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Primer and method for detecting HLA-B*5701 typing

A technology for HLA-B and sequencing primers, applied in the fields of life science and biology, can solve problems such as low incidence, and achieve the effects of simple operation, good specificity, and reduced cost and difficulty

Pending Publication Date: 2021-04-09
合肥艾迪康医学检验实验室有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although GWAS data suggest that it is highly correlated with DILI caused by flucloxacillin, due to the low incidence of this adverse reaction (8.5 / 100,000 people), the positive predictive value is only 0.0012, and an average of 13,513 patients can be detected to predict one case of DILI

Method used

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  • Primer and method for detecting HLA-B*5701 typing
  • Primer and method for detecting HLA-B*5701 typing
  • Primer and method for detecting HLA-B*5701 typing

Examples

Experimental program
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Effect test

Embodiment 1

[0047] The present invention will be further described below in conjunction with specific embodiments and accompanying drawings. It should be noted that the routine conditions and methods not described in the examples are generally used by experimenters in the field: for example, the fourth edition of the "Refined Molecular Biology Experiment Guide" edited by Osper and Kingston, Or follow the procedures and conditions recommended by the manufacturer.

[0048] A primer for detecting the HLA-B*5701 genotype, the primer is designed to span the intron of the HLA-B gene, and the specific amplification primer designed in the second and third exons, including:

[0049] The base sequences of the primers for amplifying the HLA-B*5701 gene and the primers for amplifying the internal reference gene are respectively:

[0050] HLA-B*5701-F: TGTAAAACGACGGCCAGTGAACATGAAGGCCTCCGCG

[0051] HLA-B*5701-R: AACAGCTATGGCCATACATCACCTGGATGATGTG

[0052] actin-F: CTAACTGCGCGTGCGTTCT

[0053] acti...

Embodiment 2

[0065] (1) Operation process of extracting blood genomic DNA:

[0066] 1) Add 20 μL Proteinase K solution to 200 μL blood and mix well.

[0067] 2) Add 200 μL buffer GB, mix thoroughly by inversion, place at 70°C for 10 minutes, the solution should become clear, and briefly centrifuge to remove water droplets on the inner wall of the tube cap.

[0068] 3) Add 200 μL of absolute ethanol, shake and mix well for 15 seconds, at this time flocculent precipitation may appear, centrifuge briefly to remove water droplets on the inner wall of the tube cap.

[0069] 4) Add the solution and flocculent precipitate obtained in the previous step to an adsorption column CB3 (the adsorption column is placed in the collection tube), centrifuge at 12,000rpm (13,400×g) for 30s, discard the waste liquid, and put the adsorption column CB3 back into the collection tube middle.

[0070] 5) Add 500 μL buffer GD to the adsorption column CB (please check whether absolute ethanol has been added before...

Embodiment 3

[0099] Take blood samples from 20 cases of healthy physical examination, extract genomic DNA according to the method described in Example 2, prepare reagents, and detect whether the samples have HLA-B*5701 alleles. Add 2 μl of each sample to the detection system PCR reaction solution. Amplify with a common PCR instrument for 160 minutes. After PCR amplification, the results showed that among the 20 samples, sample No. 17 had a band, and the band was single and the size was correct, indicating that sample No. 17 was HLA-B*5701; after sequencing, it was confirmed to be HLA by comparison with the standard sequence -B*5701 allele. The results of agarose gel electrophoresis of 20 samples are as follows: figure 1 As shown, the sequence comparison chart of sample No. 17 is as follows figure 2 shown.

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Abstract

The invention discloses a primer for detecting HLA-B*5701 typing. The primer comprises a primer for amplifying HLA-B*5701 and a sequencing primer, and a polymerase reaction-direct sequencing method (PCR-SBT) is adopted and can be used for rapidly detecting HLA-B*5701 alleles related to liver injury caused by flucloxacillin medication. The detection result completed by the method is accurate, and the method has important reference significance for the clinical medication safety of the flucloxacillin.

Description

technical field [0001] The invention belongs to the field of life science and biotechnology, in particular to primers for detecting HLA-B*5701 alleles, which can be used for rapid detection of liver disease caused by flucloxacillin by using polymerase reaction-direct sequencing method (PCR-SBT). The injury-related HLA-B*5701 typing can be used to guide the clinical use of flucloxacillin. Background technique [0002] The liver is one of the metabolic organs of the human body, and most drugs need to be decomposed and metabolized in the liver, so the liver has also become one of the target organs attacked by drugs and metabolites. Then, in the process of using drugs, the toxic damage caused by the drug itself or its metabolites, or the disease caused by the allergic reaction of the liver to the drugs and metabolites, is called drug-induced liver injury (DILI). ), also known as drug-induced liver disease (drug-induced liver disease, DILD), is a serious adverse drug reaction, a...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6883C12Q1/686C12Q1/6869C12N15/11
CPCC12Q1/6883C12Q1/686C12Q1/6869C12Q2600/156C12Q2600/106
Inventor 董春燕王淑一
Owner 合肥艾迪康医学检验实验室有限公司
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