CN106367494B - A kind of method of the anti-product pollution of RT-PCR one step amplification kit - Google Patents
A kind of method of the anti-product pollution of RT-PCR one step amplification kit Download PDFInfo
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- 238000003757 reverse transcription PCR Methods 0.000 title claims abstract description 84
- 238000000034 method Methods 0.000 title claims abstract description 57
- 238000012197 amplification kit Methods 0.000 title abstract description 9
- AHCYMLUZIRLXAA-SHYZEUOFSA-N Deoxyuridine 5'-triphosphate Chemical compound O1[C@H](COP(O)(=O)OP(O)(=O)OP(O)(O)=O)[C@@H](O)C[C@@H]1N1C(=O)NC(=O)C=C1 AHCYMLUZIRLXAA-SHYZEUOFSA-N 0.000 claims abstract description 56
- 238000006243 chemical reaction Methods 0.000 claims abstract description 53
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- 238000003199 nucleic acid amplification method Methods 0.000 claims abstract description 43
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- HAAZLUGHYHWQIW-KVQBGUIXSA-N dGTP Chemical compound C1=NC=2C(=O)NC(N)=NC=2N1[C@H]1C[C@H](O)[C@@H](COP(O)(=O)OP(O)(=O)OP(O)(O)=O)O1 HAAZLUGHYHWQIW-KVQBGUIXSA-N 0.000 claims abstract description 38
- 102000004190 Enzymes Human genes 0.000 claims abstract description 25
- 108090000790 Enzymes Proteins 0.000 claims abstract description 25
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- 238000007385 chemical modification Methods 0.000 claims abstract description 8
- 238000012986 modification Methods 0.000 claims description 48
- 230000004048 modification Effects 0.000 claims description 48
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- AYKYXWQEBUNJCN-UHFFFAOYSA-N 3-methylfuran-2,5-dione Chemical compound CC1=CC(=O)OC1=O AYKYXWQEBUNJCN-UHFFFAOYSA-N 0.000 claims description 5
- CURLTUGMZLYLDI-UHFFFAOYSA-N Carbon dioxide Chemical compound O=C=O CURLTUGMZLYLDI-UHFFFAOYSA-N 0.000 claims description 5
- 229960004424 carbon dioxide Drugs 0.000 claims description 5
- 235000011089 carbon dioxide Nutrition 0.000 claims description 5
- CHDFNIZLAAFFPX-UHFFFAOYSA-N ethoxyethane;oxolane Chemical compound CCOCC.C1CCOC1 CHDFNIZLAAFFPX-UHFFFAOYSA-N 0.000 claims description 5
- HUHXLHLWASNVDB-UHFFFAOYSA-N 2-(oxan-2-yloxy)oxane Chemical class O1CCCCC1OC1OCCCC1 HUHXLHLWASNVDB-UHFFFAOYSA-N 0.000 claims description 4
- WQPDQJCBHQPNCZ-UHFFFAOYSA-N cyclohexa-2,4-dien-1-one Chemical compound O=C1CC=CC=C1 WQPDQJCBHQPNCZ-UHFFFAOYSA-N 0.000 claims 1
- 230000035945 sensitivity Effects 0.000 abstract description 38
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- 241000713869 Moloney murine leukemia virus Species 0.000 description 4
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- 102100034343 Integrase Human genes 0.000 description 3
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 3
- 108010092799 RNA-directed DNA polymerase Proteins 0.000 description 3
- 230000015556 catabolic process Effects 0.000 description 3
- 238000006731 degradation reaction Methods 0.000 description 3
- 108020004707 nucleic acids Proteins 0.000 description 3
- 102000039446 nucleic acids Human genes 0.000 description 3
- 150000007523 nucleic acids Chemical class 0.000 description 3
- 125000002467 phosphate group Chemical group [H]OP(=O)(O[H])O[*] 0.000 description 3
- 238000010839 reverse transcription Methods 0.000 description 3
- 241000711549 Hepacivirus C Species 0.000 description 2
- 208000005176 Hepatitis C Diseases 0.000 description 2
- 108020004682 Single-Stranded DNA Proteins 0.000 description 2
- ISAKRJDGNUQOIC-UHFFFAOYSA-N Uracil Chemical compound O=C1C=CNC(=O)N1 ISAKRJDGNUQOIC-UHFFFAOYSA-N 0.000 description 2
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- 239000000758 substrate Substances 0.000 description 2
- YLQBMQCUIZJEEH-UHFFFAOYSA-N tetrahydrofuran Natural products C=1C=COC=1 YLQBMQCUIZJEEH-UHFFFAOYSA-N 0.000 description 2
- BUDQDWGNQVEFAC-UHFFFAOYSA-N Dihydropyran Chemical compound C1COC=CC1 BUDQDWGNQVEFAC-UHFFFAOYSA-N 0.000 description 1
- 241000282326 Felis catus Species 0.000 description 1
- 238000012408 PCR amplification Methods 0.000 description 1
- 238000002944 PCR assay Methods 0.000 description 1
- RBYGDVHOECIAFC-UHFFFAOYSA-L acetonitrile;palladium(2+);dichloride Chemical compound [Cl-].[Cl-].[Pd+2].CC#N.CC#N RBYGDVHOECIAFC-UHFFFAOYSA-L 0.000 description 1
