CN114540476B - Primer group and kit for detecting human red blood cell rare blood type genotyping - Google Patents
Primer group and kit for detecting human red blood cell rare blood type genotyping Download PDFInfo
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Abstract
The invention discloses a primer group for detecting human erythrocyte rare blood type genotyping and a kit thereof, which cover about 35 erythrocyte antigens or phenotypes. The invention designs specific primers and Taqman probes aiming at the antigen gene sequence, and the specific primers and the Taqman probes comprise the primers shown in SEQ ID No. 01-95 and the corresponding Taqman probes, and are divided into 16 primer groups. The invention also provides a kit containing the primer group and the Taqman probe combination. The invention has the following technical effects: multiple Taqman fluorescent quantitative PCR detection is adopted, one tube is used for multiple detections, and the detection requirements are met accurately and efficiently; the specificity of the primers is greatly improved by five types of designed primers, namely normal non-mispairing primers, weak-mispairing primers, medium-mispairing primers, strong-mispairing primers and combined-mispairing primers.
Description
Technical Field
The invention relates to a primer group and a kit for detecting human red blood cell rare blood type genotyping, belonging to the field of biomedical clinical molecular detection.
Background
The erythrocyte blood type is one of the main genetic characteristics of human blood, and as the erythrocyte blood type has the greatest influence on blood transfusion, the research on the erythrocyte blood type is increasingly intensive along with the development of medical science. Since 1900 Landsteiner discovered erythrocyte ABO blood group system, more than 300 kinds of human erythrocyte blood group antigens have been discovered so far, and the homoisogenic transfusion is almost impossible to obtain blood with safe blood group consistency. Fortunately, there are only 30 clinically more blood group antigens with strong immunogenicity, and erythrocytes selectively express several of them to form different erythrocyte blood group systems, and there are currently more than 30 of the human erythrocyte blood group systems formally named by the international blood transfusion society (ISBT), and besides ABO and Rh, there are some other rare blood group systems including MNS, lutheran, kell, duffy, kidd, diego, yt, scianna, domrock, colton, landstein-Wiener, lewis, P, hh, etc. The detection of the antigen gene of the erythrocyte blood group system mainly adopts the serology and molecular biology gene typing technology at present. The rare blood group system genotyping is used as an auxiliary technology, is beneficial to improving the typing accuracy of spectral red blood cells, and can be used for identifying a blood group system with scarce serum and determining a gene mutation site. The rare blood type screening and identification has important clinical significance for transfusion science. The detection of rare blood group antigen genes of a patient before transfusion can ensure the safety of clinical transfusion and can also effectively avoid immune hemolytic transfusion reactions such as hemolytic disease of the newborn and the like, hemolytic disease of the newborn, autoimmune hemolytic anemia and the like.
A Taqman fluorescence quantitative PCR method is characterized in that a fluorescence labeling probe which is complementary with sequences in two primers of a target fragment is added into a PCR reaction system, the probe is an oligonucleotide of a 5 'end report group and a 3' end quenching group, and in the PCR amplification process, when the probe is complete, because the quenching group is close to the report group, fluorescence emitted by the report group is absorbed by the quenching group, and a fluorescence signal is not emitted. When the primer is extended, the probe combined with the template is cut off by Taq enzyme (5 '→ 3' exonuclease activity), the reporter group is separated from the quencher group, a fluorescent signal is generated, the whole PCR process is monitored in real time by using fluorescent signal accumulation, and finally, qualitative analysis is carried out on the unknown template or quantitative analysis is carried out through a standard curve. Taqman fluorescence PCR usually uses a pair of primers and a probe, a nucleic acid fragment is generated through PCR amplification, and the Taqman fluorescence PCR is mainly used for identification of a single pathogenic factor and the like; the multiple Taqman fluorescence PCR is characterized in that two or more pairs of primers and two or more corresponding probes are added in the same PCR reaction system, two or more nucleic acid fragments are amplified simultaneously, and the existence of two or more target fragments is identified. Therefore, the multiple Taqman fluorescence quantitative PCR detection has multiple detections in one tube, and accurately and efficiently meets the detection requirements.
