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ARMS-qPCR (Allele Refractory Mutation System-quantitative Polymerase Chain Reaction) detection kit for KRAS (Kirsten Rat Sarcoma Viral Oncogene Homolog) gene mutation subtype and detection method

A detection kit and kit technology, applied in the field of molecular biology, can solve the problems of easy pollution, time-consuming, cumbersome procedures, etc., and achieve the effect of increasing specificity, high sensitivity, and strong specificity

Inactive Publication Date: 2012-03-07
ZHEJIANG UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0008] The technical problem to be solved in the present invention is to overcome the deficiencies in the prior art and provide a KRAS gene mutation typing ARMS-qPCR detection kit to solve the problems of time-consuming, cumbersome procedures and easy pollution in the existing mutation detection technology. question

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  • ARMS-qPCR (Allele Refractory Mutation System-quantitative Polymerase Chain Reaction) detection kit for KRAS (Kirsten Rat Sarcoma Viral Oncogene Homolog) gene mutation subtype and detection method
  • ARMS-qPCR (Allele Refractory Mutation System-quantitative Polymerase Chain Reaction) detection kit for KRAS (Kirsten Rat Sarcoma Viral Oncogene Homolog) gene mutation subtype and detection method
  • ARMS-qPCR (Allele Refractory Mutation System-quantitative Polymerase Chain Reaction) detection kit for KRAS (Kirsten Rat Sarcoma Viral Oncogene Homolog) gene mutation subtype and detection method

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Embodiment Construction

[0063] In order to make the present invention easier to understand, specific implementation cases of the present invention will be further described below.

[0064] The wax blocks of 100 patients diagnosed with colorectal adenocarcinoma (CRC) were collected. All patients had not received cetuximab (cetuximab) treatment before operation. Genomic DNA was extracted from the wax blocks for the following experimental applications. ARMS-qPCR was used to detect 7 common base substitution mutations at codons 12 and 13 of KRAS gene.

[0065] Design and screen one specific ARMS primer and one reference primer that can specifically detect the above seven base substitution mutations, design and screen a general downstream primer, design and screen a general TaqMan probe, design and screen a LNA blocking probe , the sequences of each probe and primer are as follows:

[0066] The LNA sequence is shown in SEQ ID NO A0 in the sequence listing: 5'-TGGAGCTG G TG G CGTAGGC-PO4-3'.

[0067] Th...

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Abstract

The invention relates to the field of molecular biology and aims to provide an ARMS-qPCR (Allele Refractory Mutation System-quantitative Polymerase Chain Reaction) detection kit for KRAS (Kirsten Rat Sarcoma Viral Oncogene Homolog) gene mutation subtype and a detection method. The kit comprises a qPCR hybrid reaction solution, a locked nucleic acid retardant probe, a reference primer, an ARMS primer and a positive control sample, wherein the qPCR hybrid reaction solution comprises a PCR buffer solution, dNTPs (Deoxynucleotide Triphosphates), MgCl2, GoldStarbest Taq enzyme, a universal PCR reverse primer and a universal TaqMan probe. The kit provided by the invention can be used for rapidly and accurately detecting specific locus mutation of KRAS genes in various cancer tissues with high sensitivity, has high sensitivity, and can be used for detecting genome DNA with various tissue origins, specially free DNA segments adopting cell-free systems, such as blood serum and blood plasma, orother body fluid origins, wherein the genome DNA is derived from cell systems. Compared with direct sequencing and other mutation detection technologies, the kit and the detection method thereof havethe advantages of strong specificity, high sensitivity, simplicity and rapidness in operation, high throughput, safety, definiteness and objectivity in result identification and the like for detecting the KRAS gene mutation.

Description

technical field [0001] The invention relates to the field of molecular biology, in particular to a rapid detection kit and a detection method for KRAS gene mutation typing for guiding individualized treatment of tumors. Background technique [0002] The RAS proto-oncogene was originally a transforming gene cloned from rat sarcoma virus. Since Weinberg et al. found that there was an activated HRAS gene in human bladder cancer cells in 1982, it has aroused people's attention to the occurrence and development of RAS oncogenes in human tumors. Great attention is paid to the role played in the process. There are three genes related to human tumors in the RAS gene family—HRAS, KRAS, and NRAS, which are located on chromosomes 11, 12, and 1, respectively. Among them, KRAS has the greatest impact on human cancer. It is like a molecular switch: when it is normal, it can control and regulate the path of cell growth; Continue to grow, and prevent apoptosis to cause cancer. [0003] T...

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68
Inventor 蒋微琴滕理送王伟伟于秀菊
Owner ZHEJIANG UNIV
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