K-Ras gene mutation typing fluorescence quantitative PCR detection kit and detection method thereof

A detection kit and fluorescence quantitative technology, applied in the field of molecular biology, can solve problems such as easy contamination, limited sample size, hazards to operators and the environment

Inactive Publication Date: 2010-12-01
广州达健生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0007] 1. The detection period is long (the shortest time is about 24-48 hours from the specimen submission to the result)
[0008] 2. The operation process is complicated and involves the operation after PCR amplification, so it is easy to be contaminated, resulting in unsatisfactory results
[0009] 3. Interpretation of sequencing results is complex and time-consuming (especially when the sample size is large)
[0010] 4. The sensitivity of detection is not high enough: Katsuhiko et al once reported on "Lung Cancer" ("Lung

Method used

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  • K-Ras gene mutation typing fluorescence quantitative PCR detection kit and detection method thereof
  • K-Ras gene mutation typing fluorescence quantitative PCR detection kit and detection method thereof
  • K-Ras gene mutation typing fluorescence quantitative PCR detection kit and detection method thereof

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Embodiment 1

[0088] Example 1: Fluorescent quantitative PCR (FQ-PCR) was used to detect four base substitution mutations of the 12th codon of the KRAS gene (codon 12GGT→GAT, GGT→GTT, GGT→GCT).

[0089] Design a specific Allglo that can specifically detect the above three base substitution mutations TM One fluorescent quantitative detection probe and one pair of PCR primers. The sequences of each probe and primer are as follows:

[0090] The PNA sequence is PNA probe-5'-CCTACGCCACCAGCTCC-3', as shown in SEQID NO A0 in the sequence listing;

[0091] Mutant KRAS-Allglo targeting the GAT-12 codon TM The fluorescent quantitative detection probe sequence is: 5'-MAR-TGGAGCTGATGGCG-MAR-3', as shown in SEQ ID NO A3 in the sequence listing;

[0092] Mutant KRAS-Allglo targeting the GTT-12 codon TM The fluorescent quantitative detection probe sequence is: 5'-JUP-GTTGGAGCTGTTGGCGT-JUP-3', as shown in SEQ ID NO A4 in the sequence listing;

[0093] Mutant KRAS-Allglo targeting the GCT-12 codon TM T...

Embodiment 2

[0099] Embodiment 2: Detection of a base substitution mutation (codon 13GGC→GAC) at the 13th codon of the KRAS gene by fluorescent quantitative PCR, and three base substitution mutations at the 12th codon (codon 12GGT→GCT, GGT→AGT, GGT→CGT)

[0100] Design a specific Allglo that can specifically detect the above four base substitution mutations TM One fluorescent quantitative detection probe and one pair of PCR primers. The sequences of each probe and primer are as follows:

[0101] The PNA sequence is PNA probe-5'-CCTACGCCACCAGCTCC-3', as shown in SEQID NO A0 in the sequence listing;

[0102] Mutant KRAS-Allglo targeting the GAC-13 codon TM The fluorescent quantitative detection probe sequence is: 5'-MAR-AGCTGGTGACGTAGGC-MAR-3', as shown in SEQ ID NO B3 in the sequence listing;

[0103] Mutant KRAS-Allglo targeting the TGT-12 codon TM The fluorescent quantitative detection probe sequence is: 5'-JUP-GTTGGAGCTTGTGGCGT-JUP-3', as shown in SEQ ID NO B4 in the sequence listing...

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Abstract

The invention provides a K-Ras gene mutation typing fluorescence quantitative PCR detection kit which comprises PCR mixed reaction solution, a peptide nucleic acid probe, a mutant type K-Ras-AllgloTM fluorescent probe and a positive control sample. The invention further provides a K-Ras gene mutation typing fluorescence quantitative PCR detection method which comprises the following steps: firstly designing a pair of PCR primers and the peptide nucleic acid probe according to K-Ras genes, respectively designing the AllgloTM fluorescent probe against K-Ras gene mutation sites, and adding the PCR reaction solution, the PCR primers, the peptide nucleic acid probe and the AllgloTM fluorescent probe in a reaction system for carrying out fluorescence quantitative PCR detection. The method adopts the PNA technology and is combined with the technology of the AllgloTM fluorescent probe, thereby being capable of quickly and accurately detecting the K-Ras gene mutation sites in various cancer tissues with high sensitivity, and having short time, simple operation and clear, intuitive and safe judgment and reading.

Description

technical field [0001] The invention relates to the field of molecular biology, in particular to a fluorescent quantitative PCR kit for detecting gene mutations and a detection method thereof, in particular to a fluorescent quantitative PCR detection kit for K-Ras gene mutation typing and a detection method thereof. Background technique [0002] Ras oncogene is involved in the occurrence and development of human tumors. It was originally a transforming gene cloned from two strains of rat sarcoma virus, Harvey and Kirsten, in the acute transforming retrovirus experiment. Since Weinberg et al. discovered human bladder cancer cells in 1982 After the activation of H-ras gene in human tumors, people have paid great attention to the role of ras oncogene in the development of human tumors. The ras gene family has three genes related to human tumors - H-ras, K-ras and N-ras, which are located on chromosome 11, 12 and 1 respectively. Among them, K-Ras has the greatest impact on huma...

Claims

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Application Information

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IPC IPC(8): C12Q1/68G01N21/64
Inventor 石大陆
Owner 广州达健生物科技有限公司
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