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Digital amplification detection method, detection product and detection kit for simultaneously identifying multiple gene types

A detection method, digital amplification technology, applied in biochemical equipment and methods, microbial measurement/inspection, DNA/RNA fragments, etc., can solve the problems of increasing target gene amplification results, reduce non-specific amplification, Efficient detection process and accurate detection results

Pending Publication Date: 2022-03-18
南京普济生物有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, when this technology is used for the detection of multiple gene mutations, it is still limited by the number of primers in the same system: when detecting multiple gene mutations, multiple primers need to be set, and the increase in the number of primers will increase the number of non-specific primers such as dimers. Possibility that heterosexual amplification products are produced and affect the results of target gene amplification

Method used

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  • Digital amplification detection method, detection product and detection kit for simultaneously identifying multiple gene types
  • Digital amplification detection method, detection product and detection kit for simultaneously identifying multiple gene types
  • Digital amplification detection method, detection product and detection kit for simultaneously identifying multiple gene types

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0043] Example 1 Digital amplification detection method and detection kit for various gene mutations related to lung cancer

[0044] A digital amplification detection method for simultaneously identifying multiple gene mutations for lung cancer, comprising the following steps:

[0045] S1. Preparation of extracted sample DNA and primer-probe mixture

[0046] I. Extraction of sample DNA

[0047] Enrolled cases: Patients with pulmonary nodules who visited the Thoracic Surgery Department of Shanghai Zhongshan Hospital from January 2020 to December 2020 were studied. According to the results of chest CT and blood lung cancer tumor indicators (including KRAS), it was impossible to determine whether they were lung cancer patients. Or lung disease. All patients chose to receive surgical treatment, and 50 mL of peripheral venous blood was collected before the operation. According to the postoperative pathological results of the tumor, the samples were divided into lung cancer and lu...

Embodiment 2

[0088] Example 2 Digital amplification detection method and detection kit for various gene mutations related to lung cancer

[0089] Digital amplification detection method for simultaneously identifying multiple gene mutations for lung cancer: The difference between this example and Example 1 is that the selected small molecular compound is directly added to the primer, that is, the selected primer is Obtained after modification of locked nucleic acid. In addition, in step S122, no small molecular compound is added to the buffer.

[0090] The locked nucleic acid is commercially available, and may be a locked nucleic acid oligochain synthesized from Beijing Saibaisheng Gene Technology Co., Ltd.

[0091] Others are the same as embodiment 1.

[0092] Detection kit for simultaneous identification of multiple gene mutations for lung cancer

[0093] The difference between this example and Example 1 is that the primers for PCR amplification are prepared in this example, and the ot...

Embodiment 3

[0094] Example 3 Digital amplification detection method and detection kit for various gene mutations related to lung cancer

[0095] Digital Amplification Assay for Simultaneous Identification of Multiple Gene Mutations for Lung Cancer

[0096] The difference between this example and Example 1 is that the small molecule compound is peptide nucleic acid, which is added in step S2, specifically: in PCR Mix, peptide nucleic acid with a final concentration of 0.35 μM is added. And in step S122, the small molecule compound is no longer added to the buffer.

[0097] Others are the same as embodiment 1.

[0098] Detection kit for simultaneous identification of multiple gene mutations for lung cancer

[0099] The difference between this example and Example 1 is that the primers for PCR amplification are prepared in this example, and the others are the same as Example 1.

[0100] Ultimately, this method can also be used to obtain accurate detection of lung cancer-related mutation ge...

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PUM

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Abstract

The invention relates to the technical field of gene detection, and particularly discloses a digital amplification detection method, a detection product and a detection kit for simultaneously identifying multiple gene types. The detection method comprises the following steps: S1, extracting to obtain sample DNA; a primer probe mixed solution is prepared, specifically, primers and probes are synthesized, at least two probes combined with the target genes are designed for each target gene, and four or less basic groups exist in the nucleotide sequence of each primer; the primer is modified with a small molecule compound and a nucleotide sequence capable of forming a secondary structure, and the small molecule compound is selected from one or more of thiophosphoric acid oligonucleotide, methylated oligonucleotide, peptide nucleic acid, locked nucleic acid, hypoxanthine nucleotide or a rhodium metal inserter; mixing the primer and the probe to obtain a primer and probe mixed solution; s2, carrying out PCR (Polymerase Chain Reaction) reaction; and S3, analyzing the detection data to obtain gene types of different sites. The detection method is accurate in detection and high in efficiency.

Description

technical field [0001] This application relates to the technical field of gene mutation detection, more specifically, it relates to a digital amplification detection method, detection product and detection kit for simultaneously identifying multiple gene mutations. Background technique [0002] Multiple gene detection is a difficult problem in the industry, and most current technologies can only realize the detection of limited multiple genes. In recent years, some technologies can detect more than ten genes, such as multiple ligation-dependent probe amplification (MLPA), multicolor melting curve analysis (MMCA) ) and microfluidic technology (such as Filmarray products), etc., but these detection methods all have different technical problems. Among them, MLPA technology is limited in its application due to difficult and time-consuming probe design, expensive reagents, and the need to cooperate with complex instruments such as capillary electrophoresis. Among them, microflu...

Claims

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Application Information

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IPC IPC(8): C12Q1/6858C12Q1/6886C12N15/11
CPCC12Q1/6858C12Q1/6886C12Q2600/156C12Q2531/113C12Q2525/10C12Q2525/107C12Q2563/159
Inventor 尚午王友祥杨志颉张毅良蒋正欣
Owner 南京普济生物有限公司