- 150000008064 anhydrides Chemical class 0.000 description 1
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- 238000011033 desalting Methods 0.000 description 1
- 230000006806 disease prevention Effects 0.000 description 1
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- 238000003752 polymerase chain reaction Methods 0.000 description 1
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- 229940035893 uracil Drugs 0.000 description 1
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- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6844—Nucleic acid amplification reactions
- C12Q1/6848—Nucleic acid amplification reactions characterised by the means for preventing contamination or increasing the specificity or sensitivity of an amplification reaction
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Abstract
The invention discloses a kind of method of anti-product pollution of RT-PCR one step amplification kit, this method is that dUTP, dATP, dCTP, dGTP and UNG enzyme of specific chemical modification will be added in existing One step RT-PCR amplification reaction system；The final concentration of unmodified dTTP, dATP, dCTP, dGTP are down to 200nM simultaneously hereinafter, other operations are compared with One step RT-PCR.The method of the present invention not only has in One step RT-PCR amplification and removes product pollution effect well, while also ensuring the sensitivity of RT-PCR, and existing Antipollution method can be substantially reduced the sensitivity of RT-PCR, influence the testing result of experiment.The sensitivity of the method for the present invention and the sensitivity of conventional nonreactive Pollution System are close, can achieve the detection effect and sensitivity of common One step RT-PCR, can't reduce the sensitivity of PCR.
Description
Technical field
The present invention relates to a kind of methods of anti-product pollution of PCR amplification kit, and in particular to a kind of RT-PCR one-step method
The method of the anti-product pollution of amplification kit.
Background technique
Real-time fluorescence PCR (real time PCR, TaqMan PCR) is in pathogen detection technology of today, increasingly
Performance more importantly acts on, quick, sensitive, special, since it does not use only specific primer, and uses specificity
Probe makes the specificity of this method be higher than normal PCR, greatly reduces false positive results caused by non-specific amplification.Full envelope
The Fluorescence PCR assay for closing reaction is the fluorescence signal generated during recording and analyzing PCR by computer, is realized to PCR
Amplification procedure real-time monitoring is not necessarily to electrophoresis detection, has thoroughly prevented the pollution of the Aerosol Pollution and EB of PCR product to environment.
By adding the RT-PCR of reverse transcriptase and corresponding reverse transcription reaction condition, can be not only used for for detecting
DNA can be also used for detection RNA.The fields such as RT-PCR and fluorescence RT-PCR detect in clinical RNA virus, control and prevention of disease, have
It is more and more widely used.
Since in PCR reaction process, nucleic acid is amplified at least up to a million times (20 circulations), what a little PCR product was formed
Aerosol is just enough that false positive is caused to expand.It is this by PCR product bring false positive phenomenon in order to solve, in nucleic acid amplification
In, UNG enzyme is widely applied (6,187,575, Roche Diagnostics, Thermolabile uracil-DNA-
glycosylas,process for its preparation and use for removing uracil from DNA；
5,945,313, Life Technologies, Process for controlling contamination of nucleic
acid amplification reactions)。
In PCR system, dTTP is substituted using dUTP, the single-stranded or double-stranded DNA with dUTP can be degraded by UNG enzyme, and
The single-stranded or double-stranded DNA without dUTP will not be digested.In this way, the PCR product with dUTP that the first round generates can quilt
The degradation of UNG enzyme will not generate False Positive Effect to the second wheel PCR as template.UNG enzyme itself can be in the initial denaturation step of PCR
(95 DEG C of 10min) is inactivated afterwards, so that the PCR product with dUTP of the first round will not be degraded.
