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A kind of diagnostic kit for detecting nsclc and its using method and application

A technology of diagnostic kits and reagents, which is applied in biochemical equipment and methods, recombinant DNA technology, and microbial determination/examination, etc., can solve the problem of lack of high specificity and sensitivity, difficult to cure lung tumors, and unknown biological functions. and other problems, to achieve the effect of saving time and reagents, making methods and using simple, accurate and reliable test results

Active Publication Date: 2021-05-28
YUANJIAN BIOTECH SHANGHAI CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Limited by the lack of high-specificity and sensitivity early screening methods, about 75% of patients have local or distant metastases at the time of diagnosis. Due to the wide range of lesions, middle and advanced lung tumors are often difficult to be cured, and the 5-year survival rate is still insufficient 20%
In recent years, a large number of studies have shown that circRNA is closely related to the growth and development of organisms, stress response, disease occurrence and development, etc., and predicted its application prospects in disease diagnostic markers, etc., but its biological functions are largely limited. still unknown on

Method used

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  • A kind of diagnostic kit for detecting nsclc and its using method and application
  • A kind of diagnostic kit for detecting nsclc and its using method and application
  • A kind of diagnostic kit for detecting nsclc and its using method and application

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Experimental program
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Effect test

Embodiment 1

[0035] 1. Expression of hsa_circ_0069841 in NSCLC cell lines

[0036] 1.1 Cell culture

[0037] Human normal lung epithelial cells BEAS-2B and NSCLC cell lines H1299, A549, H1975, H1650, H1972, H2073, HCC827, H226, H520, H460, H358, H1573, H2170, SK-MES-1 were used with 10% fetal DMEM high glucose culture of bovine serum based on 37°C, 5% CO 2 , cultured in an incubator with 95% saturated humidity, when the cells reached the logarithmic growth phase, they were routinely digested and passaged once, and the cells were collected separately for experiments. Cells were digested using trypsin digest solution containing 0.25% EDTA.

[0038] 1.2. RT-qPCR experiment

[0039] 1.2.1 Extraction of total cellular RNA

[0040] Use trypsin to digest the human normal lung epithelial cells BEAS-2B and 14 kinds of NSCLC cells. After digestion, the above cells were collected in 1.5ml EP tubes, put into a centrifuge, and centrifuged at 1000rpm / min at room temperature for 10min. Discard the s...

Embodiment 2

[0064] A primer design kit for the preferred hsa_circ_0069841 and GAPDH gene was used. This kit adopts a real-time fluorescent quantitative PCR amplification system, and each 20 μl reaction system includes: a pair of amplification primer sequences for detecting the copy number of the hsa_circ_0069841 gene and a pair of primer sequences for detecting the copy number of the internal reference gene GAPDH gene (see Table 2). The forward primer (10 μM) volume is 1 μl, the reverse primer (10 μM) volume is 1 μl, the volume of 2×SYBR Green qPCR Mix is ​​10 μl, the volume of 50×ROX Dye2 is 0.4 μl, and the NSCLC tissue cDNA template to be tested ( (obtained by reverse transcription after extracting RNA from tissues) 2 μl, 5.6 μl of enzyme-free deionized water. In this kit, hsa_circ_0069841 is significantly highly expressed in NSCLC, so detection of hsa_circ_0069841 can accurately diagnose NSCLC.

[0065] PCR primer sequence in the kit in table 2

[0066]

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Abstract

The application discloses a diagnostic kit for detecting NSCLC and its use method and application. An application of hsa_circ_0069841 in detecting NSCLC biomarkers is provided. Also provided is a test kit comprising a reagent for detecting hsa_circ_0069841 expression, comprising a primer pair for amplifying the hsa_circ_0069841 gene as shown in SEQ ID NO: 1 and SEQ ID NO: 2, and a sequence such as SEQ ID NO: 9 and SEQ ID NO: 9 and SEQ ID NO: 2 A primer pair for amplifying the GAPDH gene shown in ID NO:10. The present invention finds for the first time that hsa_circ_0069841 is significantly highly expressed in NSCLC. As a biomarker for diagnosing NSCLC, a kit for diagnosing NSCLC is prepared, which can facilitate clinical diagnosis of NSCLC and guide clinical treatment.

Description

technical field [0001] The application relates to the technical fields of medicine and clinical diagnosis, and in particular to a diagnostic kit for detecting NSCLC and its use method and application. Background technique [0002] Malignant tumors have become a major high-risk disease that seriously threatens human health. The morbidity and mortality of lung cancer rank first among all malignant tumors worldwide. Lung cancer is the most common cancer in my country, and it is the top priority of cancer prevention and treatment. At present, the incidence of lung cancer in my country is increasing by about 26% every year. If effective control measures are not taken in time, it is estimated that by 2025, the number of lung cancer patients in my country will reach 1 million, becoming the world's largest lung cancer country. [0003] Non-small cell lung cancer (NSCLC) is the most common pathological type of lung cancer in clinical practice, accounting for about 85% of the incide...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/6886C12N15/11
CPCC12Q1/6886C12Q2600/158C12Q2600/166C12Q2600/178
Inventor 杜飞
Owner YUANJIAN BIOTECH SHANGHAI CO LTD
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