CN107254543A - PCR primer combination, kit and real time fluorescent quantitative detection method for detecting RhD mRNA spliceosomes - Google Patents
PCR primer combination, kit and real time fluorescent quantitative detection method for detecting RhD mRNA spliceosomes Download PDFInfo
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- CN107254543A CN107254543A CN201710674747.9A CN201710674747A CN107254543A CN 107254543 A CN107254543 A CN 107254543A CN 201710674747 A CN201710674747 A CN 201710674747A CN 107254543 A CN107254543 A CN 107254543A
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- mrna
- rhd
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- 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
- C12Q1/6851—Quantitative amplification
Abstract
It is used to detect the combination of RhD mRNA spliceosome real-time fluorescence quantitative PCRs primer, kit and detection method the invention discloses one kind, belongs to technical field of biological.The present invention is not disturbed by other non-target gene for the combination of RhD mRNA spliceosome sequences Design real-time fluorescence quantitative PCRs primer, the high specificity of the primer, quick, efficiently and accurately RhD mRNA spliceosomes can be quantified.Present invention application real time fluorescence quantifying PCR method is quantitatively detected that method is simple, accurate, is polluted in no pipeline, without gel electrophoresis, the method can be with the copy number of accurate quantitative analysis Different Individual RhD mRNA spliceosomes to RhD mRNA spliceosomes.As a result favorable reproducibility, detection sensitivity is high, high specificity, available for high-throughout sample detection.
Description
Technical field
The present invention relates to a kind of PCR primer combination for being used to detect RhD mRNA spliceosomes, also relate to draw comprising this
The kit and RhD mRNA spliceosome real time fluorescent quantitative detection methods of thing combination, belong to technical field of biological.
Background technology
The complexity of RH genes is embodied directly in the polymorphism of RhD antigen presentations, this be considered as not only with RhD mRNA
It is heterogeneous related about the missing also to related gene extron, quantity expression etc. in level.Le Van Kim etc. are gone back simultaneously
It was found that：The gene transcript [1] that RHCE genes have alternative splicing and are variously formulated, same has researcher's hair
There is the 7th extron to the alternative splicing [2] of the Various Complex of the 9th extron in the individual of normal D antigen positives now,
The polymorphism of the spliceosome determines the characteristic of RHD gene expression D antigens.The genetic background of blood group gene has poor between obvious race
It is different, it is because the RhD mRNA genes of different ethnic groups the various montage of selectivity occur and occurred caused by RhD hypotypes [3].MRNA is
It, by DNA is via transcription, is that next step is translated into egg with corresponding hereditary message that messenger RNA mRNAs, which are,
White matter provides required message.Based on RHD gene polynorphisms, the method for real time fluorescent quantitative is set up herein to Different Individual
RhD mRNA spliceosomes are detected, the difference of Different Individual RHD genes is understood by monitoring the polymorphism of RhD mRNA expression.It is real
When quantitative fluorescent PCR (real-time quantitative PCR)/QRT-PCR, refer to add in PCR reaction systems glimmering
Signal thing, monitors whole PCR processes using the accumulation of fluorescence signal, unknown template is quantified by amplification curve in real time
The method of analysis.
[1]LE VAN K C,MOURO I,CHERIF-ZAHAR B,et al.Molecular cloning and
primary structure of the human blood group RhD polypeptideProe Natl.Acad Sei
USA,1992,89(22):10925-10929.
[2] Shao Chaopeng, Xiong Wen, wait to substitute the RhD mRNA. China blood transfusion magazine that shearing forms diversified forms, 2004,17
(5)：310-314.
[3] LE VAN K C, CHERIF-ZAHAR B, RAYNAL V, et al.Multiple Rh messenger RNA
isoforms are produced by alternative splicing.Blood,1992,80(4):1074-1078.
The content of the invention
In order to overcome shortcoming and defect present in prior art, RhD is detected it is an object of the invention to provide one kind
PCR primer combination, kit and the real-time fluorescence quantitative PCR detection method of mRNA spliceosomes.