But so far, there is no RT-PCR or fluorescence RT-PCR one that any one UNG enzyme is used directly for RNA
Footwork amplification.UNG handles product pollution, is there is no at present applied to RT-PCR one-step method.This is because if in one-step method system
Middle addition UNG enzyme and dUTP, substrate dUTP can mix the cDNA of MMLV synthesis, and UNG and MMLV active temperature is close, can be to inverse
The cDNA containing dUTP of transcription enzymatic synthesis synchronizes degradation, to significantly weaken the sensitivity of PCR detection.
Summary of the invention
The purpose of the present invention is to provide a kind of methods of anti-product pollution of RT-PCR one step amplification kit.
The technical solution used in the present invention is:
The method that One step RT-PCR expands anti-product pollution will be added in existing One step RT-PCR amplification reaction system
DUTP the and UNG enzyme of modification；The final concentration of unmodified dTTP is down to 200nM or less simultaneously；Other operation compared with
One step RT-PCR；
The dUTP of the modification is chemically modified to the 5 '-phosphate groups of dUTP or 3 '-hydroxyl groups, the chemistry
Modification group is Oxyranyle, tetrahydrofuran ether, tetrahydropyranyl ethers, phenyl ring, acetic anhydride, citraconic anhydride, one in carbonic anhydride
Kind.
The method that One step RT-PCR expands anti-product pollution will be added in existing One step RT-PCR amplification reaction system
DUTP, dATP, dCTP, dGTP and UNG enzyme of modification；It is simultaneously that the end of unmodified dTTP, dATP, dCTP, dGTP is dense
Degree is down to 200nM or less；Other operations are compared with One step RT-PCR；
DUTP, dATP, dCTP, dGTP of the modification be to the 5 '-phosphate groups of dUTP, dATP, dCTP, dGTP or
3 '-hydroxyl groups are chemically modified respectively, and the chemical modification group is Oxyranyle, tetrahydrofuran ether, oxinane
One of ether, phenyl ring, acetic anhydride, citraconic anhydride, carbonic anhydride.
Further, final concentration of the dUTP of the modification in One step RT-PCR amplification reaction system is not less than 400 μ
M。
Further, final concentration of the dUTP of the modification in One step RT-PCR amplification reaction system is not less than 400 μ
M；Final concentration of dATP, dCTP, the dGTP of modification in One step RT-PCR amplification reaction system is not less than 200 μM.
Further, dosage of the UNG enzyme in One step RT-PCR amplification reaction system is 0.06U~1U.
The beneficial effects of the present invention are:
The present invention provides a kind of method of anti-product pollution of RT-PCR one step amplification kit, this method is will be existing
DUTP, dATP, dCTP, dGTP and UNG enzyme of specific chemical modification are added in One step RT-PCR amplification reaction system；
The final concentration of unmodified dTTP, dATP, dCTP, dGTP are down to 200nM simultaneously hereinafter, other operations are compared with one
Footwork RT-PCR.The method of the present invention not only has in One step RT-PCR amplification and removes product pollution effect well, while
It ensure that the sensitivity of RT-PCR, and existing common Antipollution method can be substantially reduced the sensitivity of RT-PCR, influence to test
Testing result.
The sensitivity of the method for the present invention and the sensitivity of conventional nonreactive Pollution System are close, can achieve common one-step method
The detection effect and sensitivity of RT-PCR, can't reduce the sensitivity of PCR.
Detailed description of the invention
Fig. 1 is unmodified dNTP (left side) and the dNTP (right side) of oxinane modification；
Fig. 2 is the amplification curve of each group RT-PCR；
Fig. 3 is the amplification curve of each group RT-PCR；
Fig. 4 is unmodified dNTP (left side) and the dNTP (right side) of acetic anhydride modification；
Fig. 5 is the amplification curve of each group RT-PCR；
Fig. 6 is the amplification curve of each group RT-PCR.
