CN107841559A - A kind of full premix UGT1A1 multiplex PCR genetic polymorphism detection kits and method - Google Patents
A kind of full premix UGT1A1 multiplex PCR genetic polymorphism detection kits and method Download PDFInfo
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Abstract
The present invention is provided to detect full the premix multi-primerses specific PCR detection kit and method of the types of UGT1A1 genes * 6 and two position nucleotide polymorphisms of * 28 type, according to " allele specific pcr " principle, multi-primerses specific PCR is designed, two tube reactions are realized on Real-Time Fluorescent Quantitative PCR Technique platform while detect the nucleotide type of the types of UGT1A1 genes * 6 and * 28 two pleomorphism sites of type.And biglycan PCR protective agents are added using the Taq archaeal dna polymerases of modification and in reaction system, increase the stability of reaction system, realize the long-time stability that full premix detection architecture preserves.
Description
Technical field
The invention belongs to biomedical clinical Molecular Detection field, and in particular to for UGT1A1 genes * 6 and * 28 two
Full the premix multi-primerses specific PCR detection kit and method of position nucleotide polymorphic detection.
Background technology
Irinotecan（Irinotecan, CPT-11）It is a kind of change that antitumaous effect is played by consensus dna topoisomerase I
Medicine is treated, was approved listing by U.S. FDA in 1998.During DNA replication dna, the reversible incision DNA of topoisomerase I
It is single-stranded, and re-assembly and to form DNA double chain.The active metabolite SN-38 of Irinotecan is in topoisomerase I DNA compound knots
Close, tissue DNA chain re-assemblies, causes DNA double chain break, cause cell death.
However, the reduction of neutrophil cell caused by Irinotecan and serious intestines toxicity problem, turn into and limit its medication
One of key factor of dosage.The active metabolite SN-38 of Irinotecan is through liver uridine diphosphate glucuronatetransferase
（UDP-GT）Inactivation（Mainly it is metabolized by UGT1A1, UGT1A7 and UGT1A9）, so as to which the cell that protects the health is from cytotoxicity
Influence.Scientific investigations showed that the toxic side effect of Irinotecan and UGT1A1 gene pleiomorphism are closely related.Two kinds of UGT1A1
Common allele UGT1A1*6 and UGT1A1*28 can cause the miopragia of the enzyme, so that pharmaceutical active metabolite exists
Extended residence time in blood.
UGT1A1*6 is the point mutation on exons 1（G211A）.The SN-38G and SN-38AUC of UGT1A1*6 genotype
（Blood medicine-time lower curve area）The ratio between be substantially less than wild type, and UGT1A1*6 genotype patients receive irinotecan
Progression free survival rate and overall survival afterwards is all remarkably decreased.Distribution frequencies of the UGT1A1*6 in the crowd of East Asia is about 13.6%.
UGT1A1*28 is promoter TA insertion mutations.UGT1A1*28 heterozygote is similar with the enzymatic activity of wild type, but UGT1A1*28
No mutant homozygote corresponding to enzymatic activity be only the 35% of wild type.The normal homozygote in the site is the TA base-pairs A of 6 repetitions
（TA）6TAA（6/6）, no mutant homozygote is the TA base-pairs A of 7 repetitions（TA）7TAA（7/7）, heterozygous mutation genotype
In allele, one is wild type, and one is saltant type（6/7）.The UGT1A1 carrier of different genotype, is receiving Yi Li
The probability that toxic side effect is produced when being treated for health is different.Wild type UGT1A1（6/6）It will not be produced when receiving irinotecan
Raw toxic side effect, and saltant type heterozygote（6/7）The probability for producing toxic side effect is 12.5%, saltant type homozygote（7/7）Produce
The probability of toxic side effect is 50%.In asian population, the occurrence frequency of each genotype of UGT1A1 genes is that 6/6 type accounts for respectively
70.2%, 6/7 type accounts for 27.7%, and 7/7 type accounts for 2.1%.Therefore, the product of U.S. FDA approval renewal Irinotecan in 2005 is said
Bright book, it is desirable to which the hereditary difference of user is indicated in information warning will influence its reaction to the medicine, should before medicine taking prompting
Carry out UGT1A1*28 detections.
UGT1A1*6 types and * 28 type gene pleiomorphisms are analyzed, it is mostly straight using PCR-PFLP detection techniques and DNA
Connect PCR sequencing PCR.Because PCR-PFLP technologies rely on the reasons such as digestion with restriction enzyme, electrophoretic analysis, often there is result erroneous judgement
Phenomenon, its Detection accuracy is influenceed, its detection cycle is long in addition, flux is relatively low, is not also suitable for the quick screening of a large amount of crowds.
In addition, the DNA direct sequencings as genetic test goldstandard, due to the expensive instrument and equipment of its needs and complicated operation stream
The characteristics of journey and long detection cycle, it is difficult to realize large-scale promotion.
The content of the invention
The present invention is expensive for prior art detection UGT1A1*6 types and during * 28 type gene pleiomorphism, the cycle is long, low
The deficiencies of efficiency, cumbersome data analysis, there is provided a kind of UGT1A1*6 types and * based on Real-Time Fluorescent Quantitative PCR Technique platform
28 types premix multi-primerses specific PCR detection kit and method entirely.
