Primers, probes, detection system and kit for one time detection of lung cancer multiple genes
A kit, a one-time technology, applied in biochemical equipment and methods, recombinant DNA technology, microbial determination/inspection, etc., can solve the problems of small sample size, unable to meet one-by-one screening, time-consuming, etc., and achieve cost savings Effect
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Embodiment 1
[0077] According to the wild-type gene sequences of human EGFR, KRAS, BRA, ALK and ROS1 published by Cosmic data, specific primers and New probes (see Table 1 and Table 8).
[0078] Table 8 Distribution of each tube
[0079]
[0080] In this example, 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 detection of the above lung cancer genes by fluorescent PCR of the present invention.
[0081] The experiment uses the armored RNA containing the above-mentioned fusion gene and the plasmid template of each mutation respectively, and its fluorescent PCR detection includes the following steps:
[0082] (1) Treatment and extraction of plasmids and armored RNA:
[0083] The extraction of each p...
Embodiment 2
[0131] Using the present invention to detect clinical samples, from December 2013 to October 2014, multiple lung cancer gene mutations were detected on 1,100 clinical lung cancer paraffin-embedded tissue samples.
[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 from Xiamen Aide Biomedical Technology Co., Ltd. ), Cat No.ADx-FF03 or QIAGEN Paraffin Tissue DNA Extraction Kit, Cat No.56404; follow the instructions of the extraction kit. Immediately after DNA and RNA extraction, use a UV spectrophotometer to detect the concentration and purity of DNA and RNA. DNA and RNA OD260 / OD280 should be between 1.8 and 2.1; RNA concentration should be between 50 and 500 ng / μL. The DNA and RNA extracted above were used as templates for fluorescent PCR amplification of lung cancer genes.
[0133] 2. Carry out PCR amplification with the above-mentioned DNA and RNA respectively according to the following amplif...
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