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Kit for detecting HLA genotypes through fluorescent PCR melting curve assay

A melting curve and kit technology, which is applied in the field of kits for detection of HLA genotypes by fluorescent PCR melting curve method, can solve the problems that serology cannot be presented one by one, and there are many operation steps, so as to achieve the effect of quick result reference

Pending Publication Date: 2017-09-22
DEBIQI BIOTECH XIAMEN
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] In the past, serological methods were mainly used to detect HLA-ABDRDQ. There were many steps in the operation, and there was subjectivity in the interpretation of the results. The quality of serum, complement and isolated lymphocytes would all affect the accuracy of the results. With the deciphering of the gene sequence, It was found that there were only very few differences among most types, and serology could not present them one by one, making nucleic acid detection based on polymerase chain reaction (PCR) gradually become one of the mainstream methods for detecting HLA antigens

Method used

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  • Kit for detecting HLA genotypes through fluorescent PCR melting curve assay
  • Kit for detecting HLA genotypes through fluorescent PCR melting curve assay
  • Kit for detecting HLA genotypes through fluorescent PCR melting curve assay

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0029] 1. Preparation of reaction system

[0030] (1) Preparation of optical reaction plate

[0031] Take a 10mL volumetric flask, add 1500μL of 0.5OD / mL specific primer and 375μL of 0.5OD / mL internal quality control primer specified in the well according to the formula table of 96 wells, make up the volume to 10mL with sterile water, turn over to make it fully Mix well, transfer the liquid to a 15mL centrifuge tube, use the automatic pipetting workstation to dispense into the 96-well deep-well raw material plate, and then use the original plate-type dispensing workstation to dispense the primer mixture in the raw material plate into the optical reaction plate , after a brief centrifugation, dry in an environment with a temperature of 22±2°C and a humidity of 10±5%RH for 16-24 hours. After being sealed in a bag, the optical reaction plate can be used and stored at -20°C for later use.

[0032]

[0033]

[0034]

[0035] (2) Preparation of T3 reaction solution

[003...

Embodiment 2

[0054] 1. Reagent specificity verification

[0055] (1) Experimental samples

[0056] Six specific samples were taken to verify the specificity of the reagent, and the samples were purchased from the internationally recognized IHWG (http: / / www.ihwg.org / cellbank / ) standard.

[0057] (2) Experimental process

[0058] Use the HLA-ABDRDQ (fluorescence PCR melting curve method) nucleic acid detection kit to detect the above 6 specific samples respectively, analyze the detection results, and verify the specificity of the reagents.

[0059] (3) Experimental results

[0060] The test results of the 6 specific samples were all in line with expectations, indicating that the reagent has good specificity and no cross-reaction. The specific results are shown in the table below:

[0061] Specific test results

[0062]

[0063] 2. Reagent precision verification

[0064] (1) Experimental samples

[0065] One precision sample (concentration: 10ng / μL) was taken to verify the precision o...

Embodiment 3

[0085] Embodiment 3: Detect the result of 30 routine clinical samples

[0086] 1. According to the preparation method shown in Example 1, the relevant components of the kit were prepared and stored at -20°C for later use.

[0087] 2. Genomic DNA of 30 clinical samples was extracted using the nucleic acid extraction reagent (whole blood type) produced by Dobtech Biotechnology (Xiamen) Co., Ltd., and the purity of the DNA samples was tested with an ultraviolet spectrophotometer. The OD260 / OD280 of the 30 samples were all in the Between 1.6 and 2.0.

[0088] 3. According to the steps shown in Example 1, add DNA samples and perform detection on a fluorescent quantitative PCR instrument. The instrument used this time is ABI7500.

[0089] 4. According to the judgment standard shown in Example 1, the results are interpreted and counted, and the results are as follows:

[0090]

[0091]

[0092]

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Abstract

The invention provides a kit for detecting HLA genotypes through fluorescent PCR melting curve assay. The kit includes HLA-A, HLA-B, HLA-DRB1 and HLA-DQB1 genotypes, amplification is performed by using a 96-pore optical reaction plate containing primers, an amplified dual-strand DNA product is actively bonded through SYBR Green I, the low-resolution genotypes of HLA-A, HLA-B, HLA-DRB1 and HLA-DQB1 are comprehensively judged according to the result of a 96-pore fusion curve, and accordingly diagnosis of the matched types for organ transplantation is assisted.

Description

technical field [0001] The invention belongs to the field of fluorescent qualitative PCR, in particular to a kit for detecting HLA genotypes by fluorescent PCR melting curve method. Background technique [0002] The antigenic substances encoded by the major histocompatibility complex and distributed on the surface of nucleated cells of organisms are called human leukocyte antigens (human leukocyte antigen, HLA for short) for humans. The human leukocyte antigen molecular gene located on the short arm of the sixth pair of chromosomes has a high diversity. With the decryption of the human leukocyte antigen molecular gene sequence, it is found that there are only a few or even only one amine between most human leukocyte antigens. Therefore, the past serological method is usually not as effective as the nucleic acid detection method for the identification and differentiation of human leukocyte antigen types. [0003] According to years of research, almost all blood cells and tis...

Claims

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Application Information

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IPC IPC(8): C12Q1/68
CPCC12Q1/6858C12Q2531/113C12Q2527/107C12Q2563/107
Inventor 许雅筑郑毓仁邱一帆何佩勳
Owner DEBIQI BIOTECH XIAMEN
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