CN107254543B - PCR primer combination, kit and real-time fluorescent quantitative detection method for detecting RhD mRNA spliceosome - Google Patents
PCR primer combination, kit and real-time fluorescent quantitative detection method for detecting RhD mRNA spliceosome Download PDFInfo
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Abstract
The invention discloses a real-time fluorescent quantitative PCR primer combination, a kit and a detection method for detecting a RhD mRNA spliceosome, and belongs to the technical field of biological detection. The invention designs the real-time fluorescent quantitative PCR primer combination aiming at the sequence of the RhD mRNA spliceosome, the specificity of the primer is strong, the primer is not interfered by other non-target genes, and the RhD mRNA spliceosome can be quickly, efficiently and accurately quantified. The method disclosed by the invention is used for quantitatively detecting the RhD mRNA spliceosome by using a real-time fluorescence quantitative PCR method, is simple and accurate, has no pollution in a pipeline, does not need gel electrophoresis, and can be used for accurately quantifying the copy number of the RhD mRNA spliceosome of different individuals. The result reproducibility is good, the detection sensitivity is high, the specificity is strong, and the method can be used for high-flux sample detection.
Description
Technical Field
The invention relates to a PCR primer combination for detecting a RhD mRNA spliceosome, also relates to a kit containing the primer combination and a real-time fluorescence quantitative detection method of the RhD mRNA spliceosome, and belongs to the technical field of biological detection.
Background
The complexity of the RH gene is directly manifested in polymorphisms in RhD antigen expression, which is thought to be related not only to heterogeneity at the RhD mRNA level but also to deletion, quantitative expression, etc. of exons of the relevant genes. Meanwhile, Le Van Kim et al also found that: alternative splicing of the RHCE gene to form different forms of gene transcript [1], and multiple complex alternative splicing of exon 7 to exon 9 in individuals positive for normal D antigen [2], was found by the same investigators, and the polymorphism of this spliceosome determines the characteristics of the RHD gene expressing D antigen. The genetic background of blood group genes is significantly different between races due to the RhD subtype arising from alternative multiple splicing of RhD mRNA genes of different human species [3 ]. mRNA, messenger RNA, is transcribed from DNA with the corresponding genetic information that provides the information needed for further translation into protein. Based on the polymorphism of the RHD gene, the real-time fluorescence quantification method is established to detect the RhD mRNA spliceosome of different individuals, and the difference of the RHD gene of different individuals is known by monitoring the polymorphism of the RhD mRNA expression. Real-time quantitative PCR/QRT-PCR refers to a method of adding a fluorescent marker into a PCR reaction system, monitoring the whole PCR process in real time by using the accumulation of fluorescent signals, and carrying out quantitative analysis on an unknown template through an amplification curve.
[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] Shore supermo, panden, et al. 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.
Disclosure of Invention
In order to overcome the defects in the prior art, the invention aims to provide a PCR primer combination, a kit and a real-time fluorescent quantitative PCR detection method for detecting a RhD mRNA spliceosome.
The purpose of the invention is realized by the following technical scheme:
a PCR primer combination for detecting RhD mRNA spliceosome, comprising at least one of the following three primer pairs:
(1) 1 pair of 2 primers for amplifying full-length RhD mRNA spliceosome, and the sequences of the 2 primers are respectively as follows:
an upstream primer: 5'-CCCACAGCTCCATCATGGG-3' (SEQ ID NO:1),
a downstream primer: 5'-GCCAATCATGCCATTGCCGGC-3' (SEQ ID NO: 2);
(2) the sequence of 1 pair of 2 primers used for amplifying the RhD mRNA spliceosome lacking exon-7 is as follows:
an upstream primer: 5'-TGGCTGGGCTGATCTCCG-3' (SEQ ID NO:3)
A downstream primer: 5'-TGGAAGCCAATCCGGCAGGT-3' (SEQ ID NO: 4);
(3) 2 primers in total for amplifying 1 pair of RhD mRNA spliceosomes lacking exon-7,8 and 9, wherein the sequences of the 2 primers are respectively as follows:
an upstream primer: 5'-TGGCTGGGCTGATCTCCG-3' (SEQ ID NO:5)
A downstream primer: 5'-CAAATGAGGAAACGGCAGGT-3' (SEQ ID NO: 6).
Preferably, the concentration of each primer in the primer composition is 5 pmol/L.
