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CN-120758612-B - Kit and method for HLA cross-reactive group and HPA genotyping

CN120758612BCN 120758612 BCN120758612 BCN 120758612BCN-120758612-B

Abstract

The invention discloses a kit and a method for HLA cross-reaction group and HPA genotyping. The invention is based on sharing specific SNP loci, and the system for HLA cross reaction group and HPA genotyping detection is respectively designed by combining two different types of primers, which can realize quick, simple, low-cost and high-throughput HLA epitope and HPA genotyping detection, thereby more effectively preventing ineffective platelet infusion and ineffective immune platelet infusion.

Inventors

  • WANG LUNAN
  • JI HUIMIN
  • CHANG LE
  • YAN YING
  • SUN HUIZHEN
  • Feng Kaihao

Assignees

  • 北京医院

Dates

Publication Date
20260508
Application Date
20250702

Claims (7)

  1. 1. A detection system for HLA cross-reaction group CREGs and HPA genes, characterized by comprising primers with sequences shown as SEQ ID Nos. 1-100, and probes with sequences shown as SEQ ID Nos. 101-150, and further comprising a fluorophore, and comprising a plurality of independent subsystems each comprising a primer and a probe for a specific SNP site, respectively, the information of the primers and probes in the subsystems being shown in Table 3; The detection system comprises first to twelfth reaction systems, wherein the first reaction system comprises primers shown as SEQ ID Nos. 1-6 and 11-12 and probes shown as SEQ ID Nos. 101-103 and 106; the second reaction system comprises primers shown in SEQ ID Nos. 7-8, 13-14, 41-42 and 53-54 and probes shown in SEQ ID Nos. 104, 107, 121 and 127; the third reaction system comprises the primers shown in SEQ ID Nos. 9-10, 19-20, 31-32, 55-56 and the probes shown in SEQ ID Nos. 105, 110, 116, 146, the fourth reaction system comprises the primers shown in SEQ ID Nos. 15-16, 21-22, 27-28, 89-90 and the probes shown in SEQ ID Nos. 108, 111, 114, 127, the fifth reaction system comprises the primers shown in SEQ ID Nos. 23-26, 43-44, 91-92 and the probes shown in SEQ ID Nos. 112-113, 122, 127, the sixth reaction system comprises the primers shown in SEQ ID Nos. 17-18, 33-34, 39-40, 77-78, 82-83 and the probes shown in SEQ ID Nos. 109-110, 120, 139, 141, the seventh reaction system comprises the primers shown in SEQ ID Nos. 29-30, 35-38, 79-81, 85 and the probes shown in SEQ ID Nos. 115, 118-119, 141, the eighth reaction system comprises the primers shown in SEQ ID Nos. 23-26, 43-44, 91-92 and the probes shown in SEQ ID Nos. 112-113, 122, 127, the sixth reaction system comprises the primers shown in SEQ ID Nos. 17-18, 33-34, 39-40, 77-78, 82-83 and the probes shown in SEQ ID Nos. 109-110-139, 139 and 141, and the seventh reaction system comprises the primers shown in SEQ ID Nos. 35-30-35-38, 79-81, 85 and 35-35 75-76 and SEQ ID Nos. 123, 129, 133, 135, 137, a tenth reaction system comprising primers shown in SEQ ID Nos. 49-50, 61-62, 85-86, 93-94, 97-98 and probes shown in SEQ ID Nos. 125, 131, 143, 147, 149, an eleventh reaction system comprising primers shown in SEQ ID Nos. 52-53, 63-64, 87-88, 95-96, 99-100 and probes shown in SEQ ID Nos. 125, 131, 143, 147, 149, and a twelfth reaction system comprising a negative control.
  2. 2. A test kit for HLA cross-reactive group CREGs and HPA genes, comprising reagents and means for constructing the test system of claim 1, said means comprising a space capable of holding a plurality of independent subsystems.
  3. 3. The kit of claim 2, further comprising an internal standard primer and an internal standard probe.
  4. 4. The kit of claim 2, further comprising instructions for performing the detection method and interpretation rules.
  5. 5. The kit of claim 4, wherein the detection method comprises a step of performing multiplex PCR using the detection system.
  6. 6. The kit of claim 2, wherein the interpretation is based on fluorescent signals of different colors.
  7. 7. Use of the detection system of claim 1 for the preparation of a detection kit for HLA cross-reactive group CREGs and HPA genes.

