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Reagent kit for detecting CD45 gene expression

A detection kit and gene expression technology, applied in the field of molecular biology, to achieve the effect of short detection time, high signal-to-noise ratio, and good signal amplification detection

Pending Publication Date: 2020-09-04
SUREXAM BIO TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, further studies have found that the signal amplification system and detection time of the above-mentioned detection kits need to be further optimized to achieve better detection results

Method used

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  • Reagent kit for detecting CD45 gene expression
  • Reagent kit for detecting CD45 gene expression
  • Reagent kit for detecting CD45 gene expression

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0051] Embodiment 1 CD45 gene expression detection kit (in situ hybridization method)

[0052] The CD45 gene expression detection kit described in this embodiment includes in situ hybridization detection reagents for the CD45 gene and the internal reference gene ACTB, mainly including:

[0053] 1. Capture Probes

[0054] The capture probe consists of four base sequences, from the 5' end to the 3' end are the stem structure sequence, the specific P1 sequence that can bind to the target mRNA to be detected, the spacer sequence, and the amplification probe P3 sequence. Sequence-bound P2 sequences, the P2 sequences in the capture probes of the same target mRNA are the same. The spacer is used to space the capture probe P2 sequence from the target mRNA, and by setting a spacer sequence of appropriate length inside the probe, it can reduce steric hindrance, improve the efficiency of the hybridization reaction and the specificity of the hybridization reaction . The spacer arm of t...

Embodiment 2

[0082] Example 2 Using the kit described in Example 1 to detect the sample

[0083] The formula of described various solutions is as follows table 9:

[0084] Formulation of Table 9 solution

[0085]

[0086] In this embodiment, the blood samples of tumor patients are preferred, and the CD45 gene expression level of CTCs in the samples is detected, wherein the capture probe mixture, the pre-amplification probe mixture, the amplification probe mixture, and the chromogenic probe mixture are all All the probes of the kit in Example 1 were used, and the formulations described in Example 1 were used for the rapid hybridization buffer and the erythrocyte lysate.

[0087] 1. Sample pre-treatment, the cells to be tested are filtered onto the filter membrane

[0088] (1) Take 5 mL of blood from the patient's vein, add it to a separation tube containing 10 mL of erythrocyte lysate, mix for 1 minute, centrifuge at 600×g for 5 minutes, discard the supernatant to retain the cell pelle...

Embodiment 3

[0139] The application of embodiment 3 preamplification probes

[0140] 1. Design of kit preparation (use of pre-amplification probes)

[0141] The signal amplification system of the kit of the present invention includes a pre-amplification probe, an amplification probe and a labeled probe with a fluorescent group modified at the end, which is different from the traditional labeled probe with a fluorescent group modified at the amplification probe and the terminal. Compared with the composed signal amplification system, the signal amplification system of the kit of the present invention further improves the fluorescence signal intensity through the application of the pre-amplification probe, and further improves the detection sensitivity of RNA in situ hybridization.

[0142] In order to investigate the influence of the use of pre-amplified probes on the detection effect of the kit, experimental groups 1-2 were designed, in which the signal amplification system of experimental...

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Abstract

The invention provides a reagent kit for detecting CD45 gene expression. The reagent kit for detecting CD45 gene expression comprises a capturing probe and a signal amplification system, wherein the signal amplification system comprises a pre-amplification probe, an amplification probe and a marking probe; the capturing probe consists of a stem part structure sequence, a specific P1 sequence whichcan be combined with CD45 gene mRNA, a spacer arm sequence and a P2 sequence sequentially from a 5' terminal to a 3' terminal; the pre-amplification probe consists of a P3 sequence which can be in complementary pairing with the P2 sequence, a spacer arm sequence and a P4 sequence sequentially from a 5' terminal to a 3' terminal; the amplification probe consists of a P5 sequence which can be in complementary pairing with the P4 sequence, a spacer arm sequence and a P6 sequence sequentially from a 5' terminal to a 3' terminal; and the marking probe has a P7 sequence in complementary pairing with the P6 sequence, and a fluorophore is modified at a tail terminal. The reagent kit also contains a quick hybrid buffer solution. The reagent kit has the advantages of being high in sensitivity, highin accuracy and short in detection time, and can quickly and accurately detect the expression level of CD45 genes in biological samples.

Description

technical field [0001] The invention belongs to the technical field of molecular biology, and in particular relates to a CD45 gene expression detection kit. Background technique [0002] CD45, the leukocyte common antigen, is a transmembrane protein tyrosine phosphatase (PTPase) widely expressed on the surface of hematopoietic cells (except platelets and mature erythrocytes). CD45 is encoded by the PTPRC gene located on chromosome 1q31.3-1q32.1. It is an important factor regulating the activation and maturation of T cells and B cells and thymus selection. It plays a role in the development and activation of lymphocytes and the signal transduction mediated by cytokine receptors. , Bone marrow cell function plays an important role. With the continuous research and in-depth understanding of the molecular gene structure and function of CD45, CD45 has been used as an important molecular target for immunotherapy and anticancer strategy research. In the study of circulating tumor...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6886C12Q1/682C12Q1/6841
CPCC12Q1/6886C12Q1/682C12Q1/6841C12Q2600/158C12Q2600/118C12Q2537/143C12Q2563/107C12Q2527/125
Inventor 吴诗扬许嘉森黄洁芬刘志明刘芳
Owner SUREXAM BIO TECH
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