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Kit for detecting human natural killer cell immunoglobulin-like receptor KIR genotyping

A genotyping and kit technology, applied in the field of biomedical clinical molecular detection, can solve the problems of incomplete KIR gene detection, high sample DNA detection limit, and small sample detection throughput, so as to achieve good practical application value and shorten the experimental time. Time, the effect of increasing the detection flux

Active Publication Date: 2021-03-05
JIANGSU WEIHE BIOTECH
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Problems solved by technology

[0012] In view of the deficiencies in the existing technology, such as small sample detection throughput, non-specific amplification products and false negative results, long experimental process, low accuracy of results, complicated analysis, high minimum detection limit of sample DNA concentration, relative sensitivity Poor, incomplete KIR gene detection and other problems, the present invention provides a method and kit for detecting human natural killer cell immunoglobulin-like receptor KIR genotyping

Method used

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  • Kit for detecting human natural killer cell immunoglobulin-like receptor KIR genotyping
  • Kit for detecting human natural killer cell immunoglobulin-like receptor KIR genotyping
  • Kit for detecting human natural killer cell immunoglobulin-like receptor KIR genotyping

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Embodiment 1

[0042] 1. Raw materials and equipment

[0043] 1.1 Preparation of PCR main reaction solution: 0.18mM deoxynucleotide (dNTP), 1.8mM magnesium chloride (MgCl2), 60.3mM potassium chloride (KCl), 18.9mM Tris-hydrochloride (Tris-HCl) , glycerol (glycerol) 0.6% (v / v), dimethyl sulfoxide (DMSO) 5% (v / v) and formamide 2.5% (v / v), prepare 6*PCR reaction mixture 50mL according to the above ratio , Long-term storage at -20°C, temporary storage at 4°C for later use.

[0044] 1.2 Preparation of reaction plate:

[0045] 1.2.1 Preparation of primer-probe mixture: primer 0.54OD / mL, probe 1.05μM, make 6*primer-probe mixture.

[0046] 1.2.2 Plate according to the following table: primers and probes are shown in SEQ ID No.01-48, SEQ ID No.52-53 and SEQ ID No.55-56 in the following table. Internal standard primers and probes are added to each well, and the nucleotides are shown in SEQ ID No.49-51. Primers and probes were synthesized by General Biosystems (Anhui) Co., Ltd.

[0047]

[0048...

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Abstract

The invention provides 16 common genes for detecting a human natural killer cell immunoglobulin-like receptor KIR, wherein the 16 common genes comprise 14 KIR functional genes (2DL1, 2DL2, 2DL3, 2DL4,2DL5A / B, 2DS1, 2DS2, 2DS3, 2DS4, 2DS5, 3DL1, 3DL2, 3DL3 and 3DS1) and 2 pseudo genes (2DP1 and 3DP1), and a combination of a PCR amplification primer group and a Taqman probe of the common variant ofthe 2DS4; and the combination comprises six primer groups. The invention also provides a kit containing the combination of the PCR amplification primer group and the Taqman probe. The invention has the following technical effects that: an ARMS analysis method is combined with a Taqman multiplex fluorescence PCR technology to carry out qualitative typing detection on the KIR gene; furthermore, improvement is carried out on the basis of a traditional ARMS; various defects of an existing detection method are overcome; and the method disclosed by the invention has wide application prospects and clinical reference value.

Description

technical field [0001] The invention relates to a method and a kit for detecting human natural killer cell immunoglobulin-like receptor KIR genotyping, belonging to the field of biomedical clinical molecular detection. Background technique [0002] Natural killer cells (NK) are derived from bone marrow lymphoid stem cells, and their development and maturation depend on the microenvironment of bone marrow and thymus. NK cells are mainly distributed in peripheral blood and spleen, and there are also a small amount in lymph nodes and other tissues. NK cells do not express specific antigen recognition receptors, and are a type of lymphoid cells different from T and B lymphocytes. NK cells can express a variety of surface markers, most of which can also be expressed on the surface of other immune cells. NK cells are non-specific immune cells. They can directly kill some tumors and virus-infected target cells without antigen pre-sensitization, so they play an important role in t...

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Application Information

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IPC IPC(8): C12Q1/6858C12N15/11
CPCC12Q1/6858C12Q2535/137C12Q2537/143C12Q2531/113C12Q2561/101
Inventor 陈炤源王浩章婷婷王丹娜
Owner JIANGSU WEIHE BIOTECH
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