The gene pleiomorphism that the present invention detects a site for substance primer specificity PCR needs two tube reactions complete
Into using multi-primerses specific PCR detection gene pleiomorphism, two tube reaction systems can detect hCCSP T1A1 genes * 6 simultaneously
Site（G/A）Polymorphism and * 28 sites（TA insertion mutations）Polymorphism；
The reaction solution of the Taq archaeal dna polymerases of modification and the stabilizer containing PCR is premixed in the present invention, and Taq enzyme activity after premix
And reaction solution stability is unaffected, there is the advantages of easy to operate, rapid reaction, low cost, have and be widely popularized value.
To reach above-mentioned purpose, the technical solution adopted by the present invention is：
A kind of detection primer of full premix UGT1A1*6 types and * 28 type multi-primerses specific PCR genetic polymorphism detections and its
Correspondent probe, it is as follows according to UGT1A1 genes * 6 site (G/A) Design for polymorphism, sequence：
Reverse primer：
UGT1A1 6G: 5’- GTCTTCAAGGTGTAAAATGCTTC -3’ （ SEQ ID No.1）；
UGT1A1 6A: 5’- GTACGTCTTCAAGGTGTAAAATGCCCT -3’（ SEQ ID No.2）；
Forward primer：
UGT1A1 6F: 5’- ACTGTTGATCCCAGTGGATGG-3’ （ SEQ ID No.3）；
Fluorescence probe：
TM-UGT1A1 6:5 ' the fluorophor quenching groups of-CTAGCACCTGACGCCTCGTTGT -3 '（ SEQ ID No.4）.
A kind of detection primer of full premix UGT1A1*6 types and * 28 type multi-primerses specific PCR genetic polymorphism detections
And its correspondent probe, according to the sites of UGT1A1 genes * 28（TA insertion mutations）Design for polymorphism, sequence are as follows：
Reverse primer：
UGT1A1 28TA6: 5’-CCTCTCCTACTTATATATATATATATGGCA -3’ （SEQ ID No.5）；
UGT1A1 28TA7: 5’-CCTCTCCTACTTATATATATATATATATGGCA-3’（SEQ ID No.6）；
Forward primer：
UGT1A1 28F: 5’-AGTATGAAATTCCAGCCAGTTCAAC-3’ （SEQ ID No.7）；
Fluorescence probe：
TM-UGT1A1 28:5 ' fluorophor-CTCCCTGCTACCTTTGTGGACTGACAGC-3 ' quenching groups（SEQ ID
No.8）.
Present invention also offers a kind of full premix UGT1A1*6 types and * 28 type multi-primerses specific PCR gene pleiomorphisms
The internal control primer pair and corresponding fluorescence probe, sequence of detection are as follows：
Internal control primer pair：
Forward primer：ALB F: 5’- GTTGCTGTCATCTCTTGTGGGCTGT -3’（SEQ ID No.9）；
Reverse primer：ALB R: 5’-CAAACTCATGGGAGCTGCTGGTTC-3’（SEQ ID No.10）；
Internal control fluorescence probe：
TM-ALB:5 ' fluorophor-CATGCCCACACAAATCTCTCCCTGGCA-3 ' quenching groups（ SEQ ID No.11）.
The invention provides a kind of buffer solution of the biglycan of protective agent containing PCR, it is ensured that the stability of full premix reagent.
The biglycan of use, can be trehalose or sucrose.
The invention provides the method for premixing the Taq polymerase of modification and primed probe and reaction buffer, Ke Yibao
The storage stability in 8 week under the conditions of extended storage stabilities and 4 degree is spent in-the 20 of the complete mixed reagent of card.The modification of Taq polymerase
Mode can be the modification of Taq monoclonal antibodies and chemical modification.
The probe of above-mentioned site (G/A) polymorphisms of detection hCCSP T1A1 genes * 6, the sites of detection hCCSP T1A1 genes * 28
（TA insertion mutations）The fluorophor that fluorescence probe 5 ' corresponding to the probe and internal control primer of polymorphism is held is suitable for fluorescence
The conventional use of fluorescent reporter group of PCR quantitative analyses, preferably FAM, TET, VIC, HEX or ROX, the quenching group at 3 ' ends are
Suitable for the conventional use of fluorescent quenching group of fluorescent PCR quantitative analysis, preferably BHQ -1, BHQ -2, Dabcyl,
Eclipse or TAMRA, preferred scheme connect for the 5 ' ends of UGT1A1 genes * 6 site (G/A) polymorphic detection fluorescence probe
Some fluorophors are FAM, and the quenching group that 3 ' ends are connected with is BHQ1；The sites of UGT1A1 genes * 28（TA insertion mutations）It is polymorphic
Property detection fluorescence probe the 5 ' fluorophors that are connected with of end be ROX, 3 ' to hold the quenching group being connected be BHQ1；Internal control fluorescence probe
The 5 ' fluorophors that are connected with of end be HEX, 3 ' to hold the quenching group being connected be BHQ2.
