CN108642163B - Human erythrocyte ABO blood group genotyping primer set and application - Google Patents
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Abstract
The invention discloses a human erythrocyte ABO blood type genotyping primer group which comprises a primer group for respectively detecting blood types A and A205B and O(261G deletion)，O1，O2The pair of allele primers AR and AF; a. the2R and A2F; BR and BF; 0TR and OTF；O1R and O1F；O2R and O2F and an internal reference primer pair; the allele primer group designed by the invention has high power and strong specificity, and simultaneously designs an internal reference primer pair, namely an internal reference 1R and an internal reference 1F, and an internal reference 2R and an internal reference 2F according to the conservative fragment of the human albumin gene, wherein the number of pairs of the allele primer pairs in each detection hole is 2-4, and the allele primer pairs simultaneously contain 2 pairs of internal references, so that the PCR amplification quality and correctness are ensured, and the reliability of the test result is further ensured.
Description
Technical Field
The invention relates to the technical field of gene diagnosis products, in particular to a human erythrocyte ABO blood type genotyping primer set and application thereof.
Background
The ABO blood type is a classical human genetic marker and plays an important role in the fields of blood transfusion, paternity test, anthropology research, forensic material evidence inspection and the like. Genotyping of ABO blood group is a very effective method for detecting blood group, but the existing SNP typing has some defects, such as no duplication, and the PCR amplification quality and correctness need to be further improved.
Disclosure of Invention
The invention aims to overcome the defect that the correctness of the existing ABO blood type genotyping detection technology needs to be further improved, and provides a human erythrocyte ABO blood type genotyping primer set and application thereof.
In order to solve the technical problems, the invention provides the following technical scheme:
a primer group for genotyping human erythrocyte ABO blood group comprises that blood group A and blood group A are respectively detected205B and O(261G deletion)，O1，O2The pair of allele primers AR and AF; a. the2R and A2F; BR and BF; 0TR and OTF；O1R and O1F；O2R and O2F and an internal reference primer pair;
the sequences of the AR and AF are CGGCCATTGGAAGGCT and GAGGGGGAGTGAGCG, respectively;
a is described2R and A2The sequences of F are AGTGAACCTCAGCTTCC and CAGCGAGGTGGATTACAC, respectively;
the sequences of BR and BF are CGCCTGCCAGCTCCATG and TCAATGTCCACAGTCACTCGATC;
0 is describedTR and OTThe sequences of F are GAAGGATGTCCTCGCGGT and GCATGAATGACCTT, respectively;
said O is1R and O1The sequences of F are CAGAACCAAGAGTGA and GCCACGTCCCACAC, respectively.
Further, the internal reference primer pair comprises an internal reference 1R, an internal reference 1F, an internal reference 2R and an internal reference 2F;
the sequences of the internal reference 1R and the internal reference 1F are GTCCTGGAGGAGAAGAGGA and ATTGAGGACCTCTGTGTAT respectively;
the sequences of the internal reference 2R and the internal reference 2F are CCCATCACCACCTA and TACCCAGAGCCCTATCGTT respectively.
The primer pair can be applied to preparation of products for detecting human erythrocyte ABO blood type genotyping.
A kit for detecting human erythrocyte ABO blood group genotyping comprising the pair of allele primers AR and AF of claim 1; a. the2R and A2F; BR and BF; 0TR and OTF； O1R and O1F；O2R and O2F, an internal reference primer pair, dNTP-Buffer, cresol red sodium salt and SYBR Green I.
Further detection of each person comprises 6 detection holes, wherein allele primer pairs AR and AF are respectively placed in the detection holes; a. the2R and A2F; BR and BF; 0TR and OTF；O1R and O1F；O2R and O2And F, an internal reference 1R, an internal reference 1F, an internal reference 2R and an internal reference 2F are arranged in each detection hole.
Furthermore, the number of pairs of allele primers in each detection hole is 2-4, so that the PCR amplification quality and correctness are ensured.
The kit can be generally made into 16 persons/box and 64 persons/box.
A,A205B and O(261G deletion)，O1，O2The specific primers of (a) amplify only the allele complementary to the first base at the 3' end of the primer, but not the allele not complementary, thus allowing one amplification to distinguish between the alleles of ABOMelting curve analysis after amplification can confirm whether the target product is specifically amplified by melting curve analysis, and the allele of ABO is judged.