The purpose of the present invention is achieved through the following technical solutions：
A kind of PCR primer combination for being used to detect RhD mRNA spliceosomes, includes at least one pair of of following three pairs of primers：
(1) it is used to expand 1 couples of total length RhD mRNA spliceosomes totally 2 primers, the sequence of 2 primers is respectively：
Sense primer：5'-CCCACAGCTCCATCATGGG-3'(SEQ ID NO:1),
Anti-sense primer：5'-GCCAATCATGCCATTGCCGGC-3'(SEQ ID NO:2)；
(2) it is used for 1 couple totally 2 primers for expanding missing exon-7 RhD mRNA spliceosomes, the sequence difference of 2 primers
For：
Sense primer：5'-TGGCTGGGCTGATCTCCG-3'(SEQ ID NO:3)
Anti-sense primer：5'-TGGAAGCCAATCCGGCAGGT-3'(SEQ ID NO:4)；
(3) it is used to expanding missing exon-7,1 couples of 8,9 RhD mRNA spliceosomes totally 2 primers, the sequence of 2 primers
Respectively：
Sense primer：5'-TGGCTGGGCTGATCTCCG-3'(SEQ ID NO:5)
Anti-sense primer：5'-CAAATGAGGAAACGGCAGGT-3'(SEQ ID NO:6).
Preferably, it is preferable that the concentration of each primer is 5pmol/L in the Primer composition.
A kind of kit of detection RhD mRNA spliceosomes, including described PCR primer combination thing.
Preferably, the described kit for being used to detect RhD mRNA spliceosomes also includes PCR grades of H2O, Master
Mix。
A kind of real-time fluorescence quantitative PCR detection method for being used to detect RhD mRNA spliceosomes, comprises the following steps：
(3) several various concentrations standard items are prepared；
(4) PCR reaction solutions, including above-mentioned PCR primer combination thing are prepared；
(3) PCR is expanded：The standard items or testing sample of above-mentioned PCR reaction solutions and various concentrations are mixed into performing PCR respectively
Amplification；
(4) the data creating standard curve obtained using the standard items of various concentrations；
(5) concentration of testing sample is calculated using standard curve.
Preferably, in the step (1), the standard items are selected from following at least one shown nucleotide sequence：
Total length standard items：
5'-CCCACAGCTCCATCATGGGCTACAACTTCAGCTTGCTGGGTCTGCTTG
GAGAGATCATCTACATTGTGCTGCTGGTGCTTGATACCGTCGGAGCCGGCAA TGGCATGATTGGC-3'(SEQ ID
NO:7)
Lack exon-7 standard items：
5'-TGGCTGGGCTGATCTCCGTCGGGGGAGCCAAGTACCTGCCGGATTGG CTTCCA-3'(SEQ ID
NO:8)
Lack exon-7,8,9 standard items：
5'-TGGCTGGGCTGATCTCCGTCGGGGGAGCCAAGTACCTGCCGTTTCCTC ATTTG-3'(SEQ ID
NO:9)。
Preferably, in the step (1), described some one kind in 6,8,10,12,16.
Preferably, the gradient between the step (1), the adjacent concentration of standard items of the various concentrations is 1 order of magnitude,
I.e. concentration differs 10 times.
It is preferred that, in the step (2), PCR grades of H2O, Master Mix are also included in the PCR reaction solutions.
It is preferred that, in the step (3), the response procedures of PCR amplifications are：95 DEG C of pre-degeneration 10min；96 DEG C of denaturation 10s,
60 DEG C of renaturation 3s, 72 DEG C extend 3 seconds, circulate 35 times, 72 DEG C re-extend 10s.
Preferably, in the step (5), the calculating is carried out using 2nd Derivative Max calculating methods.
Main advantages of the present invention include：
The present invention for RhD mRNA spliceosome sequences Design real-time fluorescence quantitative PCRs primer combination, the primer it is special
Property it is strong, do not disturbed, quick, efficiently and accurately RhD mRNA spliceosomes can be quantified by other non-target gene.
Present invention application real time fluorescence quantifying PCR method is quantitatively detected that method is simple, essence to RhD mRNA spliceosomes
Really, polluted in no pipeline, without gel electrophoresis, the method can be with the copy of accurate quantitative analysis Different Individual RhD mRNA spliceosomes
Number.As a result favorable reproducibility, detection sensitivity is high, high specificity, available for high-throughout sample detection.
Brief description of the drawings
Fig. 1 shows that whole blood extracts high-quality, high-purity mRNA electrophoretograms.
Fig. 2 RhD mRNA spliceosome real time fluorescent quantitatives are monitored in real time.
Embodiment
Following embodiments and test example are only described in further detail to the present invention, but do not constitute any limit to the present invention
System.
