Application of hoxa7 methylation detection reagent in the preparation of lung cancer diagnostic reagent
A detection reagent and lung cancer diagnosis technology, applied in the field of genetic diagnosis, can solve problems such as limitation, patient loss, false positive rate, etc., and achieve high sensitivity, convenient sampling process, and wide application range
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Embodiment 1
[0076] Example 1: Selection of Detection Target Genes
[0077] Methylated DNA has obvious advantages as a detection target. Compared with protein markers, DNA can be amplified and easily detected; compared with mutation markers, DNA methylation sites are located in genes The specific site, generally in the promoter region, makes the detection easier and more convenient. In order to complete the present invention, the inventor screened hundreds of genes, and selected better HOXA7, SHOX2, PCDHGA12, HOXD8, GATA3 as candidate detection genes, β-actin gene as an internal reference gene, and studied the methylation of each gene According to the distribution of sites, the primers and probes designed for detection were used for real-time fluorescent quantitative methylation-specific polymerase chain reaction (real-time fluorescent quantitative methylation-specific PCR, qMSP) detection. The detection primers and probes for each gene are as follows:
[0078] The detection primers and ...
Embodiment 2
[0126] Example 2: Detection of HOXA7, SHOX2, PCDHGA12, HOXD8 and GATA3 genes in sputum
[0127] Sample information: A total of 90 cases of sputum samples were tested, including 55 cases of normal control group samples, 35 cases of cancer group control samples, 12 cases of squamous cell carcinoma, 6 cases of small cell carcinoma, 9 cases of adenocarcinoma, and large There were 2 cases of cell carcinoma and 6 cases of unclassified lung cancer.
[0128] Experimental procedure:
[0129] a. Collect sputum samples from patients diagnosed with lung cancer and non-lung cancer patients. After thickening with DTT, centrifuge to separate the pellet and separate the cells, wash them twice with PBS, and then use the DNA extraction kit (HiPure FFPE DNA Kit, D3126-03) to extract DNA.
[0130] b. Use the DNA conversion kit (EZ DNA Methylation Kit, D5002) of ZYMO RESEARCH Biological Company to carry out the bisulfite modification of DNA.
[0131] c. The liquid distribution system is as foll...
Embodiment 3
[0141] Example 3: Detection of HOXA7 and SHOX2 genes in sputum
[0142] A large number of documents show that SHOX2 can be used as a marker for the detection of lung cancer, and there are patents [CN201510203539-method and kit for diagnosing methylation of human SHOX2 gene and human RASSF1A gene-application disclosure], SHOX2 in alveolar lavage fluid, lesion sites It has a high detection rate in samples such as tissue, pleural effusion, and sputum. In order to verify the detection effect of HOXA7, the inventors simultaneously detected the detection efficiency of HOXA7 and SHOX2 gene. In this example, the detection efficiency of SHOX2 gene used the primer and probe sequences disclosed in patent CN201510203539, and the It is expressed as SHOX2_n3 to distinguish it from the SHOX2 gene detected by self-designed primers and probes in Examples 1 and 2 of the present invention.
[0143] The detection primers and probes for each gene are as follows:
[0144] The detection primers an...
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