Application of hoxa9 methylation detection reagent in the preparation of lung cancer diagnostic reagent
A HOXA9 and detection reagent technology, applied in the field of genetic diagnosis, can solve the problems of limitation, patient loss, false positive rate, etc., and achieve the effects of high sensitivity, convenient sampling process, and wide application range
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Embodiment 1
[0073] Example 1: Selection of Detection Target Genes
[0074] Using methylated DNA as the detection target has obvious advantages. Compared with protein markers, DNA can be amplified and easily detected; compared with mutation markers, DNA methylation sites are located in Specific parts of the gene, generally in the promoter region, which makes detection easier and more convenient. In order to complete the present invention, the inventors selected HOXA9, SHOX2, PCDHGA12, HOXD8, and GATA3 from hundreds of candidate genes through multiple rounds of screening as candidate detection genes, and the β-actin gene as an internal reference gene. The distribution of gene methylation sites, the primers and probes designed for detection were used for real-time fluorescent quantitative methylation-specific polymerase chain reaction (real-time fluorescentquantitative methylation-specific PCR, qMSP) detection. The detection primers and probes for each gene are as follows:
[0075] The det...
Embodiment 2
[0124] Example 2: Detection of HOXA9, SHOX2, PCDHGA12, HOXD8 and GATA3 genes in sputum
[0125] 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.
[0126] Experimental procedure:
[0127] a. Collect sputum specimens 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 Magen’s DNA extraction kit (HiPure FFPE DNA Kit, D3126-03) to extract DNA.
[0128] b. Use the DNA conversion kit (EZ DNA Methylation Kit, D5002) of ZYMO RESEARCH Biological Company to carry out the bisulfite modification of DNA.
[0129] c. The liquid distribution system is a...
Embodiment 3
[0139] Example 3: Detection of HOXA9 and SHOX2 genes in sputum
[0140] 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 HOXA9, in this example, the detection efficiency of the SHOX2 gene uses the primer and probe sequences disclosed in the patent CN201510203539, and the SHOX2 gene is expressed as SHOX2_n3 to distinguish it from the ones in Examples 1 and 2 of the present invention. The SHOX2 gene detected by self-designed primers and probes.
[0141] The detection primers and probes for each gene are as follows:
[0142] The detection primers and probes for HOXA9 are:
[0143] SEQ ID NO: 1HOXA9-F2 Primer ...
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