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Kit and method for detecting human leucocyte antigen HLA-B*1502 genetype

A technology of leukocyte antigen and HLA-B, which is applied in the field of kits for detecting the genotype of human leukocyte antigen HLA-B*1502, can solve the problems of many steps in the sequencing method, contamination of the gene amplification laboratory, etc., and achieve good results. The effect of specificity and sensitivity, simple operation

Inactive Publication Date: 2013-05-22
瀚吉康生物科技(北京)有限公司
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

This method needs to do 4 independent PCR reactions for each sample, and then PCR runs agarose gel electrophoresis, staining, and photography; this method is labor-intensive, easily causes contamination of the gene amplification laboratory, and takes a long time
As the gold standard for HLA-B*1502 gene detection, Sanger sequencing is also a more commonly used method; however, the sequencing method has many steps, long time, high cost, and open tube operation, which is also easy to cause contamination of the gene amplification laboratory
Chip method equipment is expensive, and the installed capacity is small, there are many steps, and it also needs to be opened for operation
In addition, Taiwan PharmiGen sells a real-time PCR HLA-B*1502 gene detection kit, but it cannot distinguish multiple alleles, resulting in false positives

Method used

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  • Kit and method for detecting human leucocyte antigen HLA-B*1502 genetype
  • Kit and method for detecting human leucocyte antigen HLA-B*1502 genetype
  • Kit and method for detecting human leucocyte antigen HLA-B*1502 genetype

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0028] Embodiment 1, human HLA-B*1502 genotype-specific detection

[0029] 1) Preparation of human peripheral blood genomic DNA.

[0030] According to the routine of the hospital, 1-5ml of peripheral blood was drawn from the subjects, and stored in EDTA blood collection tubes at 4°C for no more than 7 days. Genomic DNA used QIAamp DNA Blood Mini Kit from Qiagen (China). Use 0.2mL peripheral blood for each sample, and dissolve the final product DNA in 100uL sterilized TE buffer and store in a refrigerator at -20°C. Using the above method, the test samples 1, 2 and 3 were respectively obtained; at the same time, no template control (NTC) was set. Sequence analysis confirmed that samples 1 and 2 were positive.

[0031] 2) Specific primers for detecting human HLA-B*1502 genotype:

[0032] a. Upstream primer FP: 5'-CGACGCCGCGAGTCCCAGG-3' (sequence 2 in the sequence listing);

[0033] b. Downstream primer RP: 5'-CGTCGTAGGCGGACTGGTCATA-3' (sequence 3 in the sequence listing);

...

Embodiment 2

[0042] Example 2, sensitivity detection of human HLA-B*1502 genotype-specific primers

[0043] 1) Preparation of human peripheral blood genomic DNA.

[0044] Take the above-mentioned test sample 1 DNA as a test sample, and its concentration is 20ng / uL. Use sterile TE buffer to dilute the sample 10 times serially, namely 1:1, 1; 10, 1:100, 1:1000; the DNA concentrations are: 20ng / uL, 2ng / uL, 0.2ng / uL, 0.02 ng / uL.

[0045] 2) Specific primers for detecting human HLA-B*1502 genotype:

[0046] a. Upstream primer FP: 5'-CGACGCCGCGAGTCCCAGG-3'

[0047] b. Downstream primer RP: 5'-CGTCGTAGGCGGACTGGTCATA-3'

[0048] c. Probe PR: 5'-Cy5-AACACACAGATCTCCAAGACCAACACAC-p-3'

[0049] Entrust Beijing Aoke Dingsheng Company to synthesize.

[0050] 3) Reaction system for detecting human HLA-B*1502 genotype: 10xPCR buffer 2uL, 25mM MgCl21.2uL, 10mM dNTPs 0.4uL, 0.8uM upstream primer FP 2uL, 8uM downstream primer RP 2uL, 4uM probe PR 2uL, Taq enzyme 5U / uL 0.2uL, 20x SYBR fluorescent dye 1...

Embodiment 3

[0056] Embodiment 3, the comparison of human HLA-B*1502 genotype detection method and gold standard

[0057] 1. The preparation of 100 cases of normal human peripheral blood genomic DNA is the same as in Example 1.

[0058] 2. Using a 96-well special PCR plate, high-throughput HLA-B*1502 genotype detection can be performed simultaneously.

[0059] The reaction system of the HLA-B*1502 genotype, the amplification procedure, and the identification of the PCR product are the same as in Example 1.

[0060] 3. HLA-B*1502 nucleic acid sequence analysis

[0061] a) Specific primers for amplifying exon 2 of human HLA-B*1502:

[0062] a. Upstream primer FP1: 5'-CCCAGGCTCCCACTCCATGA-3' (sequence 5 in the sequence listing),

[0063] b. Downstream primer RP1: 5'-CGGCCTCGCTCTGGTTGTAGT-3' (sequence 6 in the sequence listing),

[0064] Entrust Beijing Aoke Dingsheng Company to synthesize.

[0065] b) Sequencing-specific primers for HLA-B*1502 exon 2:

[0066] a.5'-CCCAGGCTCCCACTCCATGA-...

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Abstract

The invention discloses a kit and a method for detecting human leucocyte antigen HLA-B*1502 genetype. The kit disclosed by the invention is characterized by comprising the following primer sequences: a forward primer FP: 5'-CGACGCCGCGAGTCCCAGG-3'; and a reverse primer RP: 5'-CGTCGTAGGCGGACTGGTCATA-3'. The kit further comprises the following probe sequence: a probe PR: 5'-Cy5-AACACACAGATCTCCAAGACCAACACAC-p-3'. The kit disclosed by the invention has good specificity and sensitivity for carrying out PCR (Polymerase Chain Reaction) detection of the human leucocyte antigen HLA-B*1502. The PCR detection method disclosed by the invention has the advantages of being simple for operation (closed pipe), rapid, accurate and the like and is applied to clinical molecular diagnosis.

Description

technical field [0001] The invention relates to the field of biology, in particular to a kit and a method for detecting the genotype of human leukocyte antigen HLA-B*1502. Background technique [0002] Carbamazepine is a tricyclic anticonvulsant widely used in the treatment of epilepsy, trigeminal neuralgia, and manic depression. As the drug is widely used, its adverse reactions are increasingly prominent. The milder adverse reactions are maculopapular exanthema-MPE, including Stevens-Johnson syndrome-SJS, Toxic epidermal necrolysis-TEN, drug hypersensitivity syndrome(HSS) Other serious adverse reactions can be life-threatening. [0003] Clinical pharmacogenomics studies have linked carbamazepine-induced SJS-TEN to human leukocyte antigen HLA-B*1502 genotype. In recent years, pharmacogenomics has covered different countries and regions in Asia, such as Taiwan, Hong Kong, Thailand, South Korea, Japan, and China. These studies found that 94.1-100% of patients with SJS-TEN ...

Claims

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Application Information

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IPC IPC(8): C12Q1/68
Inventor 张建东李志新谭晓利
Owner 瀚吉康生物科技(北京)有限公司
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