Draw present invention also offers a kind of premix entirely for being used to detecting hCCSP T1A1*6 types and * 28 type gene pleiomorphisms is multiple
Thing specific PCR detection kit, including site (G/A) polymorphisms of detection hCCSP T1A1 genes * 6 of the present invention and/or people
The sites of UGT1A1 genes * 28（TA insertion mutations）The detection primer of polymorphism and corresponding fluorescence probe and operation instructions.Tool
Body says, including the site G allele of detection hCCSP T1A1 genes * 6 and the site TA6 allele of hCCSP T1A1 genes * 28
Specific reaction liquid：UGT1A1 A PCR reaction solutions, and/or the site A allele of detection hCCSP T1A1 genes * 6 and people
The specific reaction liquid of the site TA7 allele of UGT1A1 genes * 28：UGT1A1 B PCR reaction solutions；Wherein PCR reaction solutions bag
Contained PCR to react outside the materials such as necessary buffer solution, magnesium ion, dNTP, also corresponding to comprising above-mentioned detection primer and probe with
And the Taq polymerase of modification, and PCR reaction protective agent biglycans.What the PCR reaction solutions of above-mentioned each specific detection included respectively
The sequence of primer and probe is as follows：
（1）UGT1A1 A PCR reaction solutions
UGT1A1 6G: 5’- GTCTTCAAGGTGTAAAATGCTTC -3’；
UGT1A1 6F: 5’- ACTGTTGATCCCAGTGGATGG-3’；
TM-UGT1A1 6:5 ' the fluorophor quenching groups of-CTAGCACCTGACGCCTCGTTGT -3 '；
UGT1A1 28TA6: 5’-CCTCTCCTACTTATATATATATATATGGCA -3’；
UGT1A1 28F: 5’-AGTATGAAATTCCAGCCAGTTCAAC-3’；
TM-UGT1A1 28:5 ' fluorophor-CTCCCTGCTACCTTTGTGGACTGACAGC-3 ' quenching groups；
（2）UGT1A1 B PCR reaction solutions
UGT1A1 6A: 5’- GTACGTCTTCAAGGTGTAAAATGCCCT -3’；
UGT1A1 6F: 5’- ACTGTTGATCCCAGTGGATGG-3’；
TM-UGT1A1 6:5 ' the fluorophor quenching groups of-CTAGCACCTGACGCCTCGTTGT -3 '；
UGT1A1 28TA7: 5’-CCTCTCCTACTTATATATATATATATATGGCA-3’；
UGT1A1 28F: 5’-AGTATGAAATTCCAGCCAGTTCAAC-3’；
TM-UGT1A1 28:5 ' fluorophor-CTCCCTGCTACCTTTGTGGACTGACAGC-3 ' quenching groups.
In the PCR reaction solution preferred schemes of mentioned reagent box, except including detection hCCSP T1A1 genes * 6 sites（G/A）It is more
State property and/or the sites of hCCSP T1A1 genes * 28（TA insertion mutations）Outside the detection primer of polymorphism and corresponding fluorescence probe, also
It is as follows including a pair of internal control primers and corresponding fluorescence probe, the sequence of internal control primer and probe：
Internal control primer：
ALB F: 5’- GTTGCTGTCATCTCTTGTGGGCTGT -3’；
ALB R: 5’-CAAACTCATGGGAGCTGCTGGTTC-3’；
Internal control fluorescence probe：
TM-ALB:5 ' fluorophor-CATGCCCACACAAATCTCTCCCTGGCA-3 ' quenching groups.
The sites of hCCSP T1A1 genes * 6 are detected in mentioned reagent box（G/A）Probe, the detection hCCSP T1A1 genes * of polymorphism
28 sites（TA insertion mutations）The fluorophor at the end of fluorescence probe 5 ' can be corresponding to the probe and internal control primer of polymorphism
Suitable for the conventional use of fluorescent reporter group of fluorescent PCR quantitative analysis, preferably FAM, TET, VIC, HEX or ROX, 3 ' ends
Quenching group be suitable for fluorescent PCR quantitative analysis conventional use of fluorescent quenching group, preferably BHQ -1, BHQ -2,
Dabcyl, Eclipse or TAMRA, preferred scheme are UGT1A1 genes * 6 site (G/A) polymorphic detection fluorescence probe
The fluorophor that 5 ' ends are connected with is FAM, and the quenching group that 3 ' ends are connected with is BHQ1；The sites of UGT1A1 genes * 28（TA insertions are prominent
Become）The fluorophor that 5 ' ends of polymorphic detection fluorescence probe are connected with is ROX, and the quenching group that 3 ' ends are connected with is BHQ1；Internal control
The fluorophor that 5 ' ends of fluorescence probe are connected with is HEX, and the quenching group that 3 ' ends are connected with is BHQ2.
Mentioned reagent box operation instructions include the description to PCR amplification conditions, and preferable PCR amplification conditions are：
The condition of pre-degeneration is：Temperature is 95 DEG C, and the time is 3 minutes；
PCR reactions are made up of first stage and second stage：
First stage is made up of 10 amplification cycles, and its condition is：
Denaturation：Temperature is 95 DEG C, and the time is 20 seconds；
Annealing extension：Temperature is 60 DEG C, and the time is 45 seconds；
Second stage is made up of 30 amplification cycles, and its condition is：
Denaturation：Temperature is 95 DEG C, and the time is 20 seconds；
Annealing extension：Temperature is 60 DEG C, and the time is 45 seconds（Fluorescence signal acquisition is set）.
Present invention also offers it is a kind of using above-mentioned detection primer and fluorescence probe or it is above-mentioned containing detection UGT1A1*
The full premix multi-primerses specific PCR detection kit of 6 types and * 28 type gene pleiomorphisms carries out UGT1A1*6 types and * 28 types
The method of genetic polymorphism detection, step include：
（1）Testing sample processing and template extraction
A. blood sample is extracted from person to be detected；
B. DNA is obtained from blood sample, it is recommended to use commercialized kit extracts peripheral blood genomic DNA；
C. DNA concentration and purity are determined, it is desirable to which concentration is more than 10ng/ μ l；OD260nm/OD280nm=1.7~2.0；
（2）Fluorescent PCR expands：
Template DNA to be detected is added containing the sites of detection hCCSP T1A1 genes * 6 of the present invention（G/A）Polymorphism and/or people
The sites of UGT1A1 genes * 28（TA insertion mutations）In the detection primer of polymorphism and corresponding fluorescent probe PCR reaction solution, glimmering
After being well mixed centrifugation in light PCR pipe, it is put into quantitative fluorescent PCR instrument and enters according to specific temperature cycles and signal acquisition program
Performing PCR reacts, and it is preferably square that template DNA to be detected preferably is added into the mentioned reagent box containing internal control primer and corresponding fluorescence probe
In the PCR reaction solutions of case, quantitative fluorescent PCR instrument is put into according to specific temperature after being well mixed centrifugation in fluorescent PCR pipe
Circulation and signal acquisition program enter performing PCR reaction, to monitor response availability.