Chinese patent CN201811024932.4, entitled "primer group for genotyping rare blood group of human red cell" and application thereof ", provides a primer group for genotyping rare blood group of human red cell and application thereof, and in the aspect of methodology, the kit utilizes a PCR dye-doping method, and shows the effect of DNA amplification through the amplification product of DNA, namely SYBR Geen I method, and the action principle is as follows: SYBR Green I is a dye that binds to the minor groove of double-stranded DNA, does not bind to the single-stranded DNA strand, does not fluoresce in the free state, and can only luminesce when incorporated into the DNA double strand. Therefore, it has a disadvantage in that it is non-specific, and when primer dimers or non-specific amplification occurs in the PCR reaction, the dye can bind to the non-specific amplification products to emit fluorescence, thereby interfering with the accurate determination and quantification of specific products.
Chinese patent CN201910228236.3, named as 'a detection method and a detection kit for human red cell rare blood type genotyping', provides a detection method and a detection kit for human red cell rare blood type genotyping, the kit obtains a target fragment by designing a specific primer and then utilizing single-tube multiplex PCR amplification reaction, designs a single-base extension primer and then carrying out single-base extension, and then accurately measures a target DNA sequence by mass spectrometry. In terms of methodology, the kit is relatively complex to operate, and a DNA sequence is analyzed by mass spectrometry after PCR is needed.
Therefore, the detection method which is simple, convenient and quick to operate, moderate in price and high in accuracy is urgently needed in the field.
Disclosure of Invention
Aiming at the problems of small sample detection flux, nonspecific and false negative results of amplification products, long experimental process, low result accuracy, complicated analysis, poor relative sensitivity, incomplete clinical detection of rare blood group genes of red blood cells and the like in the prior art, the invention provides a primer group and a kit for detecting the rare blood group genotypes of the human red blood cells.
The main technical thought of the inventor is as follows: the inventors designed five classes of primers-normal no mismatch primers, weak mismatch primers, medium mismatch primers, strong mismatch primers, and joint mismatch primers. Firstly, detecting a normal primer without mismatch, and if the detection result is completely correct, using the primer; if the detection result shows weak false positive, using weak mismatch primer, namely introducing a mismatch base within 5 bases of the 5' end of the normal primer, and if the detection result is completely correct, using the primer; if serious false positive still exists, a strong mismatch primer is used, a mismatch base is introduced within 5 bases at the 3' end of the primer, and if the detection result is completely correct, the primer can be used; if the amplification of the positive sample is weakened greatly, using a mispairing primer, namely introducing a mispairing base in the middle of the primer; if false positive still exists, the combined mispairing primer is used, namely two mispairing bases are introduced simultaneously, so that the specificity of primer detection is improved, and the method has a wide application prospect.
The invention uses multiple Taqman fluorescence PCR method, adds two or more than two pairs of primers and two or more than two corresponding probes in the same PCR reaction system, amplifies two or more than two nucleic acid segments simultaneously, and identifies the existence of two or more than two target segments. Multiple Taqman fluorescent quantitative PCR detection is adopted, one tube is used for multiple detections, and the detection requirements are met accurately and efficiently. The whole experiment process can be completed in only 1.5 hours, and the experiment time is effectively shortened. In the kit, the PCR amplification primer group and the Taqman probe are distributed in 6 holes, each sample comprises 6 detection holes, and one 96-hole plate can be used for detecting 16 samples, so that the sample detection flux is increased. The invention adopts a special process to concentrate and dry the primer probe mixed solution at the bottom of the 96-well plate, greatly improves the stability of the primer probe, is simple and convenient for a user to operate, and has better practical application value.
The experimental result can be interpreted only by Ct value and amplification curve, and the result analysis is relatively simple; the intelligent software interpretation only needs to output the ABI 7500 original result file as an Excel file and lead the Excel file into interpretation software, and the software can directly analyze the rare blood type and gene type of the sample to be detected.