A kit for detecting a RhD mRNA spliceosome comprises the PCR primer composition.
Preferably, the kit for detecting RhD mRNA spliceosome further comprises PCR-grade H2O， Master Mix。
A real-time fluorescent quantitative PCR detection method for detecting RhD mRNA spliceosome comprises the following steps:
(3) preparing a plurality of standard products with different concentrations;
(4) preparing a PCR reaction solution comprising the PCR primer composition;
(3) and (3) PCR amplification: respectively mixing the PCR reaction solution with standard products or samples to be detected with different concentrations for PCR amplification;
(4) making a standard curve by using data obtained by standard substances with different concentrations;
(5) and calculating the concentration of the sample to be measured by using the standard curve.
Preferably, in the step (1), the standard substance is a nucleotide sequence selected from at least one of the following:
full-length standard:
5'-CCCACAGCTCCATCATGGGCTACAACTTCAGCTTGCTGGGTCTGCTTG GAGAGATCATCTACATTGTGCTGCTGGTGCTTGATACCGTCGGAGCCGGCAA TGGCATGATTGGC-3'(SEQ ID NO:7)
absence of exon-7 standard:
5'-TGGCTGGGCTGATCTCCGTCGGGGGAGCCAAGTACCTGCCGGATTGG CTTCCA-3'(SEQ ID NO:8)
absence of exon-7,8,9 standard:
5'-TGGCTGGGCTGATCTCCGTCGGGGGAGCCAAGTACCTGCCGTTTCCTC ATTTG-3'(SEQ ID NO:9)。
preferably, in the step (1), the number is one selected from 6, 8, 10, 12 and 16.
Preferably, in the step (1), the gradient between adjacent concentrations of the standards with different concentrations is 1 order of magnitude, i.e., the concentrations are different by 10 times.
Preferably, in the step (2), the PCR reaction solution further includes PCR grade H2O, Master Mix.
Preferably, in the step (3), the reaction procedure of PCR amplification is: pre-denaturation at 95 ℃ for 10 min; denaturation at 96 ℃ for 10s, renaturation at 60 ℃ for 3s, extension at 72 ℃ for 3s, cycling for 35 times, and re-extension at 72 ℃ for 10 s.
Preferably, in the step (5), the calculation is performed by using a 2nd deviation Max calculation method.
The main advantages of the invention include:
the invention designs the real-time fluorescent quantitative PCR primer combination aiming at the sequence of the RhD mRNA spliceosome, the specificity of the primer is strong, the primer is not interfered by other non-target genes, and the RhD mRNA spliceosome can be quickly, efficiently and accurately quantified.
The method disclosed by the invention is used for quantitatively detecting the RhD mRNA spliceosome by using a real-time fluorescence quantitative PCR method, is simple and accurate, has no pollution in a pipeline, does not need gel electrophoresis, and can be used for accurately quantifying the copy number of the RhD mRNA spliceosome of different individuals. The result reproducibility is good, the detection sensitivity is high, the specificity is strong, and the method can be used for high-flux sample detection.
Drawings
FIG. 1 shows an electrophoretogram of mRNA extracted from whole blood with high quality and high purity.
FIG. 2 real-time fluorescence quantification real-time monitoring of RhD mRNA spliceosomes.
Detailed Description
The following examples and test examples are merely illustrative of the present invention in further detail, and are not intended to limit the present invention in any way.
Example 1
A method for preparing a PCR primer composition for detecting RhD mRNA spliceosome, the primer composition comprising at least one of the following 3 pairs of primers:
1 pair of 2 primers for amplifying full-length RhD mRNA spliceosome, and the sequences of the 2 primers are respectively as follows:
an upstream primer: 5'-CCCACAGCTCCATCATGGG-3' (SEQ ID NO:1),
a downstream primer: 5'-GCCAATCATGCCATTGCCGGC-3' (SEQ ID NO: 2);
the sequence of 1 pair of 2 primers used for amplifying the RhD mRNA spliceosome lacking exon-7 is as follows:
an upstream primer: 5'-TGGCTGGGCTGATCTCCG-3' (SEQ ID NO:3)
A downstream primer: 5'-TGGAAGCCAATCCGGCAGGT-3' (SEQ ID NO: 4); and
2 primers in total for amplifying 1 pair of RhD mRNA spliceosomes lacking exon-7,8 and 9, wherein the sequences of the 2 primers are respectively as follows:
an upstream primer: 5'-TGGCTGGGCTGATCTCCG-3' (SEQ ID NO:5)
A downstream primer: 5'-CAAATGAGGAAACGGCAGGT-3' (SEQ ID NO: 6).
