Method for qualitatively detecting HLA-B*1502 gene with PCR-SSP method and clinical kit

A technology of B1502-SSP-F2 and B1502-SSP-R5, which is applied in the field of qualitative detection of HLA-B*1502 gene and clinical detection kits, can solve problems such as damage, pollution, and mental decline of children, and achieve control of false positives , increase the pollution system, and reduce the effect of false positives

Active Publication Date: 2013-05-22
GRACELL BIOTECH SHANGHAI CO LTD
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AI Technical Summary

Problems solved by technology

At present, the PCR-SSP-based single-specific primer detection kit for HLA-B*1502 in the market is the PG1502 kit [10] from Taiwan’s Pharmigene Company, and its specificity is 98.35% (616 sample), due to the high price, it is inconvenient to promote in the mainland market
In addition, its detection primers cannot distinguish HLA-B*15:13, 15:31, 15:55, 15:88, 15:89, 18:20, 95:12, 95:21, 95:44, 95:70 Waiting for HLA-B alleles, will produce false positives
Moreover, the kit does not take measures to prevent PCR pollution, an

Method used

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  • Method for qualitatively detecting HLA-B*1502 gene with PCR-SSP method and clinical kit
  • Method for qualitatively detecting HLA-B*1502 gene with PCR-SSP method and clinical kit
  • Method for qualitatively detecting HLA-B*1502 gene with PCR-SSP method and clinical kit

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Experimental program
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Embodiment

[0036] 1. Extract the genomic DNA of the subject's whole blood, saliva or other tissues according to the manufacturer's instructions.

[0037] 2. Main reagents: HLA-B*1502 clinical kit reagents. The main components include 2×PCR Mix (Roche), dUTP, UDG enzyme (Thermo Scientific).

[0038] 3. Reaction system and PCR program.

[0039] Configure 12 μl PCR reaction system: including 11 μl kit reagents, 1 μl (1-10ng) of subject sample DNA. PCR reaction program: first incubate at 37°C for 3min, then hot start at 95°C for 10min, 1 cycle; then denature at 95°C for 20sec, extend at 68°C for 50sec, a total of 40 cycles; finally, 72°C for 5min.

[0040] 4. Separation of PCR fragments: 2% agarose gel electrophoresis detection, electric field strength not higher than 5V / cm, time 20-30min, gel imaging system imaging. If there is an HLA-B*1502-specific band with a length of 430 bp bases and an internal reference control band with a length of 219 bp bases, the tested sample is positive for ...

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Abstract

The invention belongs to the field of biotechnology, and particularly relates to a method for qualitatively detecting HLA-B*1502 gene with a PCR-SSP method and a clinical detection kit. The method comprises the following steps of: finding 6 specific areas capable of effectively identifying HLA-B*1502 allele type; designing 6 specific primers covering the 6 specific areas, and screening out a high-specificity primer pair applicable to PCR-SSP; and adding dUTP and UDG enzyme (uracil-DNA glycosylase) into a reaction system to solve the problem of PCR cross pollution and further improve the detection reliability. An HLA-B*1502 quick and convenient qualitative detection kit is researched and developed accordingly. The method and the clinical detection kit provided by the invention have the advantages of convenience in operation, short time, strong specificity, high accuracy, low cost and the like, and is suitable for realizing individual safe and reasonable drug use through HLA-B*1502 genotype detection before the Chinese or Asian epileptics take carbamazepine and phenytoin sodium.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a method for qualitatively detecting HLA-B*1502 gene and a corresponding clinical detection kit. Background technique [0002] Epilepsy is a neurological disease with a high incidence, and the number of cases abroad is 40-70 / 100,000 people every year, and the number in China is about 30 / 100,000 [1] , The main drugs for the treatment of epilepsy include conventional drugs such as phenytoin sodium, carbamazepine, ethosuximide, and sodium valproate, and new drugs such as lamotrigine, oxcarbazepine, and topiramate. Carbamazepine and phenytoin are commonly used first-line antiepileptic drugs at present, and have significant curative effect on complex partial seizure epilepsy. The side effects of antiepileptic drugs include headache, dizziness, unsteady walking, anorexia, nausea, and vomiting. Long-term use of antiepileptic drugs can cause great harm to the memory, movement sp...

Claims

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Application Information

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IPC IPC(8): C12Q1/68
Inventor 徐剑锋刘旭
Owner GRACELL BIOTECH SHANGHAI CO LTD
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