The technical scheme of the invention is as follows:
a PCR amplification primer group and Taqman probe combination for detecting rare blood type genotyping of red blood cells, the combination comprises 16 primer groups shown in the following table, and the nucleotide sequences of a primer pair and a probe contained in each primer group are shown in the following table:
in the 16 primer groups, each primer group comprises a pair of internal control primers and a corresponding probe, and the nucleotide sequence is shown as SEQ ID No. 93-95; for monitoring false negative results caused by instrument failure, reagent factors, polymerase activity or inhibitors in the sample;
the 5 'end of the Taqman probe is a report group of FAM, HEX, ROX or CY5 fluorescent label, and the 3' end of the Taqman probe is a quenching group of BHQ-1 or BHQ-2.
The invention discloses a primer group and a kit thereof for detecting rare blood type genotyping of human erythrocytes, which cover about 35 erythrocyte antigens or phenotypes and comprise MNS (M, N, vw +, hut +, S, S, U +W ),Lutheran(Lu a ,Lu b ,Au a ,Au b ),Kell(K,k,Kp a ,Kp b ,Js a ,Js b ),Duffy(Fy a ,Fy b ,Fy(a-b-),Fy x ),Kidd(Jk a ,Jk b ,Jk(a–b–)),Diego(Di a ,Di b ) Yt (Yta, ytb), dombuck (Doa, dob, hy, joa), colton (Coa, cob). The invention designs specific primers and Taqman probes aiming at the antigen gene sequence, and the specific primers and the Taqman probes comprise the primers shown in SEQ ID No. 01-95 and the corresponding Taqman probes, and are divided into 16 primer groups.
The primer is synthesized by a general biological system (Anhui) limited company, and a specific nucleotide sequence SEQ ID No. 01-95 is shown in the following table:
adding two or more pairs of primers and two or more corresponding probes in the same PCR reaction system by using a multiple fluorescence PCR technology, simultaneously amplifying two or more than two nucleic acid fragments, and identifying the existence of two or more than two genotypes.
The invention provides a kit containing the PCR amplification primer group and the Taqman probe, and the PCR amplification primer group and the Taqman probe contained in the kit are both freeze-dried at the bottom of a 96-well plate.
In the kit, a PCR amplification primer group and a Taqman probe are distributed in 16 holes, each sample comprises 16 detection holes, and one 96-hole plate can be used for detecting 6 samples.
The kit also comprises PCR reaction mixed liquor, taq enzyme and an optical sealing film.
The PCR main reaction solution comprises: 0.18mM deoxynucleotide (dNTP), 1.8mM magnesium chloride (MgCl 2), 60.3mM potassium chloride (KCl), 18.9mM Tris-HCl, 0.6% (v/v) glycerol (glycerol), 5% (v/v) dimethyl sulfoxide (DMSO), and 2.5% (v/v) formamide, wherein DMSO and formamide serve as both a PCR reaction enhancer and a stabilizer.
The invention has the following technical effects: multiple Taqman fluorescent quantitative PCR detection is adopted, one tube is used for multiple detections, and the detection requirements are met accurately and efficiently; the specificity of the primers is greatly improved through five types of designed primers, namely normal non-mismatched primers, weak-mismatched primers, medium-mismatched primers, strong-mismatched primers and combined-mismatched primers, various defects of the existing detection method are overcome, and the method has wide application prospects and clinical reference values.
Drawings
FIGS. 1 to 6 show amplification curves from well No. 1 to well No. 16 of Sample1 to Sample6, respectively.
FIG. 7 is a Sample1 sequencing diagram, the sequencing result is consistent with the detection result of the method.
FIG. 8 is a Sample2 sequencing diagram, the sequencing result is consistent with the detection result of the method.
FIG. 9 is a Sample3 sequencing diagram, the sequencing result is consistent with the detection result of the method.
FIG. 10 is a Sample4 sequencing diagram, the sequencing result is consistent with the detection result of the method.
FIG. 11 is a Sample5 sequencing chart, the sequencing result is consistent with the detection result of the method.
FIG. 12 is a Sample6 sequencing chart, the sequencing result is consistent with the detection result of the method.
