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Methylation gene related to lung cancer and detection kit of gene

A detection kit and methylation technology, which is applied in the field of methylation genes and its detection kits, can solve the problems of low sensitivity, low cancer misdiagnosis rate, low cancer missed diagnosis rate, etc., and achieve the effect of improving sensitivity

Pending Publication Date: 2019-10-11
苏州呼呼健康科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The reported methylation gene markers related to lung cancer still have the defects of low sensitivity (35.5%) and low specificity (73%), which limits their application in clinical detection.
Here, we need to briefly introduce relevant knowledge: the high sensitivity of a methylated gene marker for a certain cancer or certain cancers indicates that the corresponding cancer has a low misdiagnosis rate; similarly, its high specificity indicates that the corresponding cancer has a low misdiagnosis rate Low

Method used

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  • Methylation gene related to lung cancer and detection kit of gene
  • Methylation gene related to lung cancer and detection kit of gene
  • Methylation gene related to lung cancer and detection kit of gene

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0042] Example 1 Selection of methylated genes associated with lung cancer

[0043] In the present invention, 159 candidate genes related to the identification of benign and malignant pulmonary nodules or lung cancer are screened out by performing genome-wide 850KIllumina methylation array analysis on bronchoalveolar flushing fluid samples of patients with lung cancer and benign lung diseases, specifically including:

[0044] NFAM1, BZRAP1, MIR142, C16ORF68, MIR365-1, SPNS1, LAT, SIX1, MAB21L1, MIR548, NBEA, BIN2, ALX1, CUX2, C11ORF21, TSPAN32, WDFY4, TRIM39, LOC728264, MIR145, HOXA9, DMPR, STAB1, ARHGAP26 SCT, NDUFS2, FCER1G, SHOX2, ETV5, DPP10, PADI6, BAIAP2L1, MEIS1, DLGAP2, WDR66, JAM3, RBM24, RBM38, HOXD8, GBX2, DPP6, KCNIP4, SHANK2, CBLN1, SRGAP3, PCDHGA4, PCDHGC3, PCDHGA12, PCDHGA11, PCDHGA9,PCDHGA1,PCDHGB1,PCDHGC5,PCDHGB6,PCDHGB3,PCDHGB7,PCDHGA6,PCDHGA8,PCDHGA10,PCDHGA5,PCDHGB4,PCDHGC4,PCDHGA3,PCDHGA2,PCDHGA7,PCDHGB2,PCDHGC5,PCDHGB5,PCDHGC3,PCDHGB19P,MYCBPAP,HOXA7,FRR3...

Embodiment 2

[0078] Example 2 verifies the designed primer set

[0079] In this embodiment, real-time fluorescent PCR is used as an example to detect the methylation level of biomarker genes. The detection targets are HOXA7, HOXA9, SCT, SHOX2 genes. In this embodiment, DNA treated with bisulfite is used as a template for real-time fluorescent PCR amplification for detection.

[0080] It should be said that designing a high-quality primer set is a key factor for the success of methylated gene detection. The design principle of the present invention is different from ordinary gene detection. It requires that the primer sequence contains at least one CpG site, preferably multiple CpG sites, so as to ensure the specificity of the primer and improve the methylation. Gene base detection rate.

[0081] For HOXA7, HOXA9, SCT, and SHOX2 genes, multiple sets of primers and probe combinations can be designed, and the performance of each set of probe-primer combinations may be different, so it is n...

Embodiment 3

[0087] Example 3 Testing the Sensitivity and Specificity of Methylation Gene Detection with Bronchial Brushing Samples

[0088] In this embodiment, bronchial swabbing samples from 167 patients undergoing bronchoscopy were selected as test samples, including 95 patients with lung cancer and 72 patients with benign lung disease. DNA was extracted from these samples, and after being treated with bisulfite, the aforementioned HOXA7 primer set, HOXA9 primer set, SCT primer set 2 and SHOX2 primer set were selected, and the methylation levels of the four genes were detected by fluorescent PCR (Ct value ), and use the ROC curve to compare the methylation detection results of each gene in the respective samples of lung cancer patients and patients with benign lung diseases, and select the point with the largest Youden index on the curve to calculate the sensitivity of benign and malignant pulmonary nodules or lung cancer diagnosis , specificity, and calculate the area under the curve (...

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Abstract

The invention discloses a methylation gene related to the lung cancer and a detection kit of the gene. The gene comprises HOXA7, HOXA9, SCT and SHOX2, and methylation cytosine loci of the gene are calibrated in a sequence 1, a sequence 2, a sequence 3 and a sequence 4 in sequence. The detection kit comprises a reagent for detecting the HOXA7 gene, a reagent for detecting the HOXA9 gene, a reagentfor detecting the SCT and a reagent for detecting the SHOX2 gene. The reagent for detecting the HOXA7 gene comprises primers shown in sequences with the numbers 1-1 and 1-2 and a probe with a sequenceshown in the number 1-3; the reagent for detecting the HOXA9 gene comprises primers shown in sequences with the numbers 2-1 and 2-2 and probes shown in sequences with the number 2-3; the reagent fordetecting the SCT comprises primers shown in sequences with the numbers 3-1 and 3-2, a probe shown in a sequence with the number 3-3, primers shown in sequences with the numbers 3-4 and 3-5 and a probe shown in a sequence with the number 3-6; the reagent for detecting the SHOX2 gene comprises primers shown in sequences with the numbers 4-1 and 4-2 and a probe shown in a sequence with the number 4-3.

Description

technical field [0001] The invention relates to a reagent for detecting genes, in particular to a methylated gene related to lung cancer and a detection kit thereof. Background technique [0002] Pulmonary nodules are small focal, round-shaped shadows with increased density on imaging, which can be single or multiple, without atelectasis, hilar enlargement, and pleural effusion. There are many causes of pulmonary nodules, which are basically divided into two categories: benign nodules and malignant nodules. Benign nodules include benign tumors, tuberculosis, sarcoidosis, or inflammatory nodules. Malignant nodules include lung cancer and metastatic tumors, etc. The occurrence of lung cancer goes through a series of stages such as hyperplasia, dysplasia, carcinoma in situ, and invasive carcinoma. [0003] Lung cancer in malignant pulmonary nodules is one of the most common malignant tumors in my country. According to the latest cancer data in China released by the National Ca...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6886C12N15/11
CPCC12Q1/6886C12Q2600/154C12Q2600/118
Inventor 李丹江南刘代新
Owner 苏州呼呼健康科技有限公司
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