Primers, probe, fluorescent PCR kit and method for detecting human HLA-B*27 gene
A technology for HLA-B and detection primers, applied in the field of biomedical clinical molecular detection, can solve the problems of high price, cumbersome data analysis, long cycle, etc., and achieve the effect of ensuring authenticity and accuracy, wide promotion value, and low cost.
Patent Information
- Authority / Receiving Office
- CN · China
- Current Assignee / Owner
- Publication Date
- 2015-03-25
- Estimated Expiration
- Not applicable · inactive patent
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Abstract
Description
technical field
[0001] The invention belongs to the field of biomedical clinical molecular detection, in particular to a primer, a probe, a fluorescent PCR kit and a detection method for detection of an HLA allele type HLA-B*27 allele. Background technique
[0002] HLA antigen is the expression product of human major histocompatibility complex (Major Histocompatibility Complex, MHC). In the immune system, it is mainly responsible for the mutual recognition between cells, the induction of immune response, and the regulation of the function of immune response. HLA antigens can be divided into three categories: Class I molecules are HLA-A, -B, and -C series antigens, which are widely distributed on the surface of nucleated cells in various tissues, including platelets and reticulocytes, and mature red blood cells generally do not contain HLA antigens; Class II molecules are HLA-D / DR, -DP, and DQ series antigens, which are mainly expressed on B cells and antigen-presenting cells...
Examples
Embodiment 1
[0075] Example 1. Design of primers and probes:
[0076] According to the reference sequence of the HLA-B gene (NG_023187) published by the NCBI nucleic acid sequence database GeneBank of the National Center for Biotechnology Information in the United States (NG_023187) and the information of HLA-B*27 (HLA00223) published by the British IMG / HLA database, Primer Express of ABI Company was used 3.0 software respectively designed primers and probes for detecting HLA-B*27 and other genes. Primers and probes, as follows:
[0077] Forward primer:
[0078] HLA-B*27F: 5’-GGCTACGTGGACGACACGCTGT-3’
[0079] Reverse primer:
[0080] HLA-B*27R: 5’-GCAGGCTCTCTCGGTCAGTCTGTGCC-3’
[0081] Fluorescent probes:
[0082] TM-HLA-B*27: 5’Fam-CGTGAGGTTCGACAGCGACGCCGCG-3’BHQ1
[0083] In order to monitor the effectiveness of the reaction system, internal control primers and probes are added to the detection system. The present invention selects a sequence of the human conserved gene GAPDH (its...
Embodiment 2
[0089] Example 2. Preparation of primers
[0090] The designed primers and probe sequences are handed over to the synthesis company for synthesis, generally using automatic chemical synthesis with instruments, and a synthesis inspection report is required.
Embodiment 3
[0091] Example 3: Preparation of a kit for detecting human HLA-B*27 alleles by fluorescent PCR
[0092] Prepare a reaction solution for detecting human HLA-B*27 alleles, in which the PCR reaction solution contains not only buffers, magnesium ions, dNTPs and other substances necessary for PCR reactions, but also detection primers and probes and internal control primers and probes. The concentration of the PCR reaction solution components and the sequence information of the included primers and probes for the above-mentioned specific detection are as follows:
[0093] Table 1. Components of PCR reaction solution
[0094]
[0095] HLA-B*27 allele
[0096] HLA-B*27F: 5’-GGCTACGTGGACGACACGCTGT-3’
[0097] HLA-B*27R: 5’-GCAGGCTCTCTCGGTCAGTCTGTGCC-3’
[0098] TM-HLA-B*27: 5’Fam-CGTGAGGTTCGACAGCGACGCCGCG-3’BHQ1
[0099] GAPDH F: 5'-GCTGCTTTTTAACTCTGGTAAAGTG-3';
[0100] GAPDH R: 5'-TAGCACTCACCATGTAGTTGAG-3';
[0101] TM-GAPDH: 5'Rox-TGATGCATCTATGAACGCTTC-3'BHQ2.
[0102]