Primers, probes, detection systems and kits for one-time detection of multiple genes in lung cancer

Abstract

The present invention discloses primers, probes, a detection system and a kit for one time detection of lung cancer multiple genes, wherein the primers, the probes, and the distribution way for detecting 25 EGFR gene mutations, 7 KRAS gene mutations, 6 BRAF gene mutations, 9 NRAS gene mutations, 5 HER2 gene mutations, 2 PIK3CA gene mutations, 5 fusion genes of ALK5, 13 fusion genes of ROS1, and 6 fusion genes of RET are provided. According to the present invention, the detection kit adopts the 12 linking PCR reaction strip design, each 12 linking PCR strip detects multiple genes of a sample, and the corresponding fusion detection reagents and the internal control reagents are filled in the pipes 1-4 of the 12 linking PCR strip; and with the primers, the probes, the detection system and the kit, the one-time detection of the 24 fusions and the 54 mutations of the lung cancer can be achieved, such that the detection time is substantially shortened, the sensitivity is high, the specificity is strong, the operation is simple and rapid, and the reference for selection of tumor targeting drug therapy on lung cancer patients can be provided for clinician.

Description

Technical field

[0001] The invention belongs to the field of biotechnology, and specifically relates to primers, probes and detection systems for multiple genes of lung cancer. Background technique

[0002] Lung cancer is one of the malignant tumors with the highest morbidity and mortality. Among them, non-small cell lung cancer (NSCLC) accounts for about 85% of lung cancers. With the continuous in-depth research on tumor pathogenesis and its biological behavior, more and more people are focusing on molecular targeted therapy characterized by high specificity and low side effects. In recent years, in the field of molecular targeted therapy of lung cancer, research hotspots have mainly focused on EGFR, ALK, ROS1, KRAS, BRAF and other targets. The mutation or fusion status of these genes is related to the efficacy of targeted drugs, and the combination of multiple genes Detection can further improve the accuracy of predicting prognosis. However, at present, most of the mutations ...

Examples

Embodiment 1

[0077] The human EGFR, KRAS, BRA, ALK and ROS1 published according to Cosmic data are wild-type gene sequences. Based on the driving mutation sites and fusion sites of EGFR, KRAS, BRA, ALK and ROS1, specific primers and New probes (see Table 1 and Table 8).

[0078] Table 8 Distribution of tubes

[0079]

[0080] This example uses EML4 exon 13; ALK exon 20 (ALK fusion gene), CD74 exon 6; ROS1 exon 32 (ROS1 fusion gene), KIF5B exon 16; ROS1 exon 12 (RET fusion gene), L861Q (EGFR gene mutation), G12D (KRAS gene mutation), V600E1 (BRAF gene mutation), G776> VC (HER2 gene mutation), Q61R (NRAS gene mutation), H1047R (PIK3CA gene mutation) are examples to illustrate the fluorescent PCR detection of the above-mentioned lung cancer genes of the present invention.

[0081] The experiment uses armored RNA containing the above-mentioned fusion gene and each mutant plasmid template, and the fluorescent PCR detection includes the following steps:

[0082] (1) Plasmid and armored RNA processing a...

Embodiment 2

[0131] Using the present invention to detect clinical samples, from December 2013 to October 2014, 1100 cases of clinical lung cancer paraffin-embedded tissue samples were tested for multiple lung cancer gene mutations.

[0132] 1. Extraction of DNA and RNA from test samples: DNA and RNA can be extracted using the FFPE sample DNA / RNA co-separation kit of Xiamen Aide Biomedical Technology Co., Ltd. (Minxia Food and Drug Administration (quan) 2013 No. 1400070 ), CatNo.ADx-FF03 or QIAGEN paraffin tissue DNA extraction kit, Cat NO.56404; follow the instructions of the extraction kit. After the extraction of DNA and RNA, immediately use an ultraviolet spectrophotometer to detect the concentration and purity of DNA and RNA. DNA and RNA OD260 / OD280 should be between 1.8~2.1; RNA concentration should be between 50~500 ng / μL. The DNA and RNA extracted above are used as the template for fluorescent PCR amplification of lung cancer genes.

[0133] 2. The DNA and RNA mentioned above are resp...

Technical field

[0001] The invention belongs to the field of biotechnology, and specifically relates to primers, probes and detection systems for multiple genes of lung cancer. Background technique

[0002] Lung cancer is one of the malignant tumors with the highest morbidity and mortality. Among them, non-small cell lung cancer (NSCLC) accounts for about 85% of lung cancers. With the continuous in-depth research on tumor pathogenesis and its biological behavior, more and more people are focusing on molecular targeted therapy characterized by high specificity and low side effects. In recent years, in the field of molecular targeted therapy of lung cancer, research hotspots have mainly focused on EGFR, ALK, ROS1, KRAS, BRAF and other targets. The mutation or fusion status of these genes is related to the efficacy of targeted drugs, and the combination of multiple genes Detection can further improve the accuracy of predicting prognosis. However, at present, most of the mutations ...

Examples

Embodiment 1

[0077] The human EGFR, KRAS, BRA, ALK and ROS1 published according to Cosmic data are wild-type gene sequences. Based on the driving mutation sites and fusion sites of EGFR, KRAS, BRA, ALK and ROS1, specific primers and New probes (see Table 1 and Table 8).

[0078] Table 8 Distribution of tubes

[0079]

[0080] This example uses EML4 exon 13; ALK exon 20 (ALK fusion gene), CD74 exon 6; ROS1 exon 32 (ROS1 fusion gene), KIF5B exon 16; ROS1 exon 12 (RET fusion gene), L861Q (EGFR gene mutation), G12D (KRAS gene mutation), V600E1 (BRAF gene mutation), G776> VC (HER2 gene mutation), Q61R (NRAS gene mutation), H1047R (PIK3CA gene mutation) are examples to illustrate the fluorescent PCR detection of the above-mentioned lung cancer genes of the present invention.

[0081] The experiment uses armored RNA containing the above-mentioned fusion gene and each mutant plasmid template, and the fluorescent PCR detection includes the following steps:

[0082] (1) Plasmid and armored RNA processing a...

Embodiment 2

[0131] Using the present invention to detect clinical samples, from December 2013 to October 2014, 1100 cases of clinical lung cancer paraffin-embedded tissue samples were tested for multiple lung cancer gene mutations.

[0132] 1. Extraction of DNA and RNA from test samples: DNA and RNA can be extracted using the FFPE sample DNA / RNA co-separation kit of Xiamen Aide Biomedical Technology Co., Ltd. (Minxia Food and Drug Administration (quan) 2013 No. 1400070 ), CatNo.ADx-FF03 or QIAGEN paraffin tissue DNA extraction kit, Cat NO.56404; follow the instructions of the extraction kit. After the extraction of DNA and RNA, immediately use an ultraviolet spectrophotometer to detect the concentration and purity of DNA and RNA. DNA and RNA OD260 / OD280 should be between 1.8~2.1; RNA concentration should be between 50~500 ng / μL. The DNA and RNA extracted above are used as the template for fluorescent PCR amplification of lung cancer genes.

[0133] 2. The DNA and RNA mentioned above are resp...

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