Above-mentioned fluorescent PCR amplification scheme preferably enters performing PCR reaction using temperature below circulation and signal acquisition program：
The condition of pre-degeneration is：Temperature is 95 DEG C, and the time is 3 minutes；
PCR reactions are made up of first stage and second stage：
First stage is made up of 10 amplification cycles, and its condition is：
Denaturation：Temperature is 95 DEG C, and the time is 20 seconds；
Annealing extension：Temperature is 60 DEG C, and the time is 45 seconds；
Second stage is made up of 30 amplification cycles, and its condition is：
Denaturation：Temperature is 95 DEG C, and the time is 20 seconds；
Annealing extension：Temperature is 60 DEG C, and the time is 45 seconds；（Fluorescence signal acquisition is set）
（3）Analysis result
Under the conditions of above-mentioned PCR reaction systems and temperature cycling program, purpose detection fluorescence in specific PCR reaction system is observed
Whether signal forms logarithmic amplification " S " type curve, and the DNA sample to be checked is the spy if logarithmic amplification " S " type curve is formed
The positive of polymorphism type representated by different in nature reaction system.
Present invention also offers the sites of one kind detection hCCSP T1A1 genes * 6（G/A）Polymorphism and/or hCCSP T1A1 genes *
28 sites（TA insertion mutations）The detection primer of polymorphism and corresponding fluorescence probe and internal control primer and correspondent probe are being examined
The application surveyed in hCCSP T1A1*6 types and * 28 type gene pleiomorphisms.
Present invention also offers one kind to contain the sites of detection hCCSP T1A1 genes * 6（C/T）Polymorphism and/or hCCSP T1A1
The sites of gene * 28（TA insertion mutations）The detection primer of polymorphism and corresponding fluorescence probe and internal control primer and correspondent probe
Kit detection hCCSP T1A1*6 types and * 28 type gene pleiomorphisms in application.
Relevant content in technical scheme of the present invention is explained as follows.
1. operation principle of the present invention is：Allele specific pcr (Allele Specific PCR, ASPCR), also known as
For allele specific amplification method (Allele Specific Amplification, ASA) or amplification Refracting Mutation system
Unite (Amplification Refractory Mutation System ARMS-PCR), its general principle is due to Taq DNA
Polymerase lacks 3 ' → 5 ' 5 prime excision enzyme activities, and when entering performing PCR reaction, if the end of primer 3 ' forms mispairing, chain extension reaction will
It is obstructed because of the obstacle that 3 ', 5 '-phosphodiester bond is formed, causes PCR primer amount drastically to reduce, in certain amplification cycles,
Special amplified production is not will detect that；Conversely, 3 ' end pairings are then capable of detecting when amplified production.Then, for known more
State property site, its polymorphic base is designed at into detection primer 3 ' are held, and after amplified reaction, are passed through gel electrophoresis and are observed product
Whether there is the base type that can determine whether pleomorphism site.
Quantitative fluorescent PCR is that (Real-time PCR) is 1996 by Applied Biosystems companies of the U.S.
A kind of new quantitative experiment technology released, a specific fluorescence is added while pair of primers is added when PCR is expanded and is visited
Pin, the probe are claimed " TaqMan spies ", are an oligonucleotides, both ends respectively one reporter fluorescence group of mark and one be quenched it is glimmering
Light group.When probe is complete, the fluorescence signal of reporter group transmitting is quenched group absorptions；When just starting, probe is incorporated in
On any one of DNA is single-stranded；When PCR is expanded, 5 ' → 3 ' 5 prime excision enzyme activities of Taq enzyme degrade probe digestion, make reporter fluorescence
Group and the separation of quenching fluorescence group, so as to which fluorescence monitoring system can receive fluorescence signal, i.e., often expand a DNA, just
There is a fluorescence molecule to be formed, the accumulation and PCR primer for realizing fluorescence signal form Complete Synchronization.
By the product of real-time fluorescence PCR technology for detection " allele specific pcr " reaction system based on fluorescence probe,
According to formed amplification curve fluorescence signal in specific threshold period（Ct values）Size can determine whether in corresponding detection architecture
The base type in the corresponding purpose site of template.
2. the design of the primer and probe in technical scheme of the present invention：According in US National Biotechnology Information
The reference sequences of UGT1A1 genes disclosed in heart NCBI GenBank GeneBank（NG_002601.2）And
* 6 sites (G/A) polymorphism disclosed in dbSNP databases（SNP ID ：rs4148323）With * 28 sites (TA insertion mutations)
Polymorphism（SNP ID ：rs34983651）Information, separately designed using the softwares of Primer Express 3.0 of ABI companies
Detect the primer and probe of site (G/A) polymorphisms of UGT1A1 genes * 6 and * 28 sites (TA insertion mutations) polymorphism.In order to
The validity of reaction system is monitored, internal control primer and probe are added in detection architecture, the present invention chooses mankind's conservative gene ALB
One section of sequence（Its GeneBank reference sequences is numbered：NG_009291.1）, using the Primer Express of ABI companies
3.0 Software for Design detection primers and probe.
In technical scheme of the present invention, the primer（primer）Refer to the few nucleosides being made up of a number of dNTP
Acid sequence, it is generally artificial synthesized by DNA synthesizer, purified after synthesis through polyacrylamide gel electrophoresis or other proper methods.
