of result

When only there is Dallas's strain and Knoxville pnca gene group DNA in reaction system respectively, the fluorescent signal of the bridge-type fluorescent probe release of its correspondence can only be detected respectively, when to there is above-mentioned two kinds of hypospecificity genomic dnas simultaneously, then two kinds of fluorescent signals can be detected simultaneously.

What finally illustrate is, above preferred embodiment is only in order to illustrate technical scheme of the present invention and unrestricted, although by above preferred embodiment to invention has been detailed description, but those skilled in the art are to be understood that, various change can be made to it in the form and details, and not depart from claims of the present invention limited range.

Claims

1. the bridge-type fluorescent probe of base mismatch is mixed in a bridge-type sequence area, it is characterized in that: described bridge-type fluorescent probe 5'-end holds nucleotide sequence to comprise the recognition sequence district with mutant gene place to be detected nucleic acid array complementation successively to 3'-, bridge-type sequence area and with mutant gene 3'-to be detected hold outside complementary anchor series district, the 3'-end of described anchor series is connected with extension blocking group; Described bridge-type sequence area is the nucleotide derivative sequence containing at least one base mismatch, is marked with the fluoroscopic marker system of interaction between described recognition sequence district and bridge-type sequence area.

2. the bridge-type fluorescent probe of base mismatch is mixed in a kind of bridge-type sequence area according to claim 1, it is characterized in that: the length of nucleotides in described anchor series district is 16 ~ 50nt.

3. the bridge-type fluorescent probe of base mismatch is mixed in a kind of bridge-type sequence area according to claim 1, it is characterized in that: the GC Content in described anchor series district is 25 ~ 60%.

4. the bridge-type fluorescent probe of base mismatch is mixed in a kind of bridge-type sequence area according to claim 1, it is characterized in that: the length of nucleotides of described bridge-type sequence area is 2 ~ 16nt.

5. the bridge-type fluorescent probe of base mismatch is mixed in a kind of bridge-type sequence area according to claim 1, it is characterized in that: the length of nucleotides of described recognition sequence region sequence is 2 ~ 12nt.

6. the bridge-type fluorescent probe of base mismatch is mixed in a kind of bridge-type sequence area according to claim 1, it is characterized in that: the GC content of described recognition sequence is 10 ~ 80%.

7. the bridge-type fluorescent probe of base mismatch is mixed in a kind of bridge-type sequence area according to claim 1, it is characterized in that: the base number of described bridge-type sequence area is identical with the base number of recognition sequence spaced target sequence with anchor series.

8. the bridge-type fluorescent probe of base mismatch is mixed in a kind of bridge-type sequence area according to claim 1, it is characterized in that: described nucleotide derivative is Hypoxanthine deoxyriboside, inosine, 7-denitrification-2 '-Hypoxanthine deoxyriboside, 2-azepine-2 '-Hypoxanthine deoxyriboside, 2 '-methoxyl group inosine, 2 '-F inosine, deoxidation 3-nitro-pyrrole, 3-nitro-pyrrole, 2 '-methoxyl group 3-nitro-pyrrole, 2 '-F3-nitro-pyrrole, 1-(2 '-deoxidation-β-D-RIBOSE)-3-nitro-pyrrole, deoxidation 5-nitro-pyrrole, 5-nitroindoline, 2 '-methoxyl group 5-nitroindoline, 2 '-F5-nitroindoline, deoxidation 4-nitrobenzimidazole, 4-nitrobenzimidazole, deoxidation 4-aminobenzimidazole, 4-aminobenzimidazole, deoxidation nebularine, 2 '-F nebularine, 2 '-F4-nitrobenzimidazole, peptide nucleic acid(PNA)-5-nitroindoline, peptide nucleic acid(PNA)-nebularine, peptide nucleic acid(PNA)-inosine, peptide nucleic acid(PNA)-4-nitrobenzimidazole, peptide nucleic acid(PNA)-3-nitro-pyrrole, morpholinyl-5 nitroindoline, morpholinyl-nebularine, morpholinyl-inosine, morpholinyl-4-nitrobenzimidazole, morpholinyl-3-nitro-pyrrole, phosphoramidate-5-nitroindoline, phosphoramidate-nebularine, phosphoramidate-inosine, phosphoramidate-4-nitrobenzimidazole, phosphoramidate-3-nitro-pyrrole, 2 '-0-methoxy ethyl inosine, 2 '-0-methoxy ethyl nebularine, 2 '-0-methoxy ethyl 5-nitroindoline, one or more in 2 '-0-methoxy ethyl 4-nitro-benzoglyoxaline and 2 '-0-methoxy ethyl 3-nitro-pyrrole.