Specific embodiment
The present invention is further illustrated combined with specific embodiments below, and however, it is not limited to this.
A kind of method of the anti-product pollution of RT-PCR one step amplification kit of embodiment 1
DUTP the and UNG enzyme of modification will be added in existing One step RT-PCR amplification reaction system, makes the dUTP's of modification
Final concentration is not less than 400 μM, and the UNG enzyme dosage in each reaction system is 0.06U~1U；Simultaneously by unmodified dTTP's
Final concentration is down to 200nM hereinafter, other operations are compared with One step RT-PCR.
The dUTP of the modification is chemically modified to the 5 '-phosphate groups of dUTP or 3 '-hydroxyl groups, the chemistry
Modification group is Oxyranyle, tetrahydrofuran ether, tetrahydropyranyl ethers, phenyl ring, acetic anhydride, citraconic anhydride, one in carbonic anhydride
Kind.
Wherein the tetrahydrofuran modificationization, Oxyranyle modificationization, oxinane modificationization of the 3 ' of dUTP-hydroxyl and
The structural formula of the oxinane modificationization of the 5 ' of dUTP-phosphate group is as follows:
A kind of method of the anti-product pollution of RT-PCR one step amplification kit of embodiment 2
In PCR, dNTP (dATP, dCTP, dTTP, dGTP, dUTP) connects into DNA chain by covalent bond, this be according to
The phosphate group at its 5 ' end is relied to form phosphodiester bond with primer 3 '-hydroxyl or 3 '-hydroxyl condensations of a upper end dNTP.
If holding phosphate groups or 3 '-hydroxyls to carry out specific chemical modification to the 5 ' of dUTP, can avoid in reverse transcription step,
DUTP is introduced in cDNA synthesis；Meanwhile in order to avoid the larger possible mispairing of concentration difference, the present embodiment will be a part of
5 ' the end phosphate groups of dATP, dCTP, dGTP or 3 '-hydroxyls also carry out specific chemical modification.It is demonstrated experimentally that above-mentioned through spy
DATP, dCTP, dGTP, the dUTP for determining chemical modification are not involved in the synthesis of reverse transcription step cDNA, but after cDNA synthesis
PCR amplification program in, can normally participate in the synthesis of double-stranded DNA.
The specific operating method of this implementation is as follows:
DUTP, dATP, dCTP, dGTP and UNG of modification will be added in existing One step RT-PCR amplification reaction system
Enzyme；Make the final concentration of the dUTP of modification not less than 400 μM, the final concentration of dATP, dCTP, dGTP of modification are not less than 200 μM,
UNG enzyme dosage in each reaction system is 0.06U~1U；Simultaneously by the end of unmodified dTTP, dATP, dCTP, dGTP
Concentration is down to 200nM hereinafter, other operations are compared with One step RT-PCR.
DUTP, dATP, dCTP, dGTP of the modification be to the 5 '-phosphate groups of dUTP, dATP, dCTP, dGTP or
3 '-hydroxyl groups are chemically modified respectively, and the chemical modification group is Oxyranyle, tetrahydrofuran ether, oxinane
One of ether, phenyl ring, acetic anhydride, citraconic anhydride, carbonic anhydride (with embodiment 1).
The detection of 3 the method for the present invention sensitivity of embodiment
(1) preparation of dNTP is modified
Using tetrahydrofuran as solvent, at room temperature, respectively by dUTP, dATP, dCTP, dGTP in PdCl2(CH3CN)2
Catalysis under, dNTP 3 '-hydroxyl and dihydropyran generate tetrahydropyranyl ethers, i.e. dUTP, dATP, dCTP, dGTP 3 '-hydroxyl quilt
Oxinane modification obtains the dNTP (i.e. modification dNTP) of oxinane modification, as shown in Figure 1.
Above-mentioned product (dNTP of oxinane modification) purifies through HPLC, recycles, the oxinane for respectively obtaining purifying is repaired
DUTP, dATP, dCTP, dGTP of decorations.