In PCR, primer can be combined with region complementary therewith on purpose nucleic acid chain to be amplified, and its function is conduct
The starting point of nucleotide polymerization effect, on 3 '-OH of primer, nucleotides is synthesized in the form of diester linkage, therefore primer
3 '-OH must be free.Archaeal dna polymerase can proceed by extension by its 3 ' end, synthesize new nucleic acid chains.
In technical scheme of the present invention, the fluorescence probe is a kind of oligonucleotide probe, and fluorophor is connected to
5 ' ends of probe, and quenching group is then artificial synthesized in 3 ' ends, generally use DNA synthesizer, through polyacrylamide after synthesis
Amine gel electrophoresis or the purifying of other proper methods.Currently used fluorophor has FAM, TET, VIC, HEX, ROX etc..Base is quenched
There are BHQ -1, BHQ -2, Dabcyl, Eclipse, TAMRA etc. in group.
In technical scheme of the present invention, the Taq archaeal dna polymerases of the modification, generally there are two kinds of method of modifying, with side
Help the stability for improving enzyme：（1）Modified using Taq archaeal dna polymerases monoclonal antibody, after Taq DNA polymerase and antibody binding
Highly stable polymer is formed, it is highly stable under the conditions of normal temperature or Cord blood, when reacting startup, under the high temperature conditions
（95 degree）, Taq archaeal dna polymerases separate with antibody, start DNA formula polymerisation.
3. in technical scheme of the present invention, described internal control primer and its corresponding probe can be used for monitoring reaction
The index of validity, to judge whether that the factors such as template quality, mechanical disorder, reagent stability influence result of the test
Situation.Under normal circumstances, PCR normally expands the exponential amplification curve that can be formed, and vivid is described as " S " type amplification curve,
Show that system normally expands.When purpose detection fluorescence signal forms logarithmic amplification " S " type curve in specific PCR reaction system
When, for the positive findings of polymorphism type, also illustrate that PCR system amplification is normal, without using internal control primer and its corresponding spy
Pin carries out the checking of result.However, working as purpose detection fluorescence signal in specific PCR reaction system does not form logarithmic amplification " S "
Type curve, the negative findings of polymorphism type of the testing result representated by the specific reaction system, but it could also be possible that mould
The factors such as plate quality, mechanical disorder, reagent stability influence result of the test, in this case, can use internal control primer and its
Corresponding probe is excluded.When purpose detection fluorescence signal does not form logarithmic amplification " S " type song in specific PCR reaction system
Line, and when internal control signal forms normal logarithmic amplification " S " type curve, can verify polymorphism type negative findings it is accurate
Property.And work as purpose detection fluorescence signal in specific PCR reaction system and do not form logarithmic amplification " S " type curve, and internal control signal
When also not forming normal logarithmic amplification " S " type curve, then illustrating to go wrong on experiment condition or instrument influences result of the test, and
The negative findings of non-polymorphism type.
Because above-mentioned technical proposal is used, the present invention has following advantages and effect compared with prior art：
1. the sites of detection hCCSP T1A1 genes * 6 in technical solution of the present invention（G/A）Polymorphism and/or hCCSP T1A1 genes * 28
Site (TA insertion mutations）The detection primer of polymorphism and corresponding fluorescence probe and internal control primer and correspondent probe, its PCR
Detection specificity is very high, and uses real-time fluorescence PCR technology, and testing result interpretation is easy.
2nd, the present invention detects gene pleiomorphism for multi-primerses specific PCR, and two tube reaction systems can detect people simultaneously
The sites of UGT1A1 genes * 6（G/A）Polymorphism and * 28 sites（TA insertion mutations）Polymorphism；Reaction enzymes of the present invention are antibody simultaneously
Coated Taq archaeal dna polymerases or the Taq DNA polymerase of chemical modification, support to premix reaction enzymes and reaction solution, and premix
Taq enzyme activity and reaction solution stability are unaffected afterwards.
3rd, the detection primer in technical solution of the present invention and probe are cheap, and are not required to be sequenced in experimentation,
Using real-time fluorescence PCR technology platform, high flux detection can be achieved, while testing cost is saved, substantially reduce inspection
The cycle is surveyed, improves the efficiency of detection.
Brief description of the drawings
Accompanying drawing 1 is certain sample UGT1A1 A PCR reaction detection figures；
Accompanying drawing 2 is certain sample UGT1A1 B PCR reaction detection figures；
Accompanying drawing 3 is the site sequencer maps of certain sample UGT1A1 genes * 6；
Accompanying drawing 4 is the site sequencer maps of certain sample UGT1A1 genes * 28；
Embodiment
Used term in the present invention is unless otherwise indicated, general to have what those of ordinary skill in the art were generally understood that
Implication.
The present invention is described in further detail with reference to specific embodiment and with reference to data.It should be understood that these embodiments are only
It is rather than the scope limiting the invention in any way in order to demonstrate the invention.