Embodiment 1
The preparation method of the PCR primer combination thing of RhD mRNA spliceosomes is detected, Primer composition is drawn comprising following 3 Duis
At least one pair of in thing：
For expanding 1 couples of total length RhD mRNA spliceosomes totally 2 primers, the sequence of 2 primers is respectively：
Sense primer：5'-CCCACAGCTCCATCATGGG-3'(SEQ ID NO:1),
Anti-sense primer：5'-GCCAATCATGCCATTGCCGGC-3'(SEQ ID NO:2)；
For 1 couple totally 2 primers for the RhD mRNA spliceosomes for expanding missing exon-7, the sequence of 2 primers is respectively：
Sense primer：5'-TGGCTGGGCTGATCTCCG-3'(SEQ ID NO:3)
Anti-sense primer：5'-TGGAAGCCAATCCGGCAGGT-3'(SEQ ID NO:4)；And
For expanding missing exon-7,1 couples of 8,9 RhD mRNA spliceosomes totally 2 primers, the sequence point of 2 primers
It is not：
Sense primer：5'-TGGCTGGGCTGATCTCCG-3'(SEQ ID NO:5)
Anti-sense primer：5'-CAAATGAGGAAACGGCAGGT-3'(SEQ ID NO:6).
After upstream and downstream primer in equal volume mixing, that is, obtain Primer composition.
Preferably, the concentration of each primer is 5pmol/L in the Primer composition.
Embodiment 2
1 materials and methods
1.1 doses and instrument
MRNA extracts kits：LEV simplyRNA Blood Kit (are given birth to by Promega companies
Production)；CDNA reverse transcription reagent box：Transcriptor High Fidelity cDNA Synthesis Kit are (public by Roche
Department's production)；Real-time fluorescence quantitative PCR amplification instrument：Software is carried by German Roche Diagnostics GmbH
For.RhD mRNA spliceosome pcr amplification primer things are synthesized by TaKaRa companies.
The preparation of 1.2 standard curves
1.2.1 artificial synthesized ssDNA as standard curve each spliceosome standard items of standard items, RhD mRNA and primer sequence
Row are shown in Table 1.
1.2.2 standard items gradient dilution：Standard items are dissolved with distilled water, it is 50ng/ul to make its concentration.It is diluted to 10
The concentration of copy number, successively decrease 1 order of magnitude every time, totally 12 gradients：12 0.5ml PCR pipes are taken, since the 2nd pipe (the
One pipe is stoste pipe) often pipe add distilled water 90ul, the .. that carries out mark 1,2,3 ..., 12, add 10ul in the 2nd dilution tube
50ng/ul ssDNA libraries, outstanding whirlpool suctions out 10ul and is added to the 3rd pipe, operates successively after mixing, until being diluted to last
Pipe.The gradient dilution liquid of last pipe removes.
1.3PCR amplification operations：
1.3.1 mRNA is extracted, reverse transcription is cDNA, and using cDNA as detection sample.
1.3.2 standard items：Using respective standard items (table 1), and 3 multiple holes are set.
1.3.3 primer：The primer is that the upstream anti-sense primer described in embodiment 1 in three pairs of primers is mixed to get respectively
Three kinds of Primer compositions.
1.3.4qPCR the operation of the system of sample-adding：It is shown in Table 2.
1.3.5 system amplification condition is shown in Table 3.
The standard items and sequence of each spliceosome cDNA real time fluorescent quantitatives detections of the RhD mRNA of table 1
Each spliceosome cDNA of the RhD mRNA of table 2 PCR sample-adding systems
| Agent formulations | Volume (μ L) |
| PCR grades of H2O | 8.5 |
| Primer composition (5pmol/L) | 0.5 |
| Master Mix | 10 |
| Standard items (or cDNA) | 1 |
| React cumulative volume | 20 |
The RhD mRNA spliceosomes cDNA of table 3 shares PCR amplification conditions
| The first step | Second step (35 circulation) | 3rd step |
| 95℃ 10min | 96℃ 10s | 72℃ 10s |
| 65℃ 3s | 4℃ ∝ | |
| 68℃ 3s |
Each standard items or sample cDNA detect 3 multiple holes, finally take each hole reading and take the mean as detection
Result.
