Kit and detection method for abnormity of ROS1 and C-met genes

A technology of abnormal detection and kit, applied in the field of molecular biology, which can solve the problems of large probe length, increased detection and waiting time for results, etc., to improve detection sensitivity, shorten the time required for sufficient hybridization, and ensure the specificity of results and the effect of sensitivity

Inactive Publication Date: 2017-12-01
SUREXAM BIO TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, due to the large length of the existing ROS1 and C-met gene abnormality detection FISH probes, the hybridization time in the detection process basically takes 12-16 hours, and there is no parallel detection FISH kit for the two genes on the market , so two FISH tests are required to obtain the abnormalities of the two genes in patients, especially lung cancer patients, which greatly increases the time for testing and waiting for results

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  • Kit and detection method for abnormity of ROS1 and C-met genes
  • Kit and detection method for abnormity of ROS1 and C-met genes
  • Kit and detection method for abnormity of ROS1 and C-met genes

Examples

Experimental program
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Effect test

Embodiment 1

[0045] Example 1 A ROS1, C-met Gene Abnormal Detection Kit

[0046] The ROS1, C-met gene abnormality detection kit described in this embodiment mainly includes:

[0047] 1. Design the amplification primers

[0048] The first and second sets of amplification primers were designed for the upstream (5' end) and downstream (3' end) of the breakpoint of the ROS1 gene respectively, wherein the first group was for the amplification primer upstream (5' end) of the breakpoint of the ROS1 gene. Amplifying primers, the second group is for amplification primers downstream of the breakpoint (3' end), and the corresponding amplification products constitute the first group of probe libraries and the second group of probe libraries; respectively for the C-met gene and Design the third and fourth sets of amplification primers for the centromere region of human chromosome 7, in which the third set is the amplification primer for the C-met gene, and the fourth set is for the amplification of th...

Embodiment 2

[0079] Example 2 Detection of clinical samples from patients with lung cancer using the ROS1, C-met gene abnormality detection kit in Example 1

[0080] In this example, the detection of ROS1 and C-met gene abnormalities in circulating tumor cells of lung cancer patients was taken as an example. The ROS1 and C-met gene abnormality detection kits in Example 1 were used to examine the peripheral blood samples of 20 lung cancer patients. Proceed as follows:

[0081] 1. Sample pretreatment

[0082] 1. Enrichment of circulating tumor cells: The peripheral blood sample is lysed with commercially available red blood cells to remove red blood cells. The blood sample is mixed with the red blood cell lysate at a ratio of 1:3; the cell suspension after red blood cell lysis is added to the filter membrane ( The filter membrane has a pore size of 5-8um), and undergoes suction filtration treatment. After the suction filtration, carefully remove the filter membrane with tweezers, and place ...

Embodiment 3

[0142] Embodiment 3 The influence of different target gene selections on detection results

[0143] 1. Detection of target gene selection

[0144] In order to test the influence of different target gene selections on the sample detection results, this embodiment designs three experimental groups, wherein experimental group 1 uses all probes of the first and second groups to detect the ROS1 gene; experimental group 3 uses the third , All probes in the fourth group were used to detect the C-met gene; Experimental group 3 used all the probes in the first, second, third and fourth groups to detect ROS1 and C-met gene abnormalities in parallel. The specific test arrangement is shown in Table 6. Synthesis of probes, construction of probe library, probe labeling and experimental steps are as shown in Example 1 and Example 2.

[0145] Table 6 Selection of target genes

[0146]

[0147] 2. Sample testing

[0148] Using the kit prepared by the above design, according to the steps...

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Abstract

The invention discloses a kit and a detection method for abnormity of ROS1 and C-met genes. The kit includes a first group of probes and a second group of probes, which aim to the ROS1 gene, and/or a third group of probes and a fourth group of probes, which aim to the C-met gene. Each group of probes is labeled with a fluorescent dye, wherein the color of the fluorescent dye on the probes in the same group is same while the colors of the fluorescent dyes on the probes in different groups are different. The four groups of probes respectively are amplification products by amplifying a primer with corresponding BAC cloning as a template. The FISH probes are produced through repeated comparison and selection and then utilization of optimized BAC cloning as the template and through amplification by designing a primer aiming to a non-repeated and highly-conserved sequence, so that the kit has excellent specificity and lower background noise. The kit can achieve the optimal balance between detection specificity and hybridization time length, so that not only are specificity and sensitivity of a result ensured, but also hybridization time is reduced, thereby increasing detection efficiency.

Description

technical field [0001] The invention belongs to the field of molecular biology, relates to medicine and biotechnology, in particular to a ROS1 and C-met gene abnormality detection kit and detection method. Background technique [0002] The ROS1 gene is located on chromosome 6 6q21, with a total length of 127kb and 44 exons. The rearrangement, overexpression and mutation of ROS1 gene can lead to the disorder of ROS1 protein. Abnormal ROS1 protein kinase activity will activate multiple downstream oncogenic signaling pathways, including PI3K / AKT / mROR, STAT3, RAS-MAPK / ERK, VAV3 and SHP-1 / 2, etc., which control cell proliferation, survival and cell cycle pathways. ROS1 gene rearrangement was first found in glioblastoma multiforme (Glioblastoma multiforme, GBM) in the form of FIG-ROS1 translocation. So far, ROS1 gene rearrangement has been found in a variety of malignant tumors, including lung cancer, ovarian cancer, gastric cancer, and cholangiocarcinoma. [0003] The c-met ge...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68
CPCC12Q1/682C12Q1/6827C12Q1/6886C12Q2600/106C12Q2600/118C12Q2600/156C12Q2563/107C12Q2525/204
Inventor 廖传荣吴诗扬朱蓉
Owner SUREXAM BIO TECH
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