In the examples below, the various processes and method not being described in detail are conventional methods as known in the art, are led to
Frequently with normal condition such as Sambrook et al., molecular cloning：Laboratory manual（NewYork：Cold Spring Harbor
Laboratory Press, 1989）Described in condition, or according to the method proposed by manufacturer.It is used in the present invention
Quantitative real time PCR Instrument model ABI 7500.
The design of the primer and probe of embodiment 1.：
According to UGT1A1 genes disclosed in US National Biotechnology Information center NCBI GenBank GeneBank
Reference sequences（NG_002601.2）And * 6 sites (G/A) polymorphism disclosed in dbSNP databases（SNP ID ：
rs4148323）With * 28 sites (TA insertion mutations) polymorphism（SNP ID ：rs34983651）Information, using ABI companies
The softwares of Primer Express 3.0 separately design site (G/A) polymorphisms of detection UGT1A1 genes * 6 and * 28 site (TA
Insertion mutation) polymorphism primer and probe.In order to monitor the validity of reaction system, internal control primer is added in detection architecture
With probe, the present invention chooses mankind's conservative gene ALB one section of sequence（Its GeneBank reference sequences is numbered：NG_
009291.1）, using the Software for Design detection primers of Primer Express 3.0 and probe of ABI companies.
The sequence for detecting site (G/A) polymorphism primers of UGT1A1 genes * 6 and probe is as follows：
Reverse primer：
UGT1A1 6G: 5’- GTCTTCAAGGTGTAAAATGCTTC -3’；
UGT1A1 6A: 5’- GTACGTCTTCAAGGTGTAAAATGCCCT -3’；
Forward primer：
UGT1A1 6F: 5’- ACTGTTGATCCCAGTGGATGG-3’；
Fluorescence probe：
TM-UGT1A1 6: 5’FAM -CTAGCACCTGACGCCTCGTTGT-3’BHQ1。
The sequence for detecting site (TA insertion mutations) polymorphism primers of UGT1A1 genes * 28 and probe is as follows：
Reverse primer
UGT1A1 28TA6: 5’-CCTCTCCTACTTATATATATATATATGGCA -3’；
UGT1A1 28TA7: 5’-CCTCTCCTACTTATATATATATATATATGGCA-3’；
Forward primer
UGT1A1 28F: 5’-AGTATGAAATTCCAGCCAGTTCAAC-3’；
Fluorescence probe
TM-UGT1A1 28: 5’ROX-CTCCCTGCTACCTTTGTGGACTGACAGC-3’BHQ1
In order to monitor the validity of reaction system, internal control primer and probe are added in detection architecture, the present invention chooses the mankind and protected
Keep Gene A LB one section of sequence（Its reference sequences is numbered：NG_009291.1）, using the Primer Express of ABI companies
3.0 Software for Design detection primers and probe, particular sequence are as follows：
Internal control primer：
ALB F: 5’- GTTGCTGTCATCTCTTGTGGGCTGT -3’；
ALB R: 5’-CAAACTCATGGGAGCTGCTGGTTC-3’；
Internal control fluorescence probe：
TM-ALB: 5’HEX-CATGCCCACACAAATCTCTCCCTGGCA-3’BHQ2。
The preparation of the primer of embodiment 2.
Transfer to Synesis Company to synthesize designed primer and probe sequence, typically synthesized using automation equipment, need to provide
Synthesize qualification test report.
Embodiment 3：Full premix UGT1A1*6 types and * 28 type multi-primerses specific PCR genetic polymorphism detection kits
Preparation
Prepare the special of the site G allele of detection hCCSP T1A1 genes * 6 and the site TA6 allele of hCCSP T1A1 genes * 28
Property reaction solution：UGT1A1 A PCR reaction solutions；Detect the site A allele of hCCSP T1A1 genes * 6 and hCCSP T1A1 genes * 28
The specific reaction liquid of site TA7 allele：UGT1A1 B PCR reaction solutions；Wherein PCR reaction solutions contain PCR reaction must
Must buffer solution, magnesium ion, outside the material such as dNTP, further comprises detection primer and probe and internal control primer and probe and antibody
The Taq DNA polymerase and PCR protective agent trehaloses of modification.The PCR reaction solutions concentration of component of above-mentioned each specific detection and comprising
Primer and probe sequence information it is as follows：
The PCR reaction solution components of table 1.