(2) One step RT-PCR
1) One step RT-PCR reaction system:
One step RT-PCR reaction system is prepared by table 1, experimental group, control group 1 and control group 2 is respectively set.
Experimental group: in the reaction system of experimental group containing upper step preparation oxinane modification dUTP, dATP, dCTP,
DGTP (i.e. modification dUTP described in table 1, modification dATP, modification dCTP, modification dGTP) and unmodified dNTP (dTTP,
dATP,dCTP,dGTP)；Unmodified dNTP concentration is extremely low in reaction system, is 200nM；Modification dUTP, it modification dATP, repairs
It is relatively high to adorn dCTP, modification dGTP concentration, is 200~400 μM；Containing recombination UNG enzyme, other compositions and dosage with compare
Group 1 and 2 is identical.
Control group 1: in the reaction system of control group 1 without modification dNTP and dTTP, contain only dATP, dCTP, dGTP,
DUTP, simultaneously containing recombination UNG enzyme, other compositions and dosage are the same as experimental group and control group 2.Control group 1 is equivalent to existing routine
DUTP/UNG antipollution system.
Control group 2: in the reaction system of control group 2 without modification dNTP and dUTP, contain only dATP, dCTP, dGTP,
DTTP, without recombination UNG enzyme, other compositions and dosage are the same as experimental group and control group 1.Control group 2 is equivalent to normally without anti-pollution
The reaction system of dye.
1 One step RT-PCR reaction system of table
RNA template described in table 1 is that HCV (hepatitis C is by Hepatitis C Virus) standard items are (public purchased from Germany Qiagen
Department, article No. 1061093), HCV final concentration is 10 in each group reaction system3IU/mL。
Amplification HCV the primer 1 and 2, fluorescence probe P1 are HPLC pure in table 1, and base sequence is as follows:
Primer 1:5 '-AGCGTCTAGCCATGGCGT-3 ' (SEQ ID NO:1),
Primer 2: 5 '-GGTGTACTCACCGGTTCCG-3 ' (SEQ ID NO:2),
Fluorescence probe P1:5 ' -6Fam-MCYCCCCCTYCCGGGAGAGCCAT-3 '-BHQ1 (SEQ ID NO:3).
2) One step RT-PCR response procedures:
Above-mentioned reaction system is placed on ABI 7500fast fluorescent PCR instrument and carries out One step RT-PCR, response procedures are as follows:
45 DEG C of 15min, 95 DEG C of 15min；95 DEG C of 15s, 58 DEG C of 45s, 40 circulations, wherein it is glimmering to carry out the channel FAM at 58 DEG C
Light detection.
3) interpretation of result
The amplification curve of above-mentioned 3 groups of reaction systems is as shown in Fig. 2, as seen from the figure, experimental group amplification curve and control group 2 expand
It is very close to increase curve, almost unanimously；The sensitivity of contrast sensitivity discovery, experimental group and control group 2 is close, and compares
1 sensitivity of group decreases, and the sensitivity of PCR detection can significantly be weakened by illustrating existing UNG/dUTP antipollution system really,
Wherein reason may be since substrate dUTP can mix the cDNA of reverse transcriptase MMLV synthesis, and UNG enzyme and MMLV enzyme activity are warm-natured
Degree is close, and the cDNA containing dUTP that can be synthesized to reverse transcriptase synchronizes degradation, to significantly weaken the spirit of PCR detection
Sensitivity.And the sensitivity of experimental group (the method for the present invention) and the sensitivity of conventional nonreactive Pollution System are close, illustrate the present invention
Method can achieve the detection effect and sensitivity of common One step RT-PCR, can't reduce the sensitivity of PCR.
The antipollution effect of 4 the method for the present invention of embodiment detects
In order to further detect the effect of the anti-PCR product pollution of the method for the present invention, the present embodiment is artificial in the reaction system
Pollution products are added, to detect its resistant to pollution effect, specific experiment design is as shown in table 2.
Experimental group: being added 5 μ L embodiment, 3 experimental group RT-PCR product as pollution sources, that is, pollute template in reaction system,
Other compositions and dosage are the same as the experimental group in embodiment 3.
Positive controls: in addition to template used for other than HCV standard items, other compositions and the same experimental group of dosage.