The invention has the following beneficial effects: the allele primer group designed by the invention has high power and strong specificity, an internal reference primer pair, namely an internal reference 1R and an internal reference 1F, and an internal reference 2R and an internal reference 2F, are designed according to the conservative fragment of the human albumin gene, and the amplification product can be detected in all effective tests because the amplification product exists in all human DNA samples, the logarithm of the allele primer pair in each detection hole is 2-4 pairs, and the allele primer pair simultaneously contains 2 pairs of internal references, so that the PCR amplification quality and correctness are ensured, and the reliability of the test result is further ensured.
Drawings
The accompanying drawings, which are included to provide a further understanding of the invention and are incorporated in and constitute a part of this specification, illustrate embodiments of the invention and together with the description serve to explain the principles of the invention and not to limit the invention. In the drawings:
FIG. 1 is a diagram showing the positive results of the fluorescence PCR method of the human erythrocyte ABO blood group genotyping kit of the present invention, wherein the first peak represents the internal reference melting peak, and the second peak represents the positive melting peak;
FIG. 2 is a graph showing the positive results of the fluorescence PCR method of the kit for genotyping human erythrocyte ABO blood group of the present invention;
FIG. 3 is a diagram showing the positive result of the fluorescence PCR method of the human erythrocyte ABO blood group genotyping kit of the invention.
Detailed Description
The following description of the preferred embodiments of the present invention is provided for the purpose of illustration and description, and is in no way intended to limit the invention.
Examples
A kit for detecting human erythrocyte ABO blood group genotyping comprises an allele primer pair AR and AF; a. the2R and A2F; BR and BF; 0TR and OTF；O1R and O1F；O2R and O2F and innerA primer pair, dNTP-Buffer, cresol red sodium salt and SYBR Green I.
The sequences of AR and AF are CGGCCATTGGAAGGCT and GAGGGGGAGTGAGCG, respectively;
A2r and A2The sequences of F are AGTGAACCTCAGCTTCC and CAGCGAGGTGGATTACAC, respectively;
sequences of BR and BF are CGCCTGCCAGCTCCATG and TCAATGTCCACAGTCACTCGATC, respectively;
0Tr and OTThe sequences of F are GAAGGATGTCCTCGCGGT and GCATGAATGACCTT, respectively;
O1r and O1The sequences of F are CAGAACCAAGAGTGA and GCCACGTCCCACAC, respectively.
The internal reference primer pair comprises an internal reference 1R, an internal reference 1F, an internal reference 2R and an internal reference 2F;
the sequences of the internal reference 1R and the internal reference 1F are GTCCTGGAGGAGAAGAGGA and ATTGAGGACCTCTGTGTAT respectively;
the sequences of internal reference 2R and internal reference 2F are CCCATCACCACCTA and TACCCAGAGCCCTATCGTT, respectively.
Each person detection comprises 6 detection holes, and allele primer pairs AR and AF are respectively placed in the detection holes; a. the2R and A2F; BR and BF; 0TR and OTF；O1R and O1F；O2R and O2And F, an internal reference 1R, an internal reference 1F, an internal reference 2R and an internal reference 2F are arranged in each detection hole.
The number of pairs of allele primers in each detection hole is 2-4, so that the PCR amplification quality and correctness are ensured.
Storage condition and shelf life
All components of the kit are stored below 18 ℃ below zero, and the effective period is 6 months.
Sample requirements:
1. the required sample of the kit is whole blood, whole blood DNA extraction is required, and the concentration of the DNA sample is required to be determined.
2. When extracting DNA from whole blood, the use of heparin anticoagulated whole blood samples is not acceptable, and EDTA anticoagulation is recommended.
3. The concentration of the DNA sample is 40-70 ng/. mu.L, and the purity A260/A280 value is 1.6-2.0.
4. DNA samples that have been extracted are recommended to be stored at below-18 ℃ for no more than one year.
Inspection method
1. PCR working solution preparation (in the reagent preparation area)
Preparing 1ml according to the proportion in the following table, subpackaging, storing and adding DNA before use.
Mix by shaking for several seconds and centrifuge at 800rpm for several seconds. Other parts are prepared according to the proportion.
The PCR working solution was refrigerated at 4 ℃ for 1 day and frozen for 3 months. Repeated freeze thawing is not more than 3 times.
2. Application of sample (in sample processing zone)
2.1 mixing 60ul of PCR working solution with 6ul of sample DNA (concentration: 40-70ng/ul), mixing the working solution-DNA mixed solution by vortex, and carrying out instantaneous centrifugation at 800rpm for 5-10 seconds to ensure that residual liquid on the tube wall is gathered at the tube bottom.
