Cancer detecting method, kit and application thereof

A kit, cancer technology, applied in biochemical equipment and methods, microbial determination/inspection, etc., can solve the problems of inability to simultaneously detect samples or sites, large sample volume, unsatisfactory low-frequency mutation detection, etc.

Active Publication Date: 2015-03-25
北京圣谷同创科技发展有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The fluorescent PCR method has the characteristics of high sensitivity, and the technology is mature and widely used. However, each pair of primers can only detect one mutation, and each mutation requires a separate PCR reaction system, resulting in a large amount of samples and cannot be detected at the same time. too many samples or loci
In the Sanger sequencing method, a single pair of primers can detect multiple mutations, which has the characteristics of low cost, but also requires a large amount of samples, and the detection of low-frequency mutations is not satisfactory

Method used

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  • Cancer detecting method, kit and application thereof
  • Cancer detecting method, kit and application thereof
  • Cancer detecting method, kit and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0131] Example 1. Mutation Detection of Multiplex PCR+High-throughput Sequencing Technology for Lung Cancer Samples

[0132] The inventors of the present application selected tumor tissue samples from 136 lung cancer patients.

[0133] a) Genomic DNA extraction

[0134] According to the standard FFEP (formalin-fixed, paraffin-embedded) tumor tissue genomic DNA extraction method, using the E.Z.N.A. FFEP DNA kit (Omega Bio-Tek), according to the manufacturer's instructions, the genome was extracted from the FFEP-treated tissue DNA. The extracted DNA was subjected to gel electrophoresis and OD value measurement for quality control and quantification with a Qubit 2.0 fluorometer (Life Technologies).

[0135] b) Establishment of amplified product library

[0136]Using Ion Ampliseq Cancer Panel 2.0 (Life Technologies) and 11 pairs of primers shown in SEQ ID NO: 1-22, according to the instructions provided by the manufacturer, multiplex PCR was performed on 10 ng of genomic DNA to...

Embodiment 2

[0151] Embodiment 2. Utilize Sanger sequencing method to the result verification of the positive sample in embodiment 1

[0152] Among the mutation-positive results, four results were randomly selected for Sanger sequencing verification. Sample information and mutation detection results are as follows:

[0153]

[0154] Sanger sequencing technology, also known as next-generation sequencing technology, is the recognized gold standard for sequencing in the field. Verification result analysis:

[0155] a) L1083

[0156] Such as image 3 As shown, the left side is the detection result diagram obtained according to the method of Example 1, and the right side is the forward and reverse signal peak diagram verified by Sanger. From the Sanger diagram, it can be seen that there are overlapping peaks in the area marked by the arrow, and the analysis shows that there is a deletion of up to 15 bases with the sequence GGAATTAAGAGAAGC, and the mutation frequency is about 25%, and the...

Embodiment 3

[0164] Example 3. Detection of multi-gene multi-site mutations

[0165] The inventor of the present application selected another batch of 76 clinical paraffin samples of lung cancer, and carried out gene mutation detection according to the method of Example 1. As a result, it was found that there were as many as 5 cases of samples with multi-gene or multi-site mutations, accounting for 10% of the total number of samples. 6.6%. Some results are as follows:

[0166]

[0167] Therefore, simultaneous multi-gene and multi-locus mutation detection can provide more accurate guidance for patients' medication. Taking patient 1 as an example, due to the presence of KRAS G12C mutation, it is not recommended to use EGFR tyrosine kinase inhibitors (EGFR TKIs), but vemurafenib or dabrafenib targeting BRAF V600E can be recommended. -label drugs.

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Abstract

The invention provides a kit for detecting of cancer sample polygene and multi-site mutation for mutation-frequent-occurring genes in cancers. The kit comprises primer pairs of zones of one or more exons for amplifying any two or three or all genes of BRAF, EGFR, KRAS and PIK3CA, for example, SEQ ID NO: 11 primer pairs shown by 1-22. The kit can be used for purposes of cancer diagnosis or auxiliary diagnosis, cancer prognosis monitoring or cancer therapy pharmacy guiding and the like. The invention further provides a corresponding detecting method.

Description

field of invention [0001] The present application relates generally to the detection of genes with a high prevalence of mutations in cancer. Specifically, the present application provides a kit and detection method for multi-gene and multi-site mutation detection of cancer samples based on multiplex PCR and high-throughput sequencing technology. The detection results provided can be used for purposes such as cancer diagnosis or auxiliary diagnosis, cancer prognosis monitoring, or guiding cancer treatment and medication. Background of the invention [0002] Cancer is one of the most important non-communicable diseases in the world, and it is also the chronic disease with the highest mortality rate. The number of deaths from cancer in my country is as high as 2.7 million every year, among which malignant tumors of the digestive tract such as lung cancer, colorectal cancer, and gastric cancer are the most serious. Gynecological malignancies, led by breast cancer, are also the...

Claims

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Application Information

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IPC IPC(8): C12Q1/68
CPCC12Q1/6886C12Q2600/118C12Q2600/156C12Q2600/16
Inventor 唐川宁
Owner 北京圣谷同创科技发展有限公司
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