And mixing the upstream primer and the downstream primer in equal volume to obtain the primer composition.
Preferably, the concentration of each primer in the primer composition is 5 pmol/L.
Example 2
1 materials and methods
1.1 Agents and instruments
mRNA extraction kit:
LEV simplyRNA Blood Kit (Promega corporation); cDNA reverse transcription kit: transcriptor High Fidelity cDNA Synthesis Kit (manufactured by Roche Co.); real-time fluorescent quantitative PCR amplification instrument:
software is supplied by roche diagnostics ltd, germany. RhD mRNA spliceosome PCR amplification primers were synthesized by TaKaRa.
1.2 preparation of Standard Curve
1.2.1 artificially synthesized ssDNA as standard for the standard curve, RhD mRNA spliceosome standard and primer sequence are shown in Table 1.
1.2.2 gradient dilution of standard: the standard substance was dissolved in double distilled water to a concentration of 50 ng/ul. Dilution to 10 copy number concentration, each time decreasing by 1 order of magnitude, total 12 gradients: taking 12 PCR tubes with the volume of 0.5ml, adding 90ul of double distilled water into each tube from the 2nd tube (the first tube is a stock solution tube), marking 1, 2, 3 … and 12, adding 10ul of ssDNA library with the volume of 50ng/ul into the 2nd dilution tube, suspending, whirling, mixing uniformly, sucking 10ul of ssDNA library, adding the ssDNA library into the 3 rd tube, and sequentially operating until the final tube is diluted. The gradient dilution of the last tube was removed.
1.3PCR amplification:
1.3.1 extracting mRNA, reverse transcribing to cDNA, and using cDNA as detection sample.
1.3.2 Standard: respective standards (table 1) were used, and 3 wells were set.
1.3.3 primers: the primers used were three primer compositions obtained by mixing the upstream and downstream primers of the three pairs of primers described in example 1.
1.3.4qPCR sample-adding system operation: see table 2.
1.3.5 amplification conditions of the system are shown in Table 3.
TABLE 1 Standard substance and sequence for real-time fluorescent quantitative detection of RhD mRNA spliceosome cDNA
TABLE 2 PCR loading System for RhD mRNA spliceosome cDNA
| Reagent composition | Volume (μ L) |
| PCR grade H2O | 8.5 |
| Primer composition (5pmol/L) | 0.5 |
| |
10 |
| Standard substance (or cDNA) | 1 |
| Total volume of |
20 |
TABLE 3 common PCR amplification conditions for RhD mRNA spliceosome cDNA
| First step of | Second step (35 cycle) | The third step |
| 95℃ 10min | 96℃ 10s | 72℃ 10s |
| 65℃ 3s | 4℃ ∝ | |
| 68℃ 3s |
Each standard or sample cDNA is tested in 3 duplicate wells and the final sum of the individual well readings is averaged as the test result.
1.3.6 amplification efficiency of each spliceosome of RhD mRNA
Amplification efficiency of full-length sequence: 1.978, linear range: 104～109
Amplification efficiency of exon 7 sequence deletion: 1.951, linear range: 103～108
Amplification efficiency with deletion of exon 7/8/9 sequence: 1.878, linear range: 104～109
1.3.7 cDNA quantitative calculation method of RhD mRNA: 2nd Derivative Max calculation.
2 results
2.1 RhD mRNA extraction is required to meet the standard: ratio range of A260/A280: 1.8-2.1. mRNA concentrations > 0.8. mu.g/. mu.l, mRNA electrophoresis from whole blood is shown in FIG. 1.
2.2 real-time monitoring of the real-time fluorescence signal in each PCR amplification cycle, the real-time fluorescence signal representing the copy number of the amplified product, the accumulation of copy number being proportional to the RhD mRNA spliceosome content, the standard curve being shown in FIG. 2.
The quantitative results of the standard and the sample to be tested were calculated using the standard curve and are shown in table 4.
TABLE 4 quantification of samples using the Standard Curve
The experimental result of the standard product is required to reach the following conditions: error < 0.02;
efficiency: 1.9-2.1;
linear range: 103～109In the meantime.