1.3.6 each spliceosome amplification efficiencies of RhD mRNA
The amplification efficiency of full length sequence：1.978, the range of linearity：104~109
Lack the amplification efficiency of the sequences of exon 7：1.951, the range of linearity：103~108
Lack the amplification efficiency of the sequences of exon 7/8/9：1.878, the range of linearity：104~109
1.3.7 RhD mRNA cDNA quantitative calculation methods：2nd Derivative Max calculating methods.
2 results
2.1 RhD mRNA, which are extracted, need to reach standard：A260/A280 ratio range：Between 1.8~2.1.MRNA concentration>
0.8 μ g/ μ l, extract mRNA electrophoresis as shown in Figure 1 from whole blood.
2.2 monitor the real-time fluorescent signals in each PCR amplification cycles in real time, and real-time fluorescent signals represent amplified production
Copy number, the accumulation of copy number is directly proportional to RhD mRNA spliceosome contents, and standard curve is as shown in Fig. 2.
Using standard curve, calculating obtains standard items and the quantitative result of testing sample is as shown in table 4.
Table 4 is quantified using standard curve to sample
The experimental result requirement of standard items reaches：Error<0.02；
Efficiency：Between 1.9~2.1；
The range of linearity：103~109Between.
The experimental result of standard items is in above controlled range, and the data of actual measurement sample are valid data.
All documents referred in the present invention are all incorporated as reference in this application, independent just as each document
It is incorporated as with reference to such.In addition, it is to be understood that after the above-mentioned instruction content of the present invention has been read, those skilled in the art can
To be made various changes or modifications to the present invention, these equivalent form of values equally fall within the model that the application appended claims are limited
Enclose.
SEQUENCE LISTING
<110>Shenzhen Blood Center
<120>PCR primer combination, kit and real time fluorescent quantitative detection method for detecting RhD mRNA spliceosomes
<130> KH17-1-436
<160> 9
<170> PatentIn version 3.5
<210> 1
<211> 19
<212> DNA
<213> Artificial Sequence
<220>
<223> Whole-F
<400> 1
cccacagctc catcatggg 19
<210> 2
<211> 21
<212> DNA
<213> Artificial Sequence
<220>
<223> Whole-R
<400> 2
gccaatcatg ccattgccgg c 21
<210> 3
<211> 18
<212> DNA
<213> Artificial Sequence
<220>
<223> exon-7-F
<400> 3
tggctgggct gatctccg 18
<210> 4
<211> 20
<212> DNA
<213> Artificial Sequence
<220>
<223> exon-7-R
<400> 4
tggaagccaa tccggcaggt 20
<210> 5
<211> 18
<212> DNA
<213> Artificial Sequence
<220>
<223> exon-7,8,9-F
<400> 5
tggctgggct gatctccg 18
<210> 6
<211> 20
<212> DNA
<213> Artificial Sequence
<220>
<223> exon-7,8,9-R
<400> 6
caaatgagga aacggcaggt 20
<210> 7
<211> 113
<212> DNA
<213> Artificial Sequence
<220>
<223> Whole
<400> 7
cccacagctc catcatgggc tacaacttca gcttgctggg tctgcttgga gagatcatct 60
acattgtgct gctggtgctt gataccgtcg gagccggcaa tggcatgatt ggc 113
<210> 8
<211> 53
<212> DNA
<213> Artificial Sequence
<220>
<223> exon-7
<400> 8
tggctgggct gatctccgtc gggggagcca agtacctgcc ggattggctt cca 53
<210> 9
<211> 53
<212> DNA
<213> Artificial Sequence
<220>
<223> exon-7,8,9
<400> 9
tggctgggct gatctccgtc gggggagcca agtacctgcc gtttcctcat ttg 53
Claims (10)
1. a kind of PCR Primer compositions for being used to detect RhD mRNA spliceosomes, it is characterised in that including following three pairs of primers
At least one pair of：
（1）For expanding 1 couples of total length RhD mRNA spliceosomes totally 2 primers, the sequence of 2 primers is respectively such as SEQ ID
NO:1 and SEQ ID NO:Shown in 1,
（2）For 1 couple totally 2 primers for the RhD mRNA spliceosomes for expanding missing exon-7, the sequences of 2 primers is respectively such as
SEQ ID NO:3 and SEQ ID NO:Shown in 4；；
（3）For expanding missing exon-7,1 couples of 8,9 RhD mRNA spliceosomes totally 2 primers, the sequence point of 2 primers
Not such as SEQ ID NO:5 and SEQ ID NO:Shown in 6.