2.2 shearing the required reaction holes, and respectively adding 10 mu l of the mixed solution into each primer hole (6 holes per person);
to avoid cross-contamination between wells, it was ensured that the added sample was just over the primers (dry primers were present at the bottom of each reaction tube). The tip of the sample application gun is allowed to contact the inner wall of the hole and the liquid is allowed to slide to the bottom of the tube. Then, the mixture was centrifuged by a plate centrifuge.
2.3 sealing the membrane or adding 15-20 μ l paraffin oil into each well, and recommending plate centrifugation of 96 wells at 7500rpm for 1 min.
3. PCR amplification (performed in the amplification zone) (real-time fluorescent quantitative PCR instrument Bori 9600 Line plus)
The program is set to 3min at 96 ℃ for 1 cycle; 20 seconds at 96 ℃ and 5 cycles at 68 ℃ for 1 min; 10 cycles of 96 ℃ for 20 seconds, 66 ℃ for 45 seconds, and 72 ℃ for 30 seconds; 15 cycles of 96 ℃ for 20 seconds, 63 ℃ for 45 seconds, and 72 ℃ for 30 seconds; 2min 1 cycle at 72 ℃ and 1 cycle of dissolution curve
Melting curve collection, program selection SYBER GREEN I temperature rise 0.4 degree per second, every 0.5 degree C1 time fluorescence collection, collection temperature range 60-95 degree C.
Setting the reaction volume: 10 μ L
Quality control
The internal reference primer has a melting peak in a corresponding temperature range, and the internal reference melting peak is always visible in a negative hole as a quality control means for successful amplification; the internal reference melting peak of the positive well may be weak or non-existent because of the result of competition with the internal reference primer for the reaction raw material such as Taq enzyme during the amplification of the specific primer.
Positive/negative judgment value
1. Positive, i.e., when the well has a positive determination value of Tm (possibly simultaneously, a negative determination value), meaning that the well is determined to be positive, using intelligent analysis software. When the manual analysis is adopted, when the hole has Tm (possibly simultaneously has a negative judgment value) of the positive judgment value range and the peak fluorescence derivative value is not less than 50, the hole is judged to be positive.
2. Negative, when the well has Tm of negative judgment value (internal reference) and the peak fluorescence derivative value is not less than 50, the well is judged to be negative.
[ analysis of test results ]
1. And starting original data by real-time fluorescence quantitative PCR original factory software, and finding out positive holes by utilizing positive and negative Tm judgment value ranges.
The positive result is shown in FIG. 1. melting peak must appear in the corresponding positive temperature interval, and the highest value is more than 50 positive than the derivative fluorescence value (Y axis). When the positive melting peak has a higher derivative fluorescence value, the internal reference melting peak is lower than 50 and even disappears, the internal reference is still considered to be effective, and the result is positive.
Negative result, the complete fluorescence peak value of the internal reference melting peak, in which the melting peak appears in the corresponding temperature interval, the highest value is more than 60 higher than the derivative fluorescence value (Y axis) and no derivative fluorescence value (Y axis) is more than 50 is appeared in the interval of more than 82 ℃, is negative.
Invalid result: no distinct melting peak appears in the specific internal reference positive temperature interval. In this case, the experiment should be repeated to confirm that the reagent status and DNA concentration quality are maintained within the required range.
2. The ABO allele results were directly interpreted using the type interpretation table of the following table.
[ PRODUCT PROPERTIES INDICATOR ]
1. Positive compliance rate: the result of selecting the DNA sample identified by sequencing is as follows: A102/O01 and B101/O02 are detected with 1 artificially synthesized A205 sample, and the detection result completely accords with a positive reference product.
2. Negative coincidence rate: and 2 parts of artificially synthesized negative reference products GALC and GBLC are selected for detection, and the detection result completely accords with the negative reference products.
3. Repeatability of
1 part of DNA sample with known genotyping is selected, and the kit is used for repeating 5 times of experiments, and the genotyping results are completely consistent.
4. Effective detection range
1 DNA sample of known genotyping was diluted to a concentration of 40 ng/. mu.l and 70 ng/. mu.l, and the results of the genotyping were completely consistent.
