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CN-122012687-A - HPA genotyping detection reagent based on melting curve analysis method, detection method and application

CN122012687ACN 122012687 ACN122012687 ACN 122012687ACN-122012687-A

Abstract

The invention relates to an HPA genotyping detection reagent based on a melting curve analysis method, a detection method and application thereof, in particular to a detection reagent for HPA genotyping detection of Chinese population, a detection method and application thereof. The invention adopts a multiplex PCR technology based on fluorescent probe melting curve to arrange and combine specific primers of different HPA systems and fluorescent marked probes, and finally realizes that 14 HPA antigen systems of Chinese people can be simultaneously genotyped by using only 3 reaction tubes at a time. In addition, the detection system of the invention is simple, convenient, rapid, accurate and efficient, and has higher practical value and clinical transformation prospect.

Inventors

  • WANG LUNAN
  • SUN HUIZHEN
  • SU QIAN
  • JI HUIMIN
  • CHANG LE
  • YAN YING

Assignees

  • 北京医院

Dates

Publication Date
20260512
Application Date
20250702

Claims (10)

  1. 1. A method for HPA genotyping in chinese population comprising the step of using a detection reagent comprising the following three components: (1) A first component comprising a primer set 1 with sequences shown as SEQ ID No.1, 2, 5-8, 11, 12, 19, 20 and a probe set 1 with sequences shown as SEQ ID No.23, 25, 26, 28, 36; (2) The second component comprises a primer group 2 with sequences shown as SEQ ID No.3-6, 9, 10 and 15-18 and a probe group 2 with sequences shown as SEQ ID No.24, 27, 30, 32 and 35; (3) And the third component comprises a primer group 3 with the sequences shown as SEQ ID No.5, 6, 11-14, 21 and 22 and a probe group 3 with the sequences shown as SEQ ID No.29, 31, 33, 34 and 37.
  2. 2. HPA genotyping detection reagent based on melting curve analysis method, which is characterized by comprising the following three components: (1) A first component comprising a primer set 1 with sequences shown as SEQ ID No.1, 2, 5-8, 11, 12, 19, 20 and a probe set 1 with sequences shown as SEQ ID No.23, 25, 26, 28, 36; (2) The second component comprises a primer group 2 with sequences shown as SEQ ID No.3-6, 9, 10 and 15-18 and a probe group 2 with sequences shown as SEQ ID No.24, 27, 30, 32 and 35; (3) And the third component comprises a primer group 3 with the sequences shown as SEQ ID No.5, 6, 11-14, 21 and 22 and a probe group 3 with the sequences shown as SEQ ID No.29, 31, 33, 34 and 37.
  3. 3. The HPA genotyping test reagent according to claim 2, wherein the first component, the second component and the third component are each used to form three different reaction systems.
  4. 4. The HPA genotyping test reagent according to claim 2, wherein each of the probes in probe set 1, probe set 2 and probe set 3 has a different fluorescent group; preferably, the fluorophore is selected from the group consisting of ROX, BHQ2, CY5, VIC, BHQ1, MGB, ATTO 425, CY5.5.
  5. 5. The reagent for detecting HPA genotyping based on the melting curve analysis according to claim 2, wherein the HPA genotyping comprises HPA 1-6, 9, 12, 13, 15, 21, 27, 30, 31 gene loci.
  6. 6. The HPA genotyping test reagent according to claim 2, further comprising an internal standard gene test reagent.
  7. 7. An HPA genotyping reaction system based on a melting curve analysis method is characterized by comprising a first reaction system, a second reaction system and a third reaction system which are independently arranged: a. A first reaction system comprising a primer set 1 with sequences shown as SEQ ID No.1, 2, 5-8, 11, 12, 19, 20 and a probe set 1 with sequences shown as SEQ ID No.23, 25, 26, 28, 36; b. A second reaction system comprising a primer set 2 with sequences shown as SEQ ID Nos. 3-6, 9, 10, 15-18 and a probe set 2 with sequences shown as SEQ ID Nos. 24, 27, 30, 32, 35; c. and a third reaction system comprising a primer group 3 with the sequences shown in SEQ ID No.5, 6, 11-14, 21 and 22 and a probe group 3 with the sequences shown in SEQ ID No.29, 31, 33, 34 and 37.
  8. 8. The HPA genotyping reaction system according to claim 7, wherein the first reaction system, the second reaction system and the third reaction system each independently further comprise dNTPs, mg 2+ ; Preferably, DMSO is further included.
  9. 9. A kit for genotyping HPA based on a melting curve analysis method, comprising the detection reagent according to any one of claims 2 to 6; preferably, the kit further comprises instructions for executing the interpretation rules; preferably, the interpretation rule is: 。
  10. 10. Use of the detection reagent according to any one of claims 2 to 6 for the preparation of a kit for HPA genotyping based on a melting curve analysis; Preferably, the sample to be measured is a fluid sample; preferably, the sample to be measured is a blood sample.

