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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  • 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 d

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

Example Embodiment

[0131] Example 1. Mutation detection of lung cancer samples by multiplex PCR + high-throughput sequencing technology

[0132] The inventor 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, use EZNAFFEP DNA kit (Omega Bio-Tek), according to the manufacturer's instructions, extract the genome from the FFEP treated tissue DNA. The extracted DNA was subjected to gel electrophoresis and OD measurement for quality control, and quantified with Qubit 2.0 Fluorometer (Life Technologies).

[0135] b) Establishment of amplification product library

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

Example Embodiment

[0151] Example 2. Using Sanger sequencing method to verify the result of the positive sample in Example 1

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

[0153]

[0154] Sanger sequencing technology, also known as first-generation sequencing technology, is the recognized gold standard for sequencing in the field. Analysis of verification results:

[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 arrow-marked area. The analysis shows that there is a deletion of up to 15 bases in the sequence GGAATTAAGAGAAGC, and the mutation frequency is about 25%. The verification is successful.

[0157...

Example Embodiment

[0164] Example 3. Multi-gene multi-site mutation detection

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

[0166]

[0167] Therefore, simultaneous multi-gene and multi-site mutation detection can provide more accurate guidance for patients' medication. Take patient 1 as an example. Due to the KRAS G12C mutation, it is not recommended to use EGFR tyrosine kinase inhibitors (EGFR TKIs) drugs, but it is recommended to use Verofini or Darafenib for BRAF V600E. -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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