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A marker, kit and method for early detection of lung cancer

A detection reagent and early detection technology, applied in biochemical equipment and methods, microbiological determination/inspection, DNA/RNA fragments, etc., can solve problems such as poor signal-to-noise ratio, achieve improved accuracy, less trauma, and easy access Effect

Active Publication Date: 2021-11-30
苏州京脉生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, there are still some deficiencies in current research on circulating miRNAs as diagnostic markers for lung cancer, for example: (1) A large part of the research only selects miRNAs that have been reported to be dysregulated in lung cancer tissues as candidate indicators, and these miRNAs are in Serum is not necessarily the best choice
(2) Some studies have used microarray for preliminary screening of miRNA markers, but compared with next-generation sequencing, the signal-to-noise ratio of microarray is poor, so the miRNA markers screened by it are not necessarily the best
(3) Random tag sequences that can improve the accuracy of serum miRNA quantification by removing PCR repeats have not been widely used in such studies

Method used

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  • A marker, kit and method for early detection of lung cancer
  • A marker, kit and method for early detection of lung cancer
  • A marker, kit and method for early detection of lung cancer

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0086] Example 1: Obtain training group samples

[0087] From June 2016 to August 2018, the applicant collected peripheral venous blood samples from 102 untreated patients with early-stage lung cancer. Each sample contained 20ml of peripheral blood, including 61 males and 41 females. The age was 58.3 years, and the age distribution was 34–81 years. During the same period, the applicant collected a total of 100 samples of peripheral venous blood from healthy people (that is, healthy controls without various diseases, the same below), each containing 20ml of peripheral blood, including 60 males and 40 females, with an average age of was 57.9 years old, and the age distribution was 34–80 years old. These two groups of samples are used as training group samples, and there is no statistically significant difference in gender and age of these two groups of samples, so the principle of gender and age matching is met.

[0088] For each peripheral blood sample, sequencing library pre...

Embodiment 2

[0089] Example 2: Sequencing library preparation and next-generation sequencing

[0090] For each training group sample, the following reagents and steps were used for library preparation and next-generation sequencing:

[0091] (1) 20ml of peripheral blood samples were collected in dry blood collection tubes and left at 4°C for more than half an hour, then 400g of free RNA was obtained, centrifuged at 4°C for 10 minutes to take the supernatant, and then centrifuged at 1800g for 10 minutes at 4°C to take the supernatant , to obtain serum samples, stored in -80 ℃ refrigerator;

[0092] (2) Use Qiagen miRNeasy Serum / Plasma Kit (Cat. No.: 217184) to extract 50–200 ng of serum free RNA from the above serum sample, and dilute it to a total volume of 5 μl with ultrapure water (no DNase and RNase, the same below). , and placed in a 200μl thin-walled PCR tube;

[0093] (3) Add 1 μl of adapter RA3 with a concentration of 10 μM to the solution obtained in step (2), mix well, react at ...

Embodiment 3

[0105] Embodiment 3: obtain the expression level RPM of miRNA

[0106] For the off-machine data of the training group samples, the following steps are used for data analysis to obtain the expression level RPM of each miRNA in the samples:

[0107] (1) For the off-machine data of the sample, use FastQC, Cutadpat and Trimmomatic for data quality control and preprocessing (using default parameters) to obtain effective data with low-quality sequences and sequencing adapters removed;

[0108] (2) Remove the random label sequence S2 and the fixed base S3 in RA5 from the 5' end of the sequence of the valid data, and then use the sequence alignment software Bowtie to re-align the obtained sequence to the human reference genome sequence (allowing up to 1 base mismatch), to obtain the location information mapped to the human reference genome;

[0109] (3) Comparing the obtained sequence alignment position information with the corresponding random tag sequence S2, and performing PCR rep...

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Abstract

The present application provides a marker, kit and method for early detection of lung cancer, wherein the miRNA markers mainly include: hsa-miR-15b-3p, hsa-miR-1246, hsa-miR-1285-3p, hsa-miR-1285-3p, hsa-miR-1285-3p, miR‑181b‑5p, hsa‑miR‑2276‑3p, hsa‑miR‑301a‑3p, hsa‑miR‑31‑5p, hsa‑miR‑3152‑3p, hsa‑miR‑448, hsa‑miR‑505‑ 3p, and hsa-miR-92a-3p; the kit is used for sample sequencing library preparation, and when analyzing next-generation sequencing data, the S2 random tag sequence of the adapter RA5 is used as a quantitative tag to remove PCR Repeated sequences can improve the accuracy of detection; the method is to obtain the expression level of each miRNA in the test sample through data analysis, and based on the expression level of the miRNA markers, use the formula S1 to calculate the probability of the test sample suffering from early lung cancer, In order to determine whether the sample suffers from early-stage lung cancer.

Description

technical field [0001] The invention relates to the field of biotechnology, more specifically, to a marker, kit and method for early detection of lung cancer. Background technique [0002] Lung cancer is the malignant tumor with the largest number of morbidity and death cases worldwide. According to statistics from the China Cancer Center in 2016, lung cancer accounted for 733,000 of the 4.29 million new cancer cases, and 610,000 of the 2.8 million cancer deaths. Among them, non-small cell lung cancer accounts for about 80% of all lung cancers. The early symptoms of lung cancer are not obvious, so about 75% of the patients are already in the advanced stage of lung cancer when they are found to have cancer, with local invasion and distant metastasis. The five-year survival rate of advanced lung cancer is very low, less than 5%. However, the 5-year survival rate of patients with early lung cancer can be as high as 90%. Therefore, early diagnosis of lung cancer is an import...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/6886C12Q1/6869C12N15/113
CPCC12Q1/6869C12Q1/6886C12Q2600/158C12Q2600/178C12Q2531/113C12Q2521/107C12Q2535/122C12Q2537/165
Inventor 李华胡延平郭子文沈益行
Owner 苏州京脉生物科技有限公司
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