2 One step RT-PCR reaction system of table
(hepatitis C is by the Hepatitis C Virus) standard items of HCV described in table 2 are purchased from Qiagen company, Germany, article No.
1061093。
Amplification HCV the primer 1 and 2, fluorescence probe P1 are HPLC pure in table 2, and base sequence is as follows:
Primer 1:5 '-AGCGTCTAGCCATGGCGT-3 ' (SEQ ID NO:1),
Primer 2: 5 '-GGTGTACTCACCGGTTCCG-3 ' (SEQ ID NO:2),
Fluorescence probe P1:5 ' -6Fam-MCYCCCCCTYCCGGGAGAGCCAT-3 '-BHQ1 (SEQ ID NO:3).
2) One step RT-PCR response procedures:
Above-mentioned reaction system is placed on ABI 7500fast fluorescent PCR instrument and carries out One step RT-PCR, response procedures are as follows:
45 DEG C of 15min, 95 DEG C of 15min；95 DEG C of 15s, 58 DEG C of 45s, 40 circulations, wherein it is glimmering to carry out the channel FAM at 58 DEG C
Light detection.
3) interpretation of result
The amplification curve of above-mentioned 2 groups of reaction systems is as shown in figure 3, take HCV standard items as the sun of RNA template as seen from the figure
Property control group have apparent amplification curve, illustrating experimental system, there is no problem, can carry out One step RT-PCR well
Amplification；However there is no amplification curve using the experimental group that the method for the present invention carries out RT-PCR one step amplification, illustrate present invention side
Method can effectively remove product pollution (template polluted).
In conclusion the method for the present invention not only has in One step RT-PCR amplification and removes product pollution effect well,
The sensitivity of RT-PCR is also ensured simultaneously, and existing common Antipollution method can be substantially reduced the sensitivity of RT-PCR, shadow
Ring the testing result of experiment.
The detection of 5 the method for the present invention sensitivity of embodiment
(1) preparation of dNTP is modified
Appropriate acetic anhydride powder is taken, methanol is dissolved in, is slowly added to dUTP, dATP, dCTP, dGTP.300mM Tris- is added
It after Cl, is refrigerated at 0 DEG C, every 5min shakes once, continues 45min, is sufficiently mixed acetic anhydride and dUTP, dATP, dCTP, dGTP.
Finally with Sephdex G-15 desalting column remove excess anhydride, deepfreeze, the thermal starting purified acetic anhydride modification
DUTP, dATP, dCTP, dGTP, as shown in Figure 4.
(2) One step RT-PCR
1) One step RT-PCR reaction system:
One step RT-PCR reaction system is prepared by table 3, experimental group, control group 1 and control group 2 is respectively set.
Experimental group: in the reaction system of experimental group containing upper step preparation acetic anhydride modification dUTP, dATP, dCTP,
DGTP (i.e. modification dUTP described in table 3, modification dATP, modification dCTP, modification dGTP) and unmodified dNTP (dTTP,
dATP,dCTP,dGTP)；Unmodified dNTP concentration is extremely low in reaction system, is 200nM；Modification dUTP, it modification dATP, repairs
It is relatively high to adorn dCTP, modification dGTP concentration, is 200~400 μM；Containing recombination UNG enzyme, other compositions and dosage with compare
Group 1 and 2 is identical.
Control group 1: in the reaction system of control group 1 without modification dNTP and dTTP, contain only dATP, dCTP, dGTP,
DUTP, simultaneously containing recombination UNG enzyme, other compositions and dosage are the same as experimental group and control group 2.Control group 1 is equivalent to existing routine
DUTP/UNG antipollution system.
Control group 2: in the reaction system of control group 2 without modification dNTP and dUTP, contain only dATP, dCTP, dGTP,
DTTP, without recombination UNG enzyme, other compositions and dosage are the same as experimental group and control group 1.Control group 2 is equivalent to normally without anti-pollution
The reaction system of dye.
