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sybr Green I detection method of hla-b*1502 gene and its special primers and kits

An HLA-B, detection method technology, applied in DNA/RNA fragments, recombinant DNA technology, fluorescence/phosphorescence, etc., can solve the problems of low specificity and sensitivity, time-consuming and labor-intensive, and achieve wide applicability and simple operation procedures. , the effect of improving specificity

Inactive Publication Date: 2011-12-28
BEIJING SEARCH BIOTECH
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  • Abstract
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AI Technical Summary

Problems solved by technology

[0007] The present invention provides a SYBR Green I detection method of HLA-B*1502 gene to solve the problems of low specificity and sensitivity, time-consuming and labor-intensive detection methods of HLA-B*1502 gene

Method used

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  • sybr Green I detection method of hla-b*1502 gene and its special primers and kits
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  • sybr Green I detection method of hla-b*1502 gene and its special primers and kits

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Effect test

Embodiment 1

[0039] Example 1. Design and screening of primers for SYBR Green I detection of HLA-B*1502 gene

[0040] Referring to the full sequence of the HLA-B*1502 gene included in GenBank, the 2nd and 3rd exons of the HLA-B*1502 gene were selected to design synthetic primers. In primer design, 2 or less degenerate bases are allowed at the same variable site.

[0041] In the screening process of primers, it is necessary to select primers with higher PCR amplification efficiency and SYBR Green I dye binding efficiency in the target conserved region (the PCR primers used are synthesized by Shanghai Invitrogen Company, and the primers require PAGE purification. The primers are dry powder when they arrive. , after reconstitution with sterile water, the dry powder is assayed, and the primers are stored at 0.4um / ul for later use). For the HLA-B*1502 gene, 2 sets of upstream and downstream primers (SYBR-1 and SYBR-2) were designed, and the upstream and downstream primers for detecting beta-ac...

Embodiment 2

[0055] Embodiment 2, the optimization of the reaction system that carries out SYBR Green I detection to HLA-B*1502 gene

[0056] 1. Optimization of Primer Consumption

[0057] Primer concentration is a key factor affecting PCR reactions. If the primer concentration is low, the reaction will be incomplete. If there are too many primers, the possibility of mismatches and non-specific products increases. The amount of primers for the upstream and downstream were chosen to be the same. Primer concentrations range from 0.1-1um. In the experiment, three different primer concentrations of 0.3um, 0.5um, and 1um were used to detect SYBR Green I against the same template (peripheral blood genomic DNA of patients carrying the HLA-B*1502 gene). The conditions are the same as in Example 1.

[0058] The result is as Figure 2A-Figure 2D as shown ( Figure 2A It is the SYBR Green I amplification curve under the primer concentration of 0.3um and 0.5um (curve 1 is 0.5um, curve 2 is 0.3u...

Embodiment 3

[0063] The SYBR Green I detection of embodiment 3, HLA-B*1502 gene

[0064] 1. Construction of the recombinant plasmid p-HLA-B*1502 carrying the HLA-B*1502 gene

[0065] The HLA-B*1502 gene sequence was synthesized by Shanghai Yingjie Company, and cloned into the pGEM-T Easy vector (purchased from Promega Company) between the NoT I and Eco RI restriction sites of the vector multiple cloning site to obtain the recombinant vector p- HLA-B*1502.

[0066] 2. Detection of 10 clinical samples by SYBR Green I method of HLA-B*1502 gene

[0067] The peripheral blood genomic DNA of 5 confirmed patients carrying the HLA-B*1502 gene and the peripheral blood genomic DNA of 5 patients without the HLA-B*1502 gene were used as templates (with p-HLA-B*1502 as the positive control, With sterile water as a negative control), the SYBR Green I method detection of the HLA-B*1502 gene was carried out with the SYBR-2 primers in Example 1 and the beta-actin internal reference gene primers. Each sam...

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Abstract

The invention provides a SYBR Green I detection method for detecting HLA-B*1502 gene and its special primers and kit. The primers are designed according to the 2nd and 3rd exon regions of the HLA-B*1502 gene, and are used for qualitative detection of the HLA-B*1502 gene in the sample to be tested. The upstream primer of the primers has the nucleotide sequence of SEQ ID NO: 1 in the sequence listing, and the downstream primer has the nucleotide sequence of SEQ ID NO: 2 in the sequence listing. The invention can quickly and accurately detect the HLA-B*1502 gene, and is of great significance for ensuring human health and the drug safety of carbamazepine drugs. The detection method and the kit of the present invention can be used for nucleic acid detection of the HLA-B*1502 gene in various clinical samples (bone marrow, peripheral blood or tissue), and have broad application prospects.

Description

technical field [0001] The invention relates to a molecular biology detection method of an objective gene in the field of biotechnology, in particular to a SYBR Green I detection method of the HLA-B*1502 gene and its special primers and kit. Background technique [0002] Adverse drug reaction (ADR) refers to the harmful and irrelevant reactions that occur when the normal dose of drugs is used to prevent, diagnose, treat diseases or regulate physiological functions. Some ADRs are mild, but some ADRs can lead to prolonged hospital stays, permanent disability, and even death. Antiepileptic drugs are a common cause of cutaneous adverse reactions. Skin-type adverse reactions include mild maculopapular rashes and life-threatening severe adverse reactions such as Stevens Johnson syndrome (Stevens Johnson syndrome, SJS) and toxic epidermal necrolysis (toxicepidermal necrolysis, TEN). Carbamazepine is a drug widely used clinically to treat epilepsy, manic depression and neuropathic...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12N15/11G01N21/64
Inventor 徐玉现毛吉扬吴赓
Owner BEIJING SEARCH BIOTECH
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