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Use of multiple target nucleic acid detection method using clamping probe and detection probe

A detection method and a detection probe technology, applied in biochemical equipment and methods, microbial measurement/inspection, etc., can solve problems such as inability to confirm variants

Pending Publication Date: 2017-03-22
PANAGENE INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although pyrosequencing is more sensitive and faster than Sanger sequencing, and it is convenient for data interpretation, it cannot confirm all variants (Tan et al.World J Gastroenterol2012)

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0129] Detection of epidermal growth factor receptor mutation in patients with non-small cell lung cancer

Embodiment 1-1

[0130] Example 1-1. Specimen collection

[0131] Plasma samples isolated from tissue and blood of 192 patients with non-small cell lung cancer who participated in a randomized phase 2 trial were obtained. In the case of tissue specimens, the base sequence analysis method was used to obtain the results, and the method for simultaneously detecting multiple target nucleic acids of the present invention was used to conduct an epidermal growth factor receptor test in plasma.

Embodiment 1-2

[0132] Example 1-2. Extraction of deoxyribonucleic acid

[0133] In this experiment, the QIAGEN Circulating Nucleic Acid Kit (Cat#55114) to extract DNA. A brief description of this is as follows. Add 800 μl of buffer (Buffer) ACL containing 100 μl of proteinase K (Proteinase K) and 1.0 μg of carrier nucleic acid to 1 ml of plasma (plasma) specimen, and after stirring well, heat at 60° C. under treatment for 1 hour. After incubation, 1.8 ml of ACB buffer was added, followed by standing on ice for 5 minutes. After putting the above solution into a column by vacuum, washing was performed in this order with 600 μl of buffer ACW1, 750 μl of buffer ACW2, and 750 μl of 100% ethanol. The column was separated in a vacuum system to centrifuge at 14000 rpm for 3 minutes, and then treated at 56° C. for 10 minutes to remove residual ethanol. After adding 100 µl of buffer AVE to the column and performing centrifugation at 14000 rpm for 1 minute, deoxyribonucleic acid was eluted. The e...

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PUM

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Abstract

The present invention relates to an application of a target nucleic acid detection method using a clamping probe and a detection probe. The method of the present invention can effectively detect a small amount of variation or a specific gene sequence contained in a sample by selective amplification and detection of a trace amount of a target gene to be detected while inhibiting amplification of wild-type genes or undesired genes. Also, it is possible to determine a large number of genotypes at the same time through a melting curve analysis. In particular, the method can be used for diagnosis, prognosis and monitoring of the medical condition of a disease, treatment efficacy evaluation, and for aiding nucleic acid and protein delivery studies and so on, through a very small amount of a mutant genotype that is confirmed at a high detection sensitivity. The method of the present invention comprises a step for evaluating the detection of biomarkers such as EGFR, KRAS, NRAS etc. and the presence of mutations of biomarkers using invasive specimens such as tissues as well as non-invasive specimens (blood, urine, sputum, stool, saliva, and cells). The presence of the biomarker and mutations provides a method used for monitoring of the entire cycle of a related disease, disease prognosis and prediction, decision of disease treatment strategy, disease diagnosis / early diagnosis, disease prevention, and development of disease therapeutics.

Description

technical field [0001] The present invention relates to a detection method and application of a target nucleic acid using clamping probes and detection probes. It can be used in various applications, especially for the detection of gene mutations, along with rapid and sensitive detection of a small amount of mutation contained in a gene sample. Methods and provide a variety of information needed for the diagnosis or research of diseases related to gene mutations. [0002] Furthermore, the present invention relates to a method for sensitively detecting mutations contained in small amounts in genes, thereby assisting in the diagnosis, prognosis, monitoring of diseases, evaluation of therapeutic efficacy, nucleic acid and protein signal transduction research, etc. of diseases or medical conditions. Also, the present invention relates to methods of monitoring the onset of various diseases (eg, cancer) and methods of monitoring recurrence in a subject. Background technique [00...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68
CPCC12Q1/68C12Q1/6858C12Q2537/163C12Q1/6818C12Q1/6886C12Q2600/106C12Q2600/156C12Q2600/158
Inventor 崔在真尹志惠金秀南金成基金镇羽
Owner PANAGENE INC
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