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Kit for rapidly detecting HLA-B*1502

An HLA-B and kit technology, applied in the biological field, can solve the problems of requiring expensive, complex steps, and high temperature control requirements, and achieve the effects of effective judgment, wide application prospects, and reduced detection costs.

Active Publication Date: 2021-06-18
广州海思医疗科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, Sanger requires complicated steps, expensive instruments, and takes a long time
The PCR-fluorescent probe method requires high temperature control and requires expensive and sophisticated instruments

Method used

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  • Kit for rapidly detecting HLA-B*1502
  • Kit for rapidly detecting HLA-B*1502
  • Kit for rapidly detecting HLA-B*1502

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0037] Example 1 A kit and method for detecting HLA-B*1502

[0038] The kit includes: dry powder reaction tubes, A Buffer, Bbuffer, HLA-B*1502 Primer Mix, LbaCas12a (Cpf1), crRNA, ssDNA, colloidal gold test strips, positive quality control products, negative quality control products, instructions. The main components of each component in the kit are described in Table 1:

[0039] Each component and its main composition in the kit of the present invention in table 1

[0040] serial number component name main components total content 001 Dry powder reaction tube Recombinase, single-strand DNA binding protein (SSB), strand-displacing DNA polymerase 30 tubes 002 A Buffer Tris-AC, KAC, etc. 1.4mL 003 B Buffer Magnesium acetate 85μL 004 HLA-B*1502 Primer Mix target gene primer 140μL 005 LbaCas12 (Cpf1) Cas protein 70μL 006 crRNA RNA fragment 70μL 007 ssDNA ssDNA 70μL 008 Colloidal gold test str...

Embodiment 2

[0047] Embodiment 2 specific detection

[0048] HLA-B*1502 positive human DNA1 (concentration greater than or equal to 0.01ng / μL), HLA-B*1502 negative human DNA2 (concentration greater than or equal to 0.01ng / μL), Escherichia coli DNA, mouse DNA, negative and positive quality controls As a sample, use this kit to detect:

[0049] 1. RPA reaction: Add 10 μL of A Buffer, 2 μL of HLA-B*1502 Primer Mix, 3 μL of B Buffer, and 5 μL of Template to the dry powder reaction tube in sequence; shake and mix; centrifuge at 3000-5000 rpm for 5 seconds; place at constant temperature Metal bath, 37 degrees, 20 minutes; the primer Mix is ​​mixed with HLA-B*1502-F and HLA-B*1502-R in a volume ratio of 1:1, and diluted with double distilled water to 0.4 μmol / L before use;

[0050] 2. Cas12a-crRNA reaction: Add 2 μL of LbaCas12 (Cpf1), 2 μL of crRNA and 2 μL of ssDNA to the reaction tube, shake and mix; centrifuge at 4000 rpm for 5 seconds; place in a constant temperature metal bath at 37 degree...

Embodiment 3

[0056] The detection of embodiment 3 sensitivity

[0057] Dilute HLA-B*1502-positive human DNA1 to 10ng / μL, 1ng / μL, 0.1ng / μL, 0.01ng / μL, respectively, and use this kit for detection:

[0058] 1. Add 10 μL of A Buffer, 2 μL of HLA-B*1502 Primer Mix, 3 μL of BBuffer, and 5 μL of Template to the dry powder reaction tube in turn; shake and mix; centrifuge at 3000 rpm for 5 seconds; place in a constant temperature metal bath at 37 degrees, 20 minutes; the primer Mix was mixed with HLA-B*1502-F and HLA-B*1502-R at a volume ratio of 1:1, and diluted to 0.3 μmol / L with double distilled water before use;

[0059] 2. Add 2 μL of LbaCas12 (Cpf1), 2 μL of crRNA and 2 μL of ssDNA to the reaction tube, shake and mix; centrifuge at 3000-5000 rpm for 5 seconds; place in a constant temperature metal bath at 37 degrees for 15 minutes; the LbaCas12 ( Cpf1) was diluted to 70 nmol / L with double distilled water before use; the crRNA was diluted to 0.5 μmol / L with double distilled water before use;...

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Abstract

The invention belongs to the technical field of biology, and particularly relates to a kit for rapidly detecting HLA-B*1502 and a detection method thereof. The kit provided by the invention comprises the following components: a dry powder reaction tube, A Buffer, B Buffer, an HLA-B*1502 primer Mix, LbaCas12a (Cpf1), crRNA, ssDNA, a colloidal gold test strip, a positive quality control product, a negative quality control product and the like. When the kit provided by the invention is used for detecting the HLA-B*1502, a result can be clearly, conveniently and effectively judged on the test strip, so that the detection result is visible to naked eyes, meanwhile, expensive and complex instruments are not needed, and all that is needed is an instrument capable of stably heating at 37 DEG C is needed.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a kit for rapidly detecting HLA-B*1502. Background technique [0002] Epilepsy is a chronic brain disease caused by repeated and sudden excessive abnormal discharge of brain neurons caused by various etiologies. It occurs in people of all ages, regions, and races, with a higher incidence in children and adolescents. According to statistics, the average annual incidence of epilepsy in my country is about 35 / 100,000, and the prevalence rate is about 5‰. Antiepileptic drug therapy is the most important and basic treatment, and it is often the first choice for the treatment of epilepsy. [0003] Carzepine / Oxcarbazepine as first-line drugs for idiopathic generalized epilepsy; epilepsy with generalized tonic-clonic seizures only; benign epilepsy in children with centrotemporal spikes, Panayiotopoulos syndrome or late-onset childhood occipital lobe epilepsy, etc. However, abo...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6844
CPCC12Q1/6844C12Q2531/119C12Q2545/113C12Q2521/327C12Q2565/625
Inventor 夏昊强张天海陈曦曹治家
Owner 广州海思医疗科技有限公司
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