Detailed Description
Example 1
1. Feedstock and apparatus
1.1 preparation of PCR main reaction solution: 0.18mM deoxynucleotide (dNTP), 1.8mM magnesium chloride (MgCl 2), 60.3mM potassium chloride (KCl), 18.9mM Tris-HCl (Tris-HCl), glycerol (glycerol) 0.6% (v/v), dimethyl sulfoxide (DMSO) 5% (v/v) and formamide 2.5% (v/v) were prepared in the above-mentioned proportions to prepare a 6X PCR reaction mixture of 50mL, stored at-20 ℃ for a long period, and stored at 4 ℃ for temporary use.
1.2 preparation of reaction plate:
1.2.1 preparation of primer and probe mixed solution: primer 0.54OD/mL, probe 1.05. Mu.M, prepared 6 primer probe mixture.
1.2.2 the following table points for the panels:
1.2.3 drying of reaction plates: the reaction plate was freeze dried according to the following procedure. Packaging in a light-proof tin foil bag, and storing at-20 deg.C.
1.3 sources of samples
1) Blood sample collection
Blood samples were collected using blood collection tubes containing anticoagulant Sodium citrate (Sodium citrate), ethylenediaminetetraacetic acid (EDTA) or Heparin (Heparin), and fresh or cryopreserved whole blood samples that were not repeatedly frozen and thawed were used as experimental samples.
2) Nucleic acid sample extraction
Nucleic acid can be extracted from a sample containing nucleated cells such as whole blood or a leukocyte layer by a precipitation method, a column method or a magnetic bead method, and a sufficient amount of nucleic acid with qualified quality can be obtained for the polymerase chain reaction.
3) Nucleic acid sample quantification
The extracted nucleic acid sample must be dissolved in sterile water or other suitable solution (e.g., TE Buffer) at a concentration of 10-40 ng/. Mu.l.
4) Nucleic acid sample quality specification
The A260/A280 ratio of the nucleic acid sample should be between 1.6 and 2.0.
1.4 required Experimental Equipment
A fluorescent quantitative PCR instrument, pipettors with different ranges, a small-sized desk centrifuge and a 96-pore plate centrifuge.
2. Genotyping process
2.1 configuration of the reaction system: each sample needs 16 independent real-time fluorescent PCR reactions, that is, each person can detect the sample by 16 detection holes, and one reaction plate can detect 6 samples. The mixed mother liquors were prepared according to the following table (see table 1):
table 1: PCR reaction system
Because the primer probe is lyophilized into the reaction plate in advance, 18 mu L of the prepared mixed solution is taken and subpackaged into each hole of the reaction plate, an optical sealing film is attached, and the reaction plate is placed into a fluorescent PCR instrument after short-time centrifugation.
2.2PCR reaction procedure (see Table 2):
table 2: PCR reaction procedure
Setting programs according to the operation manual of automatic cycle temperature controllers of various types, and setting a fluorescence signal acquisition point at 65 ℃; the fluorescence signal acquisition wavelength is set to FAM, HEX, ROX and CY5.
2.3 analysis of experimental results:
the experimental result analysis can adopt manual interpretation or intelligent software interpretation.
2.3.1 Intelligent software interpretation:
and under the conditions that the CY5 Ct values of the internal standard genes in all the holes of the detection sample are less than 35 and the amplification curve is normal, the result interpretation can be continued. Outputting the ABI 7500 original result file into an Excel file, and introducing the Excel file into interpretation software, wherein the software can directly analyze the rare blood type gene type of the sample to be detected.
2.3.2 Manual interpretation
The internal standard gene CY5 has a normal amplification curve and Ct less than 35. The amplification curve of the labeled probe is normal and Ct is less than 35, and the labeled probe is judged to be a positive reaction; if no amplification curve rises or Ct is more than or equal to 35, the labeled probe is judged to be negative reaction.
And (4) conclusion: the whole experimental process only needs 1.5 hours, the experimental result is accurate, and the kit can accurately judge the rare blood type and the genotype of the red blood cells of the experimental sample.