| Reaction solution component | Concentration |
| ddH2O | -- |
| Buffer solution | 10× |
| Magnesium ion | 25mM |
| dNTP MIX | 10mM |
| Upstream detection primer 1 | 10µM |
| Downstream detector primer 1 | 10µM |
| Detection signal probe 1 | 10µM |
| Upstream detection primer 2 | 10µM |
| Downstream detector primer 2 | 10µM |
| Detection signal probe 2 | 10µM |
| Upstream internal control primer | 10µM |
| Downstream internal control primer | 10µM |
| Internal control signal probe | 10µM |
| Taq archaeal dna polymerases | 5U/μL |
（1）UGT1A1 A PCR reaction solutions
UGT1A1 6G: 5’- GTCTTCAAGGTGTAAAATGCTTC -3’；
UGT1A1 6F: 5’- ACTGTTGATCCCAGTGGATGG-3’；
TM-UGT1A1 6: 5’FAM-CTAGCACCTGACGCCTCGTTGT -3’BHQ1；
UGT1A1 28TA6: 5’-CCTCTCCTACTTATATATATATATATGGCA -3’；
UGT1A1 28F: 5’-AGTATGAAATTCCAGCCAGTTCAAC-3’；
TM-UGT1A1 28: 5’ROX-CTCCCTGCTACCTTTGTGGACTGACAGC-3’BHQ1；
ALB F: 5’- GTTGCTGTCATCTCTTGTGGGCTGT -3’；
ALB R: 5’-CAAACTCATGGGAGCTGCTGGTTC-3’；
TM-ALB: 5’HEX-CATGCCCACACAAATCTCTCCCTGGCA-3’BHQ2。
（2）UGT1A1 B PCR reaction solutions
UGT1A1 6A: 5’- GTACGTCTTCAAGGTGTAAAATGCCCT -3’；
UGT1A1 6F: 5’- ACTGTTGATCCCAGTGGATGG-3’；
TM-UGT1A1 6: 5’FAM-CTAGCACCTGACGCCTCGTTGT -3’BHQ1；
UGT1A1 28TA7: 5’-CCTCTCCTACTTATATATATATATATATGGCA-3’；
UGT1A1 28F: 5’-AGTATGAAATTCCAGCCAGTTCAAC-3’；
TM-UGT1A1 28: 5’ROX-CTCCCTGCTACCTTTGTGGACTGACAGC-3’BHQ1；
ALB F: 5’- GTTGCTGTCATCTCTTGTGGGCTGT -3’；
ALB R: 5’-CAAACTCATGGGAGCTGCTGGTTC-3’；
TM-ALB: 5’HEX-CATGCCCACACAAATCTCTCCCTGGCA-3’BHQ2。
Embodiment 4：The method of full premix UGT1A1*6 types and * 28 type multi-primerses specific PCR genetic polymorphism detections
The first step：Prepare DNA
（1）Blood sample is extracted from person to be detected；
（2）DNA is obtained from blood sample, it is recommended to use commercialized kit extracts peripheral blood genomic DNA；
（3）DNA concentration and purity are determined, it is desirable to which concentration is more than 10ng/ μ l；OD260nm/OD280nm=1.7~2.0。
Second step：PCR reaction systems are prepared
In the special pipe of quantitative fluorescent PCR PCR reaction systems are prepared according to following table
Table 2.PCR reaction systems form
| PCR reaction solutions | 23μl |
| Genomic DNA | About 80ng |
Above-mentioned PCR reaction solutions are anti-using the UGT1A1 A PCR reaction solutions in the kit of the preparation of embodiment 3, UGT1A1 B PCR
Liquid is answered, the compound method that can also be provided according to the table 1 of embodiment 3 is prepared containing the sites of detection hCCSP T1A1 genes * 6（G/A）It is more
State property and the sites of detection hCCSP T1A1 genes * 28（TA insertion mutations）The detection primer and probe of polymorphism simultaneously contain internal control primer
With the PCR reaction solutions of probe;
3rd step：Upper machine testing
According to following table, temperature cycles and signal acquisition program are set
The PCR response procedures of table 3.
Note：" * " place sets FAM, ROX and HEX triple channel collection fluorescence signal.
4th step：Analysis result
Under the conditions of above-mentioned PCR reaction systems and temperature cycling program, normal logarithmic amplification " S " type curve is formed in internal control signal
Precondition under, observe specific PCR reaction system in purpose detection fluorescence signal whether formed logarithmic amplification " S " type song
Line, the DNA sample to be checked is the polymorphism class representated by the specific reaction system if logarithmic amplification " S " type curve is formed
The positive of type.Such as：Certain sample to be checked is in UGT1A1 A PCR reaction systems and UGT1A1 B PCR reaction systems detection fluorescence
Signal（FAM passages）Logarithmic amplification " S " type curve is respectively formed, then it is the G/A heterozygous of UGT1A1 * 6 to prompt the sample.
Accompanying drawing 1 is certain sample UGT1A1 A reaction systems detection figure, and detection figure is shown in UGT1A1 A reaction systems
Internal control fluorescence signal is qualified, and detection fluorescence signal in UGT1A1*6 G reaction systems（FAM passages）Form logarithmic amplification " S "
Type curve, then judge that the sample contains UGT1A1*6 G allele；Fluorescence letter is detected in UGT1A1*28 TA6 reaction systems
Number（ROX passages）Logarithmic amplification " S " type curve is formed, then judges that the sample contains UGT1A1*28 TA6 allele.
Accompanying drawing 2 is certain sample UGT1A1 B reaction systems detection figure, and detection figure is shown in UGT1A1 B reaction systems
Internal control fluorescence signal is qualified, and detection fluorescence signal in UGT1A1*6 A reaction systems（FAM passages）Amplification curve is not formed,
Then judge that the sample is free of UGT1A1*6 A allele；Fluorescence signal is detected in UGT1A1*28 TA7 reaction systems（ROX
Passage）Logarithmic amplification " S " type curve is formed, then judges that the sample contains UGT1A1*28 TA7 allele.
Accompanying drawing 3 is the site sequencer maps of sample UGT1A1 genes * 6 in accompanying drawing 1 and accompanying drawing 2, and sequencing result shows the sample not
UGT1A1*6 GG wild types.Kit testing result is consistent with sequencing result.
Accompanying drawing 4 is the site sequencer maps of sample UGT1A1 genes * 28 in accompanying drawing 1 and accompanying drawing 2, and sequencing result shows that the sample is
UGT1A1*28 TA6/7 are mutated heterozygous.Kit testing result is consistent with sequencing result.
Pay attention to：
The above embodiments merely illustrate the technical concept and features of the present invention, and its object is to allow person skilled in the art's energy
Solution present disclosure much of that is simultaneously implemented according to this, and it is not intended to limit the scope of the present invention.It is all spiritual according to the present invention
The equivalent change or modification that essence is made, should all be included within the scope of the present invention.