Description

Kit and method for HLA cross-reactive group and HPA genotyping Technical Field The invention relates to the field of gene detection, in particular to a kit and a method for HLA cross-reaction group and HPA genotyping. Background Currently there are 35 HPA antigens, designated HPA-1 to HPA-35 by time of discovery, and are characterized genetically by co-dominant bi-alleles. HLA is a platelet-non-specific antigen present on the surface of platelets that is common to other cells or tissues, where HLA-class I antigen is an HLA antigen that is expressed primarily on the surface of platelets. While in HLA-class I antigens, infusion of platelets with matching HLA-a and HLA-B genotypes can significantly improve platelet infusion inefficiency, but the probability of finding a perfectly matched HLA allele for a patient is only one ten thousandth due to the high polymorphism of HLA. Thus, establishing a large pool of platelet donors is difficult to perform, both economically and from a donor source. It was found that recipients produced anti-donor HLA antibodies only against certain epitopes of their HLA antigens, and not the whole HLA antigen of the donor. Further studies demonstrated that matching of HLA epitopes of the donor can avoid the production of donor-specific antibodies. Clinical studies have shown that infusion of HLA epitope matched platelets based on HLA antigen composition cross-reactive group (CREGs) is equivalent to infusion of HLA antigen matched platelets in patients who have been alloimmunized with HLA and in patients who have not been infused with platelets, providing direct evidence that HLA epitope matching can replace HLA antigen matched infusion. Therefore, the method has important clinical significance for the recipients to develop the detection of HPA genes and HLA-AB epitopes in a targeted manner and infuse compatible platelets matched with the HPA genes and the HLA epitopes. The main methods currently used for HLA and HPA genotyping include polymerase chain reaction-sequence specific primers (PCR-SSP), polymerase chain reaction-sequence specific oligonucleotide probes (PCR-SSO), polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) DNA chip method, sequencing-based typing (SBT) and Next Generation Sequencing (NGS). PCR-SSP is a typing method based on PCR technology, which uses the highly specific amplification of PCR and DNA sequence specific hybridization to type target DNA, but the PCR-SSP needs to be analyzed by gel electrophoresis, and has complex operation and easy laboratory pollution. The principle of PCR-SSO is that according to the base sequences of various HLA alleles, oligonucleotides complementary with the base sequences, namely SSO probes, are artificially synthesized to hybridize with HLA in genomic DNA of a specimen to be detected, and the HLA type of the specimen to be detected is judged by observing whether hybridization occurs or not. The typing accuracy of PCR-RFLP typing is far higher than that of serological methods, but the operation steps are numerous, and the judgment of the result is complex, especially in the case of certain heterozygotes. In addition, this method requires the use of multiple restriction endonucleases for the enzymatic analysis, which is costly and time consuming. The DNA chip method is based on the technical design of anchored oligonucleotides, a series of oligonucleotide probes are introduced on the chip, the probes can be combined with DNA sequences with high specificity of different mutation types on HLA/HPA genes, and when the probes are hybridized with target DNA successfully, the probes at specific positions on the chip display fluorescent signals, which indicate that the DNA in a sample is completely matched with the probes, but the method has the disadvantages of complex technology, high cost, need of professional experiment technology and data analysis capability, and difficulty in wide clinical application. Sanger sequencing and NGS are gold standards which are clinically applied at present, HLA/HPA gene specific sequences are amplified by using PCR, then Sanger sequencing is carried out on amplified products, or sample whole genome DNA is extracted to carry out sequencing typing on HLA genes by a high-throughput sequencing technology, but Sanger sequencing flux is relatively low, only tens to hundreds of target sequences can be sequenced in a single experiment, and the target sequences are required to be cloned independently, so that a plurality of HLA alleles cannot be identified simultaneously, NGS requires higher operation technical level, large data amount, special instruments and equipment are required, and professional data analysis technology is not suitable for large-scale clinical development, and cost is high. At present, HLA typing kits approved by the national drug administration are HLA typing kits developed by One lambda company according to the principles of PCR-SSP and PCR-SSO and HLA nucleic aci