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Primer combination, kit and method for performing PCR-SSP typing detection on KIR gene

A primer combination and detection method technology, applied in the field of genetic engineering, can solve the problems of expensive consumables, missed judgments, and difficult to judge results, and achieve the effects of simple and stable operation, improved accuracy and reliability, and easy to judge results.

Inactive Publication Date: 2016-12-14
BLOOD TRASFUSION INST CHINESE ACAD OF MEDICAL SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, these methods have certain limitations, mainly in that PCR-SSOP and RT-PCR methods require specific instruments, and reagents and consumables are expensive
Although, generally speaking, PCR-SSP is a relatively simple, convenient, and highly specific method, and there is currently a kit for detecting KIR with a single specific primer based on the PCR-SSP method, but it is still due to the complexity of the KIR gene itself. , when the specimen is of certain genotypes, there are technical problems such as misjudgment, missed judgment or difficulty in judging the result, unstable detection, etc.

Method used

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  • Primer combination, kit and method for performing PCR-SSP typing detection on KIR gene
  • Primer combination, kit and method for performing PCR-SSP typing detection on KIR gene
  • Primer combination, kit and method for performing PCR-SSP typing detection on KIR gene

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Effect test

Embodiment 1

[0036] Example 1: Preparation of a kit for rapid detection of KIR gene

[0037] (1) Synthesis of primers:

[0038] The primers were synthesized by Shanghai Yingjun Company, and the sequence list is shown as SEQ ID NO.1-65.

[0039] The specific sequence is shown in Table 1:

[0040] Table 1. Primers for rapid detection of KIR genes

[0041]

[0042]

[0043] The kit for multiplex PCR-SSP typing detection of KIR gene in the present invention includes GoTaq GreenMastermix (Promega, WI, USA) and the above-mentioned primer set, and the total amount of GoTaq Green Mastermix is ​​65ul.

[0044] (2) Pretreatment and preparation of primers:

[0045] Centrifuge at 10,000g for about 30s before primer dilution, and carefully open the cover to avoid the dry primer powder flying out. Dilute the primers to 10 μM with TE (10 mM Tri-HCl, pH 8.0, 1 mM EDTA), and mix thoroughly.

[0046] Primer pair preparation for a single site: mix the forward and reverse primers for each site at a r...

Embodiment 2

[0055] Example 2: Typing detection of KIR gene in samples

[0056] Positive DNA samples covering 16 KIR genotypes were selected. Add to PCR tubes separately, and add Taq enzyme to each tube at the same time. After adding the sample, mix the reaction mixture evenly, centrifuge briefly, and carry out the PCR reaction.

[0057] Wherein, the PCR reaction system is:

[0058] After the primers and DNA samples were taken out from -20°C, they were placed at room temperature to equilibrate for 30 minutes, thoroughly shaken and mixed, and centrifuged briefly, and the PCR reaction system was prepared according to the following system according to the detection site.

[0059] All-site PCR reaction system: distribute the primers into 96-well plates, 2 μl of primers per well. Each sample requires 12 wells (such as A1-A12), and each 96-well plate can complete 8 specimen typing (A-H, one specimen per row). For the convenience of adding samples with an eight-channel pipette when narrow-wel...

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Abstract

The invention belongs to the technical field of genetic engineering, relates to a primer combination, a kit and a method for performing PCR-SSP (polymerase chain reaction-sequence specific primer) typing detection on a KIR (killer cell immunoglobulin-like receptor) gene. The primer combination consists of detection primers shown in SEQ ID NO. 1-65, wherein detection primers 2DL1 and 2DL2 form a group, 2DS3 and 2DP1 form a group, DRB1, 3DS1 and 3DL1 form a group, 3DP1, 2DL5 and 2DS1 form a group, 2DL3 and 2DL4 form a group, 2DL3, 2DS3 and 3DS1 form a group, 2DS4, 3DL1 and 2DL1 form a group, 2DS4 and 2DS5 form a group, 3DL3, 2DS5 and 3DL2 form a group, 2DP1 and 2DS2 form a group, 2DS2 forms a group, 2DL4, 3DL2 and 3DL3 form a group, and all the detection primers form 12 primer groups. Through the primer combination, the kit and the method, the accuracy and the reliability are high, the operation is simple and stable, and a result is easily judged.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering, and in particular relates to a primer combination, a kit and a method for KIR gene PCR-SSP typing detection. Background technique [0002] Killer cell immunoglobulin-like receptors (KIR) are a group of receptors expressed on the surface of NK cells and some T cells that specifically recognize human major histocompatibility antigen MHC class I molecules, and play an important role in the effector function of NK cells. Immunomodulatory effect. KIR is encoded by a group of polymorphic-rich multi-gene families located on human chromosome 19q13.4, and its structure and function are diverse. Each individual contains different types and numbers of KIR genes, and the same bit The alleles of the point have rich polymorphisms, and 15 KIR genes have been found, including (KIR2DL1, KIR2DL2 / L3, KIR2DL4, KIR2DL5A, KIR2DL5B, KIR2DS1, KIR2DS2, KIR2DS3, KIR2DS4, KIR2DS5, KIR3DL1 / S1, KIR3DL2 , KIR3DL3...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12N15/11
CPCC12Q1/6883C12Q1/686C12Q2600/16C12Q2537/143
Inventor 王珏欧国进纪欣陈强苏品璨刘忠
Owner BLOOD TRASFUSION INST CHINESE ACAD OF MEDICAL SCI
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