Sequence table
<110> OrganizationName :Suzhou Kuangyuan Molecular Biotechnology Co., Ltd.
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<120> Title :A kind of full premix UGT1A1 multiplex PCR genetic polymorphism detection kits and method
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Claims (11)
1. the detection primer of a kind of full premix UGT1A1*6 types and * 28 type multi-primerses specific PCR genetic polymorphism detections and
Its correspondent probe, it is characterised in that the detection primer and probe are according to UGT1A1 genes * 6 site (G/A) Design for polymorphism, sequence
Arrange as follows：
Reverse primer：
UGT1A1 6G: 5’- GTCTTCAAGGTGTAAAATGCTTC -3’；
UGT1A1 6A: 5’- GTACGTCTTCAAGGTGTAAAATGCCCT -3’；
Forward primer：
UGT1A1 6F: 5’- ACTGTTGATCCCAGTGGATGG-3’；
Fluorescence probe：
TM-UGT1A1 6:5 ' the fluorophor quenching groups of-CTAGCACCTGACGCCTCGTTGT -3 '.
2. the detection primer of a kind of full premix UGT1A1*6 types and * 28 type multi-primerses specific PCR genetic polymorphism detections and
Its correspondent probe, it is characterised in that the detection primer and probe are according to the sites of UGT1A1 genes * 28（TA insertion mutations）It is polymorphic
Property design, sequence is as follows：
Reverse primer：
UGT1A1 28TA6: 5’-CCTCTCCTACTTATATATATATATATGGCA -3’；
UGT1A1 28TA7: 5’-CCTCTCCTACTTATATATATATATATATGGCA-3’；
Forward primer：
UGT1A1 28F: 5’-AGTATGAAATTCCAGCCAGTTCAAC-3’；
Fluorescence probe：
TM-UGT1A1 28:5 ' the fluorophor quenching groups of-CTCCCTGCTACCTTTGTGGACTGACAGC -3 '.
A kind of 3. internal control primer pair of full premix UGT1A1*6 types and * 28 type multi-primerses specific PCR genetic polymorphism detections
And corresponding fluorescence probe, it is characterised in that the internal control primer pair and the sequence of corresponding fluorescence probe are as follows：
Internal control primer pair：
Forward primer：ALB F: 5’- GTTGCTGTCATCTCTTGTGGGCTGT -3’；
Reverse primer：ALB R: 5’-CAAACTCATGGGAGCTGCTGGTTC-3’；
Internal control fluorescence probe：
TM-ALB:5 ' fluorophor-CATGCCCACACAAATCTCTCCCTGGCA-3 ' quenching groups.
4. primer pair and corresponding fluorescence probe as described in claim 1-3, it is characterised in that the fluorescence probe 5 ' is held glimmering
Light group is FAM, TET, VIC, HEX or ROX, the quenching groups at 3 ' ends are BHQ -1, BHQ -2, Dabcyl, Eclipse or
TAMRA。
A kind of 5. modification of full premix UGT1A1*6 types and * 28 type multi-primerses specific PCR genetic polymorphism detection kits
The PCR buffer solutions of reaction enzymes and the composition containing biglycan premix mode.
6. a kind of full premix UGT1A1*6 types and * 28 type multi-primerses specific PCR genetic polymorphism detection kits, its feature
It is that the kit includes detection primer described in claim 1 and its correspondent probe and/or includes inspection described in claim 2
Survey primer and its correspondent probe.
7. a kind of full premix UGT1A1*6 types as claimed in claim 6 and * 28 type multi-primerses specific PCR gene pleiomorphisms
Detection kit, it is characterised in that the kit also includes internal control primer pair as claimed in claim 3 and corresponding fluorescence probe.
8. the detection method of a kind of full premix UGT1A1*6 types and * 28 type multi-primerses specific PCR gene pleiomorphisms, the side
Primer and probe of the method described in including the use of claim 1 and/or claim 2.
9. a kind of full premix UGT1A1*6 types as claimed in claim 8 and * 28 type multi-primerses specific PCR gene pleiomorphisms
Detection method, it is characterised in that methods described is also including the use of the primer and probe described in claim 3.
10. a kind of method of full premix UGT1A1*6 types and * 28 type multi-primerses specific PCR genetic polymorphism detections, described
Kit of the method described in including the use of claim 6-7.
11. the primer and probe described in claim 1-3 are in a kind of full premix UGT1A1*6 types and * 28 type multi-primerses specificity
Application in pcr gene polymorphic detection.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109295171A (en) * | 2018-10-22 | 2019-02-01 | 北京华夏时代生物工程有限公司 | The nucleic acid molecule combination and application of detection SNP are sequenced for fluorescence in situ hybridization |
| CN109971841A (en) * | 2019-04-16 | 2019-07-05 | 北京和合医学诊断技术股份有限公司 | The method and its application of gene mononucleotide polymorphism are detected by LC-MS |
-
2017
- 2017-09-28 CN CN201710899416.5A patent/CN107841559A/en active Pending
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109295171A (en) * | 2018-10-22 | 2019-02-01 | 北京华夏时代生物工程有限公司 | The nucleic acid molecule combination and application of detection SNP are sequenced for fluorescence in situ hybridization |
| CN109971841A (en) * | 2019-04-16 | 2019-07-05 | 北京和合医学诊断技术股份有限公司 | The method and its application of gene mononucleotide polymorphism are detected by LC-MS |
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Application publication date: 20180327 |