Nucleic acid extraction kit and nucleic acid extraction method

A nucleic acid extraction reagent and nucleic acid extraction technology, applied in the field of molecular biology, can solve problems such as the influence of relational quantity recovery, low microbial extraction efficiency, difficulty in microbial extraction, etc., to ensure accuracy and stability, and to avoid mixed blood and secretions. interference, protection stability and complete effect

Pending Publication Date: 2020-04-21
阿吉安(福州)基因医学检验实验室有限公司
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  • Abstract
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Problems solved by technology

[0003] Alveolar lavage fluid is one of the most important sample types for studying lower respiratory tract infection. It is an ideal sample for detection of viral pneumonia, bacterial pneumonia and fungal pneumonia, and its positive detection rate is higher than that of sputum; Sampling, such as mixed blood during BAL operation, mixing of large airway secretions, suction negative pressure, will have an impact on the recovery of related quantities, and there will be great differences in the amount of microorganisms sampled. In addition, the alveolar lavage fluid is relatively viscous. The extraction of microorganisms is difficult
[0004] The extraction of microbial nucleic acid is the primary factor for the accurate detection of pathogenic bacteria. Few of the nucleic acid extraction kits currently on the market are specifically designed for samples such as alveolar lavage fluid, and the extraction of microbial nucleic acid from BAL is even rarer. This will cause the extraction efficiency of microorganisms in BAL to be low, and false negatives are likely to occur

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  • Nucleic acid extraction kit and nucleic acid extraction method

Examples

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Effect test

Embodiment 11

[0039] A method for extracting nucleic acid specifically includes the following steps:

[0040] Step 1. Mix the lysate and the sample to be extracted at 10°C for 5 minutes, then add the nucleic acid attachment and mix at 40°C for 5 minutes to obtain the nucleic acid attachment that has adsorbed nucleic acid;

[0041] The sample to be extracted is an alveolar lavage fluid from non-affected patients; the nucleic acid attachment is magnetic beads;

[0042] Step 2. Use washing liquid I to soak the nucleic acid attachments with nucleic acid at 40°C, and use magnetic separation to perform solid-liquid separation to obtain supernatant I and the nucleic acid attachments after one wash;

[0043] Use washing solution II to soak the nucleic acid attachments after one wash at 10°C for 1 min, and use magnetic separation for solid-liquid separation to obtain supernatant II and the nucleic acid attachments after the second cleaning;

[0044] Use the eluate to soak the nucleic acid attachments after th...

Embodiment 12

[0047] A method for extracting nucleic acid specifically includes the following steps:

[0048] Step 1. Mix the lysate and the sample to be extracted at 40°C for 2 minutes, then add the nucleic acid attachment and mix at 10°C for 2 minutes to obtain the nucleic acid attachment that has adsorbed nucleic acid;

[0049] The sample to be extracted is an alveolar lavage fluid from non-affected patients; the nucleic acid attachment is magnetic beads;

[0050] Step 2. Use washing solution I to soak the nucleic acid attachments with nucleic acid adsorbed at 10°C for 5 minutes, and use magnetic separation for solid-liquid separation to obtain supernatant I and the nucleic acid attachments after cleaning once;

[0051] Use washing solution II to soak the nucleic acid attachments after washing once at 40°C for 5 minutes, and use magnetic separation for solid-liquid separation to obtain supernatant II and the nucleic acid attachments after the second washing;

[0052] Use the eluate to soak the nuc...

Embodiment 13

[0055] A method for extracting nucleic acid specifically includes the following steps:

[0056] Step 1. Mix the lysate and the sample to be extracted at 25°C for 3 minutes, then add the nucleic acid attachment and mix for 3 minutes at 25°C to obtain the nucleic acid attachment that has adsorbed nucleic acid;

[0057] The sample to be extracted is an alveolar lavage fluid derived from a non-affected patient (definitely not suffering from a lower respiratory tract disease); the nucleic acid attachment is a magnetic bead;

[0058] Step 2. Use washing liquid I to soak the nucleic acid attachments with nucleic acid adsorbed at 25°C for 3 minutes, and use magnetic separation to perform solid-liquid separation to obtain supernatant I and the nucleic acid attachments after one wash;

[0059] Use washing solution II to soak the nucleic acid attachments after one wash at 10°C for 2 minutes, and use magnetic separation for solid-liquid separation to obtain supernatant II and the nucleic acid atta...

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Abstract

The invention discloses a nucleic acid extraction kit and a nucleic acid extraction method, and belongs to the field of molecular biology. The nucleic acid extraction kit comprises a lysis solution, awashing solution I and a washing solution II, and the lysis solution comprises Tris, EDTA, Tween20, dithiothreitol and guanidinium isothiocyanate. The nucleic acid extraction method comprises the following steps: mixing the lysis solution, a nucleic acid attachment and a sample to be extracted to obtain the nucleic acid attachment adsorbed with the nucleic acid, cleaning the nucleic acid attachment adsorbed with the nucleic acid by using the cleaning solution I, the cleaning solution II and an eluent in sequence, carrying out solid-liquid separation after each cleaningprocess, and collectingthe supernatant to obtain the nucleic acid. According to the nucleic acid extraction kit and the nucleic acid extraction method provided by the invention, cells can be fully and effectively cracked, the cracking efficiency is high, nucleic acid with higher concentration and higher purity is obtained, the accuracy of subsequent operation is ensured, operation such as centrifugation is not needed, the operation is simple, one-time extraction is carried out within 1 hour, and the extraction efficiency is improved.

Description

Technical field [0001] The invention belongs to the field of molecular biology, and specifically relates to a nucleic acid extraction kit and a method for extracting nucleic acid. Background technique [0002] According to the ranking of the global burden of disease published by the World Health Organization (WHO) in 2012, lower respiratory tract infections rank fourth, second only to ischemic heart disease, stroke and chronic obstructive pulmonary disease; and lower respiratory tract infections in low-income countries Ranked the first cause of death. At present, the international research organization for long-term dynamic monitoring of pneumonia-the United States and the European Pneumonia Research Network (CAPO and CAPNEZ) has been established for nearly 20 years, providing pneumonia epidemiology, pathogen diagnosis, treatment methods and prevention There is a lot of evidence-based medicine, but my country still lacks long-term dynamic research in this area, especially the und...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/10
CPCC12N15/1006C12N15/1013
Inventor 郭文浒张书祖薛怡婷陈浩
Owner 阿吉安(福州)基因医学检验实验室有限公司
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