Finally, it should be noted that: although the present invention has been described in detail with reference to the foregoing embodiments, it will be apparent to those skilled in the art that changes may be made in the embodiments and/or equivalents thereof without departing from the spirit and scope of the invention. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
<110> Ji Wan Tai biomedical Co., Ltd, Jiangsu
<120> human erythrocyte ABO blood group genotyping primer group and application
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Claims (6)
1. A human erythrocyte ABO blood group genotyping primer group is characterized in thatComprises the detection of blood types A and A respectively205B and O(261G deletion)，O1The pair of allele primers AR and AF; a. the2R and A2F; BR and BF; 0TR and OTF；O1R and O1F and an internal reference primer pair;
the sequences of the AR and AF are CGGCCATTGGAAGGCT and GAGGGGGAGTGAGCG, respectively;
a is described2R and A2The sequences of F are AGTGAACCTCAGCTTCC and CAGCGAGGTGGATTACAC, respectively;
the sequences of BR and BF are CGCCTGCCAGCTCCATG and TCAATGTCCACAGTCACTCGATC;
0 is describedTR and OTThe sequences of F are GAAGGATGTCCTCGCGGT and GCATGAATGACCTT, respectively;
said O is1R and O1The sequences of F are CAGAACCAAGAGTGA and GCCACGTCCCACAC, respectively.
2. The set of human red blood cell ABO blood group genotyping primers of claim 1, wherein the pair of internal reference primers comprises internal reference 1R and internal reference 1F and internal reference 2R and internal reference 2F;
the sequences of the internal reference 1R and the internal reference 1F are GTCCTGGAGGAGAAGAGGA and ATTGAGGACCTCTGTGTAT respectively;
the sequences of the internal reference 2R and the internal reference 2F are CCCATCACCACCTA and TACCCAGAGCCCTATCGTT respectively.
3. Use of the primer set of claim 1 for the preparation of a product for testing human red blood cell ABO blood group genotyping.
4. A kit for detecting human erythrocyte ABO blood group genotyping, comprising the pair of allele primers AR and AF of claim 1; a. the2R and A2F; BR and BF; 0TR and OTF；O1R and O1F, an internal reference primer pair, dNTP-Buffer, cresol red sodium salt and SYBR Green I.
5. The method of claim 4The kit is characterized in that the detection of each person comprises 6 detection holes, and allele primer pairs AR and AF are respectively placed in the detection holes; a. the2R and A2F; BR and BF; 0TR and OTF；O1R and O1F; and each detection hole is internally provided with the internal reference primer pair of claim 2, which comprises an internal reference 1R and an internal reference 1F, and an internal reference 2R and an internal reference 2F.
6. The kit of claim 5, wherein the number of pairs of allele primers in each detection well is 2 to 4 pairs.
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Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN1900314A (en) * | 2006-07-19 | 2007-01-24 | 辽宁省刑事科学技术研究所 | Fluorescence labelling ABO gene typing method and its reagent kit |
| CN102115788A (en) * | 2010-12-02 | 2011-07-06 | 公安部物证鉴定中心 | SNP composite detection system and detection method |
| WO2012171949A2 (en) * | 2011-06-16 | 2012-12-20 | Gendiag.Exe, S.L. | Thromboembolic disease |
| CN104004854A (en) * | 2014-06-18 | 2014-08-27 | 天津市秀鹏生物技术开发有限公司 | Primer set and kit for detecting genetic typing of ABO blood types of human red blood cells |
| CN108624663A (en) * | 2017-03-23 | 2018-10-09 | 上海市血液中心 | A kind of human erythrocyte's abo blood group antigen multiplex PCR classifying method and kit |
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| CN1900314A (en) * | 2006-07-19 | 2007-01-24 | 辽宁省刑事科学技术研究所 | Fluorescence labelling ABO gene typing method and its reagent kit |
| CN102115788A (en) * | 2010-12-02 | 2011-07-06 | 公安部物证鉴定中心 | SNP composite detection system and detection method |
| WO2012171949A2 (en) * | 2011-06-16 | 2012-12-20 | Gendiag.Exe, S.L. | Thromboembolic disease |
| CN104004854A (en) * | 2014-06-18 | 2014-08-27 | 天津市秀鹏生物技术开发有限公司 | Primer set and kit for detecting genetic typing of ABO blood types of human red blood cells |
| CN108624663A (en) * | 2017-03-23 | 2018-10-09 | 上海市血液中心 | A kind of human erythrocyte's abo blood group antigen multiplex PCR classifying method and kit |
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| CN108642163A (en) | 2018-10-12 |
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