3 One step RT-PCR reaction system of table
RNA template described in table 3 is influenza A virus H5N1 hypotype standard items (purchased from Harbin dimension section biology), each group
HCV final concentration is 10 in reaction system3IU/mL。
Amplification H5N1 the primer 3 and 4, fluorescence probe P2 are HPLC pure in table 3, and base sequence is as follows:
Primer 3:5 '-CTTCTAACCGAGGTCGAAACGTA-3 ' (SEQ ID NO:4),
Primer 4:5 '-GGTGACAGGATTGGTCTTGTCTTTA-3 ' (SEQ ID NO:5),
Fluorescence probe P2:5 ' -6Fam-TCAGGCCCCCTCAAAGCCGAG-3 ' BHQ1-3 ' (SEQ ID NO:6).
2) One step RT-PCR response procedures:
Above-mentioned reaction system is placed on ABI 7500fast fluorescent PCR instrument and carries out One step RT-PCR, response procedures are as follows:
45 DEG C of 15min, 95 DEG C of 15min；95 DEG C of 15s, 58 DEG C of 45s, 40 circulations, wherein it is glimmering to carry out the channel FAM at 58 DEG C
Light detection.
3) interpretation of result
As seen from the figure, experimental group amplification curve and 2 amplification curve of control group are close, and control group 1 can then reduce reaction
Sensitivity.Illustrate the method in the present invention, can achieve the detection sensitivity of common fluorescent RT-PCR.
The amplification curve of above-mentioned 3 groups of reaction systems is as shown in figure 5, as seen from the figure, experimental group amplification curve and control group 2 expand
It is very close to increase curve, almost unanimously；The sensitivity of contrast sensitivity discovery, experimental group and control group 2 is close, and compares
1 sensitivity of group is substantially reduced, and the spirit of PCR detection can significantly be weakened by further illustrating existing UNG/dUTP antipollution system really
Sensitivity.And the sensitivity of experimental group (the method for the present invention) and the sensitivity of conventional nonreactive Pollution System are close, illustrate the present invention
Method can achieve the detection effect and sensitivity of common One step RT-PCR, can't reduce the sensitivity of PCR.
The antipollution effect of 6 the method for the present invention of embodiment detects
In order to further detect the effect of the anti-PCR product pollution of the method for the present invention, the present embodiment is artificial in the reaction system
Pollution products are added, to detect its resistant to pollution effect, specific experiment design is as shown in table 4.
Experimental group: being added 5 μ L embodiment, 5 experimental group RT-PCR product as pollution sources, that is, pollute template in reaction system,
Other compositions and dosage are the same as the experimental group in embodiment 5.
Positive controls: in addition to template used for other than influenza A virus H5N1, other compositions and the same experimental group of dosage.
4 One step RT-PCR reaction system of table
RNA template described in table 4 is influenza A virus H5N1 hypotype standard items purchased from Harbin dimension section biology.
Amplification H5N1 the primer 3 and 4, fluorescence probe P2 are HPLC pure in table 4, and base sequence is as follows:
Primer 3:5 '-CTTCTAACCGAGGTCGAAACGTA-3 ' (SEQ ID NO:4),
Primer 4:5 '-GGTGACAGGATTGGTCTTGTCTTTA-3 ' (SEQ ID NO:5),
Fluorescence probe P2:5 ' -6Fam-TCAGGCCCCCTCAAAGCCGAG-3 ' BHQ1-3 ' (SEQ ID NO:6).
2) One step RT-PCR response procedures:
Above-mentioned reaction system is placed on ABI 7500fast fluorescent PCR instrument and carries out One step RT-PCR, response procedures are as follows:
45 DEG C of 15min, 95 DEG C of 15min；95 DEG C of 15s, 58 DEG C of 45s, 40 circulations, wherein it is glimmering to carry out the channel FAM at 58 DEG C
Light detection.
3) interpretation of result
The amplification curve of above-mentioned 2 groups of reaction systems is as shown in fig. 6, take H5N1 standard items as the sun of RNA template as seen from the figure
Property control group have apparent amplification curve, illustrating experimental system, there is no problem, can carry out One step RT-PCR well
Amplification；However there is no amplification curve using the experimental group that the method for the present invention carries out RT-PCR one step amplification, illustrate present invention side
Method can effectively remove product pollution (template polluted).
