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Lung cancer detection kit and application thereof

A kit and technology for lung cancer, applied in the biological field, can solve the problems of inability to meet the needs of real-time detection of scattered specimens, limited specificity and sensitivity, high cost and price, and achieve rapid and convenient detection, wide application range and low cost. Effect

Inactive Publication Date: 2015-11-11
ZHEJIANG ACAD OF TRADITIONAL CHINESE MEDICINE +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The content of miRNA in serum is much lower than that in tissues, so a very sensitive and specific detection method is required; and because of the short sequence of miRNA primers, primer design is difficult, and no targeted software can be used directly, so both primers and systems need to be screened and tested. optimization process
At present, chip technology is generally used for high-throughput screening of miRNA, which has high cost, limited specificity and sensitivity, and cannot meet the needs of real-time detection of scattered samples; real-time detection of a small number of miRNAs mostly uses Taqman probe method, and some of them have been commercialized Kit products, but the cost and selling price are high

Method used

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  • Lung cancer detection kit and application thereof
  • Lung cancer detection kit and application thereof
  • Lung cancer detection kit and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0046] Embodiment 1. Preparation of diagnostic reagents and kits

[0047] (1) Raw material configuration

[0048] Probes (miR205-3P, miR21-3P, cel-miR-39-3P) were designed and synthesized by the inventors of the present invention; reverse transcription stem-loop primers (miR205-1RP, miR21-1RP, cel-miR-39-1RP ), PCR amplification primers (miR205-2F, miR21-2F, cel-miR-39-2F, miR-UR) were designed by the inventors of the present invention and synthesized by Invitrogen.

[0049]

[0050]

[0051] Reverse transcription neck loop primers (miR205-1RP, miR21-1RP, cel-miR-39-1RP): configured into a 10 μM solution, and then diluted 40 times to a 0.25 μM solution for use.

[0052] PCR amplification primers (miR205-2F, miR21-2F, cel-miR-39-2F, miR-UR): prepared as a 10 μM solution for later use.

[0053] cel-miR-39: purchased from QIAGEN, 10 pmol per tube. Add 300 μl RNase-FreeddH 2 O is configured as 2×10 10 copies / μl, then diluted 25 times to 1.6×10 8 copies / μl for later use...

Embodiment 2

[0063] Example 2. Sample detection

[0064] (1) sample

[0065] 29 cases of plasma samples from healthy people (168-182,183-197); 27 cases of plasma samples before lung cancer surgery (1P-27P); 26 cases of plasma samples after lung cancer surgery (1P-26P).

[0066] (2) Detection reagent

[0067] Serum / plasma miRNA was extracted using miRcutemiRNAIsolationKit (TIANGEN, Cat.#DP501).

[0068] The reverse transcription kit uses MicroRNA ReverseTranscription Kit (Life Technologies, cat. no. 4366596).

[0069] And the kit prepared in embodiment 1.

[0070] (3) Extraction of plasma miRNA

[0071] Use miRcutemiRNAIsolationKit (TIANGEN, Cat.#DP501) to extract plasma miRNA, the specific steps are as follows:

[0072] 1) Sample processing: add an equal volume of lysate MZ to 200 μl plasma, shake and mix for 30 seconds, and place at room temperature for 5 minutes;

[0073] 2) Centrifuge at room temperature at 12000rpm for 10min, take the supernatant, and discard the precipitate; ...

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Abstract

The invention relates to the technical field of biotechnology, in particular to a lung cancer detection kit which takes miR-21 and miR-205 in serum as markers and is used for lung cancer screening and postoperative monitoring. The miR-21 and miR-205 in serum / blood plasma are selected to jointly serve as lung cancer markers, and the lung cancer diagnostic kit which has rapidness and convenience in detection, is high in accuracy and can be used for achieving lung cancer early screening and prognostic auxiliary diagnoses is developed through a qRT-PCR method. The kit has rapidness and convenience in detection and is high in detection sensitivity and specificity and low in cost, can meet the early screening and prognosis judgment detection requirements of a majority of cancer patients, is extremely wide in application range, and is high in accuracy according to clinical verification prediction. The detection sensitivity of the kit in distinguishing lung cancer samples and samples of able-bodied persons is 96.3%, and the specificity is 89.7%.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a lung cancer detection kit using serum miR-21 and miR-205 as markers, which is used for lung cancer screening and postoperative monitoring. Background technique [0002] Lung cancer is a very common cancer worldwide. Due to its advanced clinical manifestations, it has a high mortality rate (Jemaletal, 2010; Duffyetal, 2007). About 75% of lung cancer patients are diagnosed at an advanced stage or cancer cells have metastasized, so the 5-year survival rate of lung cancer patients is less than 15% (Aberleetal, 2011). Some screening strategies for reducing lung cancer mortality have also been applied. For example, compared with chest X-ray, LDCT screening for heavy smokers can reduce mortality by 20%, but high false positives and high costs make LDCT Can not be a good screening method, we still need to find other screening methods. [0003] In recent years, microRNAs (miRN...

Claims

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Application Information

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IPC IPC(8): C12Q1/68
CPCC12Q1/6886C12Q2600/118C12Q2600/158C12Q2600/178
Inventor 王伟李芳琼孟冠敏刘琴姜凯杨双彪陈文华张捷
Owner ZHEJIANG ACAD OF TRADITIONAL CHINESE MEDICINE
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