9. the bridge-type fluorescent probe of base mismatch is mixed in a kind of bridge-type sequence area according to claim 1, it is characterized in that: the base mismatch that bridge-type sequence area is mixed is GA, CT, TT, CC, AA, GG, CA and GT mispairing.

10. the bridge-type fluorescent probe of base mismatch is mixed in a kind of bridge-type sequence area according to claim 9, it is characterized in that: the base mismatch that bridge-type sequence area is mixed is GA, CT, TT mispairing.

11. according to claim 1 a kind of bridge-type sequence area mix the bridge-type fluorescent probe of base mismatch, it is characterized in that: the fluoroscopic marker system of described interaction is made up of fluorescent reporter group or quenching group.

12. mix the bridge-type fluorescent probe of base mismatch according to bridge-type sequence area a kind of described in claim 11, it is characterized in that: the fluoroscopic marker system of described interaction is mark fluorescent reporter group and quenching group recognition sequence district while, or at recognition sequence district mark fluorescent reporter group, bridge-type sequence area mark quenching group mark; Also or in recognition sequence district quenching group is marked, bridge-type sequence area mark fluorescent reporter group.

13. according to claim 11 or 12 a kind of bridge-type sequence area mix the bridge-type fluorescent probe of base mismatch, it is characterized in that: described fluorescent reporter group is Fluoresceincarboxylic acid, chlordene fluorescein, Tetrachlorofluorescein, carboxyl-4 ', 5 '-two chloro-2 ', 7 '-dimethoxyfluorescein, VIC, fluorescein isothiocyanate, indoles dicarboxyl cyanines, tetramethyl 6 carboxylic rhodamine and ROX dyestuff; Described quenching group is fluorescence class quencher or non-fluorescence class quencher, and described fluorescence class quencher is TAMRA, ROX dyestuff, and described non-fluorescence class quencher is DABCYL, BHQ1 or BHQ2.

14. according to claim 1 a kind of bridge-type sequence area mix the bridge-type fluorescent probe of base mismatch, it is characterized in that: described blocking group modifies phosphate group, amino group in the 3'-terminal bases 3'-hydroxyl position in anchor series district, or the 3'-end in anchor series district uses reversion base or dideoxy nucleotide.

The application of bridge-type fluorescent probe in the reagent of the single nucleotide variation of preparation detection of base mismatch is mixed in bridge-type sequence area described in 15. any one of claim 1 ~ 14.

16. application according to claim 15, is characterized in that: described single nucleotide variation is single base deletion, sudden change, insertion or SNP.

The application of bridge-type fluorescent probe in the reagent of preparation detection high homology nucleic acid target molecule of base mismatch is mixed in bridge-type sequence area described in 17. any one of claim 1 ~ 14.

18. application according to claim 17, is characterized in that: described high homology nucleic acid target molecule is that region to be detected separate target nucleic acid subsequence exists the different nucleic acid target molecule of one or more bases.

The bridge-type fluorescent probe that base mismatch is mixed in bridge-type sequence area described in 19. any one of claim 1 ~ 14 is preparing the application in digital pcr detection probes, it is characterized in that: adopt micro-fluidic or droplet method, be dispersed to by reaction mixture and comprise in several microreactors of droplet or micro-reacting hole, often kind of target molecule number in each microreactor is less than or equals 1.

20. mix the test kit of the bridge-type fluorescent probe of base mismatch containing bridge-type sequence area described in any one of claim 1 ~ 14.

21. test kits according to claim 20, is characterized in that: described test kit also comprises the pair of primers in amplification target sequence district, described bridge-type fluorescent probe land.

22. test kits according to claim 21, is characterized in that: described test kit also comprises hot resistant DNA polymerase, dNTP, ion component and the buffer system with 5' → 3' exonuclease activity.

Test kit described in 23. claims 20 ~ 22 detects the application in single nucleotide variation or high homology nucleic acid target molecule at real-time fluorescence quantitative PCR.