In conclusion further illustrating that the method for the present invention in One step RT-PCR amplification, not only there is good remove to produce
Object pollution effects, while also ensuring the sensitivity of RT-PCR, and existing common Antipollution method can be substantially reduced RT-PCR
Sensitivity, influence experiment testing result.
The above embodiment is a preferred embodiment of the present invention, but embodiments of the present invention are not by above-described embodiment
Limitation, other any changes, modifications, substitutions, combinations, simplifications made without departing from the spirit and principles of the present invention,
It should be equivalent substitute mode, be included within the scope of the present invention.
<110>Guangzhou Wei Baixin Biotechnology Co., Ltd
<120>a kind of method of the anti-product pollution of RT-PCR one step amplification kit
<130>
<160> 6
<170> PatentIn version 3.5
<210> 1
<211> 18
<212> DNA
<213>artificial sequence
<400> 1
agcgtctagc catggcgt 18
<210> 2
<211> 19
<212> DNA
<213>artificial sequence
<400> 2
ggtgtactca ccggttccg 19
<210> 3
<211> 23
<212> DNA
<213>artificial sequence
<400> 3
mcycccccty ccgggagagc cat 23
<210> 4
<211> 23
<212> DNA
<213>artificial sequence
<400> 4
cttctaaccg aggtcgaaac gta 23
<210> 5
<211> 25
<212> DNA
<213>artificial sequence
<400> 5
ggtgacagga ttggtcttgt cttta 25
<210> 6
<211> 21
<212> DNA
<213>artificial sequence
<400> 6
tcaggccccc tcaaagccga g 21
Claims (2)
1. the method that One step RT-PCR expands anti-product pollution, it is characterised in that: by existing One step RT-PCR amplified reaction
DUTP, dATP, dCTP, dGTP and UNG enzyme of modification are added in system；The dUTP of the modification expands in One step RT-PCR
The final concentration increased in reaction system is not less than 400 μM, and dATP, dCTP, dGTP of modification are in One step RT-PCR amplified reaction body
Final concentration in system is not less than 200 μM；The final concentration of unmodified dTTP, dATP, dCTP, dGTP are down to simultaneously
200nM or less；Other operations are compared with One step RT-PCR；
DUTP, dATP, dCTP, dGTP of the modification are the 5 '-phosphate groups or 3 '-hydroxyls to dUTP, dATP, dCTP, dGTP
Base group is chemically modified respectively, and the chemical modification group is Oxyranyle, tetrahydrofuran ether, tetrahydropyranyl ethers, benzene
One of ring, acetic anhydride, citraconic anhydride, carbonic anhydride.
2. according to the method described in claim 1, it is characterized by: the UNG enzyme is in One step RT-PCR amplification reaction system
In dosage be 0.06U~1U.
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5536649A (en) * | 1993-05-11 | 1996-07-16 | Becton, Dickinson And Company | Decontamination of nucleic acid amplification reactions using uracil-N-glycosylase (UDG) |
| CN102321611A (en) * | 2011-08-28 | 2012-01-18 | 苏州旷远生物分子技术有限公司 | Application of UNG-dUTP product contamination-resistant system in whole blood PCR reaction system |
| CN103966356A (en) * | 2013-01-25 | 2014-08-06 | 上海星耀医学科技发展有限公司 | Human immunodeficiency virus type 1 one-step fluorescence quantitative RT-PCR detection kit |
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2016
- 2016-08-30 CN CN201610768052.2A patent/CN106367494B/en active Active
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5536649A (en) * | 1993-05-11 | 1996-07-16 | Becton, Dickinson And Company | Decontamination of nucleic acid amplification reactions using uracil-N-glycosylase (UDG) |
| CN102321611A (en) * | 2011-08-28 | 2012-01-18 | 苏州旷远生物分子技术有限公司 | Application of UNG-dUTP product contamination-resistant system in whole blood PCR reaction system |
| CN103966356A (en) * | 2013-01-25 | 2014-08-06 | 上海星耀医学科技发展有限公司 | Human immunodeficiency virus type 1 one-step fluorescence quantitative RT-PCR detection kit |
Non-Patent Citations (1)
| Title |
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| Simple enzymatic means to neutralize DNA contamination in nucleic acid amplification;John Ashkenas et al.;《BioTechniques》;20051231;69-73 |
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