The experimental result of the standard sample is in the controllable range, and the actually measured data of the sample is effective data.
All documents referred to herein are incorporated by reference into this application as if each were individually incorporated by reference. Furthermore, it should be understood that various changes and modifications of the present invention can be made by those skilled in the art after reading the above teachings of the present invention, and these equivalents also fall within the scope of the present invention as defined by the appended claims.
SEQUENCE LISTING
<110> Shenzhen City blood center
<120> PCR primer combination, kit and real-time fluorescent quantitative detection method for detecting RhD mRNA spliceosome
<130> KH17-1-436
<160> 9
<170> PatentIn version 3.5
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cccacagctc catcatggg 19
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gccaatcatg ccattgccgg c 21
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cccacagctc catcatgggc tacaacttca gcttgctggg tctgcttgga gagatcatct 60
acattgtgct gctggtgctt gataccgtcg gagccggcaa tggcatgatt ggc 113
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tggctgggct gatctccgtc gggggagcca agtacctgcc ggattggctt cca 53
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<223> exon-7,8,9
<400> 9
tggctgggct gatctccgtc gggggagcca agtacctgcc gtttcctcat ttg 53
Claims (2)
1. A real-time fluorescent quantitative PCR assay for detection of RhD mRNA spliceosomes for non-diagnostic and non-therapeutic purposes, comprising the steps of:
(1) preparing 12 standard substances with different concentrations, wherein the gradient between adjacent concentrations is 1 order of magnitude, namely the difference between the concentrations is 10 times;
(2) preparing a PCR reaction solution:
PCR grade H2O 8.5μL
5pmol/L primer composition 0.5. mu.L
Master Mix 10μL
1 mu L of standard substance or cDNA sample to be detected
Wherein the primer composition comprises the following three pairs of primers:
(a) 1 pair of 2 primers in total for amplifying the full-length RhD mRNA spliceosome, the sequences of the 2 primers are respectively shown as SEQ ID NO. 1 and SEQ ID NO. 2,
(b) 2 primers in total are used for amplifying 1 pair of the RhD mRNA spliceosome lacking exon-7, the sequences of the 2 primers are respectively shown as SEQ ID NO. 3 and SEQ ID NO. 4,
(c) 2 primers in total are used for amplifying 1 pair of RhD mRNA spliceosomes which lack exon-7,8 and 9, and the sequences of the 2 primers are respectively shown as SEQ ID NO. 5 and SEQ ID NO. 6;
(3) and (3) PCR amplification: respectively mixing the PCR reaction solution with standard products or samples to be detected with different concentrations for PCR amplification, wherein the reaction procedure of the PCR amplification is as follows: pre-denaturation at 95 ℃ for 10 min; denaturation at 96 ℃ for 10s, renaturation at 60 ℃ for 3s, extension at 72 ℃ for 3s, circulation for 35 times, and re-extension at 72 ℃ for 10 s;
(4) making a standard curve by using data obtained by standard substances with different concentrations;
(5) calculating the concentration of the sample to be measured by using the standard curve,
wherein in the step (1), the standard substance is a nucleotide sequence shown as SEQ ID NO 7-9.
2. The real-time fluorescent quantitative PCR detection method for detecting RhD mRNA spliceosome according to claim 1, wherein in the step (5), the calculation is performed by using 2nd Derivative Max calculation method.
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| 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 |
| 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 |
| 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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| CN1552918A (en) * | 2003-12-15 | 2004-12-08 | 深圳市血液中心 | Primer, reagent box and sizing method for Chinese the Han nationality crowd differential RHD gene sizing |
| CN102282176A (en) * | 2008-07-18 | 2011-12-14 | 诺华有限公司 | Non-invasive fetal rhd genotyping from maternal whole blood |
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| CN1552918A (en) * | 2003-12-15 | 2004-12-08 | 深圳市血液中心 | Primer, reagent box and sizing method for Chinese the Han nationality crowd differential RHD gene sizing |
| CN102282176A (en) * | 2008-07-18 | 2011-12-14 | 诺华有限公司 | Non-invasive fetal rhd genotyping from maternal whole blood |
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| Weak D alleles in Japanese: a c.960G>A silent mutation in exon 7 of the RHD gene that affects D expression;Ogasawara K等;《Vox sanguinis》;20150904;第110卷(第2期);第180页左栏第2段至第181页右栏第2段，图1，图3，表1 * |
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