2. the PCR Primer compositions according to claim 1 for being used to detect RhD mRNA spliceosomes, it is characterised in that institute
State primer concentration respectively 5pmol/L.
3. a kind of kit for being used to detect RhD mRNA spliceosomes, it is characterised in that combined including described PCR primers
Thing.
4. the kit according to claim 3 for being used to detect RhD mRNA spliceosomes, it is characterised in that also including PCR
Level H2O, Master Mix.
5. a kind of real-time fluorescence quantitative PCR detection method for being used to detect RhD mRNA spliceosomes, it is characterised in that including such as
Lower step：
Prepare several various concentrations standard items；
Prepare PCR reaction solutions, including above-mentioned PCR primer combination thing；
（3）PCR is expanded：The standard items or testing sample of above-mentioned PCR reaction solutions and various concentrations are mixed into performing PCR respectively to expand
Increase；
（4）The data creating standard curve obtained using the standard items of various concentrations；
（5）The concentration of testing sample is calculated using standard curve.
6. the real-time fluorescence quantitative PCR detection method according to claim 5 for being used to detect RhD mRNA spliceosomes, it is special
Levy and be, the step（1）In, the standard items are selected from SEQ ID NO:At least one in nucleotide sequence shown in 7-9
Kind.
7. the real-time fluorescence quantitative PCR detection method according to claim 5 for being used to detect RhD mRNA spliceosomes, it is special
Levy and be, the step（1）In, described some one kind in 6,8,10,12,16, it is preferable that the various concentrations
Gradient between the adjacent concentration of standard items is that 1 order of magnitude, i.e. concentration differ 10 times.
8. the real-time fluorescence quantitative PCR detection method according to claim 5 for being used to detect RhD mRNA spliceosomes, it is special
Levy and be, the step（2）In, PCR grades of H2O, Master Mix are also included in the PCR reaction solutions.
9. the real-time fluorescence quantitative PCR detection method according to claim 5 for being used to detect RhD mRNA spliceosomes, it is special
Levy and be, the step（3）In, the response procedures of PCR amplifications are：95 DEG C of pre-degeneration 10min；96 DEG C of denaturation 10s, 60 DEG C multiple
Property 3s, 72 DEG C extend 3 seconds, circulate 35 times, 72 DEG C re-extend 10s.
10. the real-time fluorescence quantitative PCR detection method according to claim 5 for being used to detect RhD mRNA spliceosomes, its
It is characterised by, the step（5）In, the calculating is carried out using 2nd Derivative Max calculating methods.
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| CN111304211A (en) * | 2020-03-10 | 2020-06-19 | 无锡市第五人民医院 | RHD-T268A mutant and detection thereof |
| CN114032320A (en) * | 2021-10-21 | 2022-02-11 | 南京林业大学 | Real-time fluorescence quantitative PCR detection method for plant alternative splicing and application |
| CN114317685A (en) * | 2022-01-05 | 2022-04-12 | 苏州贝康医疗器械有限公司 | Kit, library construction method and sequencing method for detecting mRNA variable shearing variation |
| CN116297120A (en) * | 2023-03-30 | 2023-06-23 | 深圳市血液中心(深圳市输血医学研究所) | Method for detecting drug antibody in sample |
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Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111304211A (en) * | 2020-03-10 | 2020-06-19 | 无锡市第五人民医院 | RHD-T268A mutant and detection thereof |
| CN111304211B (en) * | 2020-03-10 | 2020-12-01 | 无锡市第五人民医院 | RHD-T268A mutant and detection thereof |
| CN114032320A (en) * | 2021-10-21 | 2022-02-11 | 南京林业大学 | Real-time fluorescence quantitative PCR detection method for plant alternative splicing and application |
| CN114317685A (en) * | 2022-01-05 | 2022-04-12 | 苏州贝康医疗器械有限公司 | Kit, library construction method and sequencing method for detecting mRNA variable shearing variation |
| CN114317685B (en) * | 2022-01-05 | 2023-10-20 | 苏州贝康医疗器械有限公司 | Kit for detecting mRNA variable shear variation, library building method and sequencing method |
| CN116297120A (en) * | 2023-03-30 | 2023-06-23 | 深圳市血液中心(深圳市输血医学研究所) | Method for detecting drug antibody in sample |
| CN116297120B (en) * | 2023-03-30 | 2023-12-01 | 深圳市血液中心(深圳市输血医学研究所) | Method for detecting drug antibody in sample |
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