FIGS. 1 to 6 show amplification curves from well No. 1 to well No. 16 of samples 1 to 6, respectively. The internal standard gene CY5 has a normal amplification curve and Ct less than 35. The labeled probe is judged to be a positive reaction when the amplification curve of the labeled probe is normal and Ct is less than 35; if no amplification curve rises or Ct is more than or equal to 35, the labeled probe is judged to be negative reaction.
FIG. 7 is a Sample1 sequencing diagram, the sequencing result is consistent with the detection result of the method.
FIG. 8 is a Sample2 sequencing diagram, the sequencing result is consistent with the detection result of the method.
FIG. 9 is a Sample3 sequencing diagram, the sequencing result is consistent with the detection result of the method.
FIG. 10 is a Sample4 sequencing diagram, the sequencing result is consistent with the detection result of the method.
FIG. 11 is a Sample5 sequencing diagram, the sequencing result is consistent with the detection result of the method.
FIG. 12 is a Sample6 sequencing chart, the sequencing result is consistent with the detection result of the method.
The above embodiments are merely illustrative of the technical ideas and features of the present invention, and the purpose thereof is to enable those skilled in the art to understand the contents of the present invention and implement the present invention, and not to limit the protection scope of the present invention. All equivalent modifications or changes made according to the spirit of the present invention should be covered within the protection scope of the present invention.
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<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 44
caaccatgcc ctccaggaag c 21
<210> 45
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 45
ccttccaaga tggagactat gg 22
<210> 46
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 46
ccttccaaga tggagactat ga 22
<210> 47
<211> 16
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 47
<210> 48
<211> 19
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 48
caacctggaa gcagctgcc 19
<210> 49
<211> 23
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 49
catgcttttc agacctctct acc 23
<210> 50
<211> 23
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 50
catgcttttc agacctctct act 23
<210> 51
<211> 19
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 51
gcatagcacc ctagcagca 19
<210> 52
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 52
<210> 53
<211> 23
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 53
cctcattagt ccttggctca tat 23
<210> 54
<211> 23
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 54
cctcattagt ccttggctca tac 23
<210> 55
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 55
ggcataggga taagggactc t 21
<210> 56
<211> 19
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 56
cacaggcgct gacagccgt 19
<210> 57
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 57
<210> 58
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 58
<210> 59
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 59
agatcctgta gtcatgagca g 21
<210> 60
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 60
ctactttgga ctctggggtt tcaac 25
<210> 61
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 61
<210> 62
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 62
<210> 63
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 63
<210> 64
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 64
ctgcattgca atgggaggaa tgttc 25
<210> 65
<211> 19
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 65
gcacttgaga aaattgggc 19
<210> 66
<211> 19
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 66
gcacttgaga aaattcggt 19
<210> 67
<211> 16
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 67
<210> 68
<211> 24
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 68
tgcatgtgac aaagcaggct gaca 24
<210> 69
<211> 18
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 69
<210> 70
<211> 18
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 70
<210> 71
<211> 19
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 71
gccagaacct agacaccag 19
<210> 72
<211> 18
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 72
ctggccctgc ccttcgtc 18
<210> 73
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 73
<210> 74
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 74
<210> 75
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 75
<210> 76
<211> 24
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 76
taactagtgg ctagctggcg tgaa 24
<210> 77
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 77
<210> 78
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 78
<210> 79
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 79
