ROS1 fusion gene ARMS fluorescent quantitative PCR typing detection kit

A detection kit and gene fusion technology, which is applied in the field of molecular biology detection, can solve the problems such as the inability to obtain amplification products, and achieve the effects of simplified experiments, shortened lengths, and high resolution

Active Publication Date: 2016-04-20
ANHUI DAJIAN MEDICAL TECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

That is, during PCR amplification, the extension of the primer starts from its 3' end, and the extension requires that the bases at the 3' end of the primer must be completely paired with the template, only in this way can the primer be extended and the amplification can proceed If the 3' end of the primer cannot be paired with the template, the extension of the primer will be blocked and the corresponding amplification product will not be obtained.

Method used

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  • ROS1 fusion gene ARMS fluorescent quantitative PCR typing detection kit
  • ROS1 fusion gene ARMS fluorescent quantitative PCR typing detection kit
  • ROS1 fusion gene ARMS fluorescent quantitative PCR typing detection kit

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0065] Example 1: Design and screening of primers and probes for RT-ARMS-qPCR detection of ROS1 fusion gene variants

[0066] 1. ARMS primer design

[0067] When designing ARMS primers in the present invention, Taq enzyme lacks 3'-5' exonuclease activity. When the 3' end of the primer cannot be completely matched with the template, PCR amplification cannot be performed. The 3' end of the primer is designed to only match the mutant template, but not the normal template. The primer can only amplify the mutant template, but not the normal template, so as to achieve the purpose of typing.

[0068] On the basis of conventional ARMS primers, the present invention introduces a mismatched base at the 3rd base and the 7th base from the 3' end of the primer respectively, so as to enhance the specificity of the primer and suppress non-specific amplification.

[0069] The position of the mutation site is crucial to the specificity of the primer, and some mutation sites cannot prevent the...

Embodiment 2

[0100] Example 2: Specific investigation of primers and probes for RT-ARMS-qPCR detection of ROS1 fusion gene variants

[0101] Using DNA plasmids containing ROS1 fusion gene Variant1, Variant2, Variant3, Variant4, Variant5, Variant6, Variant7, Variant8, Variant9 and Variant10 genomic fragments as sample templates, the primers and probe combinations screened in Example 1 were used for RT-ARMS -qPCR detection.

[0102] The detection reaction system is: 1 μL of primer and probe mixture (concentration: 5uM), 2 μL of sample template DNA (100-300 ng / μL), 15 μL of 2*Taqmanuniversal PCR MasterMix (purchased from Applied Biotech, USA), ddH 2 07 μL.

[0103] PCR reaction conditions: pre-denaturation at 95°C for 30 seconds; 20 cycles of amplification at 95°C for 15 seconds and 20 seconds at 62°C; 40 cycles of amplification at 95°C for 15 seconds and 60°C for 34 seconds.

[0104] The result is: the primer and probe combination (comprising the forward primer shown in SEQIDNO.1 and the r...

Embodiment 3

[0115] Example 3: ROS1 fusion gene ARMS fluorescent quantitative PCR typing detection kit

[0116] The ROS1 fusion gene ARMS fluorescent quantitative PCR typing detection kit of the present invention includes the fluorescent quantitative reaction premix (PCRMasterMix), fluorescent quantitative reaction solution A, fluorescent quantitative reaction solution B and fluorescent quantitative reaction solution C of independent packaging respectively; And positive control Sample A, positive control sample B and positive control sample C are assembled together in an outer packaging box.

[0117] Wherein, the fluorescent quantitative reaction solution A includes three forward primers, a common reverse primer and a fluorescent probe for detecting ROS1 fusion gene variants Variant1, Variant9 and Variant10 screened in Example 1;

[0118] Fluorescent quantitative reaction solution B, including four forward primers, a common reverse primer and a fluorescent probe for detecting ROS1 fusion g...

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PUM

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Abstract

The invention discloses a ROS1 fusion gene ARMS fluorescent quantitative typing detection kit. The kit comprises a positive primer for detecting a ROS1 fusion body variant, a common reverse primer and a fluorescence probe, wherein the positive primer is at least one of ten single-chain DNAs as shown in SEQ ID NO.1 to SEQ. ID NO. 10; the common reverse primer is at least one of three single-chain DNAs as shown in SEQ ID NO.11 to SEQ ID NO.13; and the fluorescence probe is at least one of three single-chain DNAs as shown in SEQ ID NO.14 to SEQ ID NO.16. The invention further discloses a method for detecting the ROS1 fusion gene variant. The specific primers and fluorescence probe are designed for the ROS1 fusion gene variant, so that the sensitivity and specificity for ROS1 fusion gene variant detection are improved, and the false positivity is low.

Description

technical field [0001] The invention relates to a ROS1 fusion gene ARMS fluorescent quantitative PCR typing detection kit, which belongs to the technical field of molecular biology detection. Background technique [0002] Lung cancer is a malignant tumor with the highest morbidity and mortality in the world. Lung cancer is divided into small cell lung cancer (SCLC) and non-small cell lung cancer (NSCLC). NSCLC includes squamous cell carcinoma, adenocarcinoma, adenosquamous carcinoma, large cell carcinoma, carcinoid, etc. NSCLC accounts for 80-90% of all lung cancer cases. There are about 1.5 million new cases of lung cancer every year, which seriously threatens human health. The treatment of NSCLC includes surgery, chemotherapy, radiotherapy, molecular targeted therapy and biological immunotherapy and other methods. Surgical treatment is the best treatment for NSCLC, but when NSCLC is discovered, only 20-30% of the cases have indications for surgery, and the postoperative ...

Claims

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Application Information

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IPC IPC(8): C12Q1/68
CPCC12Q1/6851C12Q1/6886C12Q2600/158C12Q2531/113C12Q2545/113C12Q2563/107C12Q2561/113
Inventor 钟明李香梅
Owner ANHUI DAJIAN MEDICAL TECH CO LTD
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