Description

HPA genotyping detection reagent based on melting curve analysis method, detection method and application Technical Field The invention relates to the field of gene detection, in particular to an HPA genotyping detection reagent based on a melting curve analysis method, a detection method and application thereof, and particularly relates to a reagent, a detection system, a detection method, a kit and application for HPA genotyping detection of Chinese population. Background Platelets are produced by megakaryocytes in bone marrow and released into the blood, playing a vital role in hemostasis and thrombosis. The surface of platelet membrane has complex antigen system, which can be divided into two kinds according to its distribution characteristics, the first kind includes sugar chain antigen (such as ABO, H, lewis) and human leukocyte antigen (HLA-I antigen), which are called platelet nonspecific antigen or platelet-related antigen. The second is a relatively specific platelet alloantigen system (human platelet alloantigen, HPA), consisting of platelet-specific antigenic determinants, which are platelet-specific antigens. The HPA system exhibits significant genetic polymorphisms and is expressed on platelets and their precursor megakaryocytes. The genetic polymorphism of HPA is mainly derived from Single Nucleotide Polymorphism (SNP) in platelet membrane glycoprotein structural gene. Such polymorphisms can result in a single amino acid change at a particular position, thereby creating a different antigenic phenotype. HPA is a double dominant co-allele, the nomenclature of which follows the unified principle of "HPA" as a prefix followed by a number, the alleles being distinguished by the letters "a" and "b", where "a" denotes alleles with a gene frequency of greater than 50% and "b" denotes alleles with a gene frequency of less than 50%. Rapid and accurate typing of the HPA antigen system is of great importance in both clinical and transfusion medicine, and genetic and ergonomic research. The major serological methods of HPA antigen typing include a hybrid passive hemagglutination assay (MPHA), a platelet antigen monoclonal specific antibody immobilization assay (MAIPA), an enzyme-linked immunosorbent assay (ELISA), and a simple Sensitized Erythrocyte Platelet Serological Assay (SEPSA). However, due to factors such as limited sources of antisera and difficult platelet acquisition of patients, the application of serology methods is greatly limited and difficult to widely popularize. With the continuous progress of molecular biology technology, HPA genotyping has become increasingly popular, becoming a more efficient and reliable means of typing, and has been applied to group investigation, genotyping of clinical cases, and establishment of platelet donor libraries of known HPA types. The genotyping of HPA is mostly based on Polymerase Chain Reaction (PCR), and among the numerous genotyping technologies of HPA, the PCR-SSP technology is most common, but electrophoresis identification needs to be carried out after PCR amplification, the operation is complicated, the time is relatively consuming, the result cannot be obtained automatically, the subjectivity is provided, and the accuracy is required to be improved, so that the application of the HPA in a large number of samples is limited. As for the PCR-RFLP technique, incorrect typing results may be caused if the enzymatic hydrolysis is not complete during the operation, and not every HPA allele has a suitable cleavage site. The direct sequencing typing method has high accuracy, is a gold standard for HPA genotyping, but has higher detection cost and higher requirements on instruments and equipment, and has complicated operation steps. In addition, other genotyping methods have more or less cost or technical problems. The information in the background section is only for the purpose of illustrating the general background of the invention and is not to be construed as an admission or any form of suggestion that such information forms the prior art that is well known to those of ordinary skill in the art. Disclosure of Invention Aiming at least part of the technical problems in the prior art, the invention can realize the effective genotyping detection of 14 HPA antigen systems of Chinese people simultaneously at a time by reasonably grouping and combining with a fluorescent probe melting curve analysis technology by using 3 systems. Specifically, the present invention includes the following. In a first aspect, the invention provides a reagent for detecting HPA genotyping based on a melting curve analysis method, in particular to a reagent for detecting HPA genotyping of Chinese population, which comprises the following three components: (1) A first component comprising a primer set 1 with sequences shown as SEQ ID No.1, 2, 5-8, 11, 12, 19, 20 and a probe set 1 with sequences shown as SEQ ID No.23, 25, 26, 28, 36; (2) The second component comprises a primer grou