tggattttct ctaatctatc agttc 25
<210> 80
<211> 24
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 80
ttgaggtcaa ctgggaacct gagc 24
<210> 81
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 81
cttagcctgg cttaaccaag g 21
<210> 82
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 82
cttagcctgg cttaaccaag t 21
<210> 83
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 83
tgtctttcct cagcagctag at 22
<210> 84
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 84
ccccagaaca tgactaccac acacg 25
<210> 85
<211> 23
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 85
caatatagcc acagcgtgtg tgg 23
<210> 86
<211> 23
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 86
caatatagcc acagcgtgtg tga 23
<210> 87
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 87
<210> 88
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 88
tggggtagaa cttttccttg gttaa 25
<210> 89
<211> 17
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 89
gggaacaacc agaaggc 17
<210> 90
<211> 17
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 90
gggaacaaca agacggt 17
<210> 91
<211> 17
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 91
gcacggaaga tgctgat 17
<210> 92
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 92
tgaaggtgtc gctggccttc 20
<210> 93
<211> 18
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 93
<210> 94
<211> 17
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 94
acacatggaa gaccaca 17
<210> 95
<211> 18
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 95
Claims (5)
1. The PCR amplification primer group and Taqman probe combination for detecting the rare blood type genotyping of human red blood cells is characterized by comprising 16 primer groups shown in the following table, wherein the nucleotide sequences of a primer pair and a probe contained in each primer group are shown in the following table:
in the 16 primer groups, each primer group comprises a pair of internal control primers and a corresponding probe, and the nucleotide sequence is shown as SEQ ID No. 93-95; for monitoring false negative results caused by instrument failure, reagent factors, polymerase activity or inhibitors in the sample;
the 5 'end of the Taqman probe is a report group of FAM, HEX, ROX or CY5 fluorescent label, and the 3' end of the Taqman probe is a quenching group of BHQ-1 or BHQ-2.
2. A kit comprising the PCR amplification primer set of claim 1 and a Taqman probe combination.
3. The kit of claim 2, wherein the kit further comprises a PCR reaction mixture, taq enzyme and an optical sealing membrane.
4. The kit according to claim 3, wherein the PCR reaction mixture comprises: 0.18mM deoxynucleotide dNTP, 1.8mM magnesium chloride, 60.3mM potassium chloride, 18.9mM Tris-HCl, glycerol 0.6% v/v, 5% dimethyl sulfoxide and 2.5% formamide, wherein DMSO and formamide act as both a PCR reaction enhancer and a stabilizer.
5. The kit of claim 2, wherein the PCR amplification primer set and the Taqman probe are dried on the bottom of a 96-well plate.
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Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101597642A (en) * | 2009-04-23 | 2009-12-09 | 陕西省血液中心 | A kind of multiple PCR method and test kit that detects human RhD blood type and gene type |
| CN109136354A (en) * | 2018-09-04 | 2019-01-04 | 江苏中济万泰生物医药有限公司 | A kind of human erythrocyte's rare blood type genotyping primer group and application |
| CN110079590A (en) * | 2019-03-25 | 2019-08-02 | 为康（苏州）基因科技有限公司 | A kind of detection method and detection kit of human erythrocyte's rare blood type Genotyping |
| CN110964799A (en) * | 2019-12-20 | 2020-04-07 | 江苏伟禾生物科技有限公司 | Kit for detecting genotyping of human platelet surface antigens HPA and HLA-AB |
-
2021
- 2021-12-23 CN CN202111588714.5A patent/CN114540476B/en active Active
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101597642A (en) * | 2009-04-23 | 2009-12-09 | 陕西省血液中心 | A kind of multiple PCR method and test kit that detects human RhD blood type and gene type |
| CN109136354A (en) * | 2018-09-04 | 2019-01-04 | 江苏中济万泰生物医药有限公司 | A kind of human erythrocyte's rare blood type genotyping primer group and application |
| CN110079590A (en) * | 2019-03-25 | 2019-08-02 | 为康（苏州）基因科技有限公司 | A kind of detection method and detection kit of human erythrocyte's rare blood type Genotyping |
| CN110964799A (en) * | 2019-12-20 | 2020-04-07 | 江苏伟禾生物科技有限公司 | Kit for detecting genotyping of human platelet surface antigens HPA and HLA-AB |
Non-Patent Citations (2)
| Title |
|---|
| 中国哈尔滨地区满族人群11个红细胞血型系统24种稀有血型抗原的基因多态性研究;刘颖等;《中国实验血液学杂志》;20171220(第06期);132 * |
| 多个红细胞稀有血型系统抗原基因筛检与建库研究;孙昂等;《实用预防医学》;20201015(第10期);85 * |
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