A kit for extracting pathogen nucleic acids from a phlegm sample in a high-flux and automatic manner

A medium- and high-throughput technology for samples, applied in the field of biochemistry, can solve the problems of unfavorable nucleic acid detection automation, loss of nucleic acid, complex operation, etc., to achieve the effect of easy automation, increased value, and high purity

Inactive Publication Date: 2017-06-20
AUTOBIO DIAGNOSTICS CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

This process requires repeated centrifugal washing, the operation is very complicated, and it is easy to lose a large amount of nucleic acid during the digestion and extraction process. At the same time, the removal rate of impurities is low, resulting in false negative detection; repeated centrifugation is not conducive to th

Method used

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  • A kit for extracting pathogen nucleic acids from a phlegm sample in a high-flux and automatic manner
  • A kit for extracting pathogen nucleic acids from a phlegm sample in a high-flux and automatic manner
  • A kit for extracting pathogen nucleic acids from a phlegm sample in a high-flux and automatic manner

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0018] 1. The high-throughput automated kit for extracting pathogen nucleic acid from sputum samples of the present invention includes the following reagents:

[0019] 1) Sputum digestive juice: sodium hydroxide, concentration 1M;

[0020] 2) Lysis solution: pH<1.0, including

[0021] Guanidine isothiocyanate, concentration 4M,

[0022] Trizma® HCl, concentration 50mM,

[0023] Triton X-100 (v / v,%), concentration 10%,

[0024] EDTA, concentration 10mM,

[0025] HCl, concentration 0.1M;

[0026] 3) Washing solution: sodium citrate, concentration 10 mM, pH 6.0;

[0027] 4) Eluent: pH 8.0, including

[0028] Trizma®Base, 10 mM concentration,

[0029] EDTA, concentration 1 mM;

[0030] 5) Magnetic beads 20ul.

[0031] 2. Take sputum specimens from patients whose clinical sputum culture was identified as positive for Candida albicans, and use the above reagents to extract nucleic acid from sputum on an automated pipetting workstation. The specific steps include:

[0032] ...

Embodiment 2

[0046] 1. The high-throughput automated kit for extracting pathogen nucleic acid from sputum samples of the present invention includes the following reagents:

[0047] 1) Sputum digestive juice: sodium hydroxide, concentration 1M;

[0048] 2) Lysis solution: pH<2.0, including

[0049] Guanidine isothiocyanate, concentration 4M,

[0050] Trizma® HCl, concentration 50mM,

[0051] Triton X-100 (v / v,%), concentration 10%,

[0052] EDTA, concentration 10mM,

[0053] HCl, concentration 50mM;

[0054] 3) Washing solution: sodium citrate, concentration 10 mM, pH 6.0;

[0055] 4) Eluent: pH 8.0, including

[0056] Trizma®Base, 10 mM concentration,

[0057] EDTA, concentration 1 mM;

[0058] 5) Magnetic beads 20ul.

[0059] 2. Take sputum samples from patients with respiratory syncytial virus infection identified by clinical PCR, and use the above reagents to extract nucleic acids from sputum on an automated pipetting workstation. The specific steps include:

[0060] 1) Add 3 ti...

Embodiment 3

[0074] 1. The high-throughput automated kit for extracting pathogen nucleic acid from sputum samples of the present invention includes the following reagents:

[0075] 1) Sputum digestive juice: sodium hydroxide, concentration 1M;

[0076] 2) Lysis solution: pH<2.0, including

[0077] Guanidine isothiocyanate, concentration 4M,

[0078] Trizma® HCl, concentration 50mM,

[0079] Triton X-100 (v / v,%), concentration 10%,

[0080] EDTA, concentration 10mM,

[0081] HCl, concentration 50mM;

[0082] 3) Washing solution: sodium citrate, concentration 10 mM, pH 6.0;

[0083] 4) Eluent: pH 8.0, including

[0084] Trizma®Base, 10 mM concentration,

[0085] EDTA, concentration 1 mM;

[0086] 5) Magnetic beads 20ul.

[0087] 2. Take sputum samples from patients with mycoplasma pneumonia infection identified by clinical PCR, and use the above reagents to extract nucleic acids from sputum on an automated pipetting workstation. The specific steps include:

[0088] 1) Add 3 times t...

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Abstract

A kit for extracting pathogen nucleic acids from a phlegm sample in a high-flux and automatic manner is disclosed. The kit includes a phlegm digestion agent, a lysis liquid, magnetic beads, a washing liquid and an eluant. The phlegm digestion agent is a NaOH solution having a concentration of 1-6 M. The lysis liquid is an acidic solution the pH of which is not more than 3, and which is prepared from a guanidine salt having a concentration of 2-8 M, a surfactant, a pH buffer medium and a metal chelating agent. Centrifugation is not needed during extraction of nucleic acids of pathogens (including bacteria, viruses and fungi) in the phlegm sample by using the kit, thus facilitating automatic operation. The kit can extract DNAs and RNAs of bacteria, viruses and fungi in the phlegm sample so that the kit is high in universality. An extraction process is free of organic and toxic solvents so that the kit is safer. Extracted nucleic acids are high in purity so that value of a pathogen clinical sample in disease diagnosis is improved.

Description

technical field [0001] The invention relates to the technical field of biochemistry, in particular to a high-throughput kit for automatically extracting pathogen nucleic acid from sputum samples. Background technique [0002] The inner walls of the human trachea and bronchi are covered with a layer of mucous membrane, and the submucosa contains more mucus glands and serous glands. Under normal circumstances, goblet cells and glands secrete a small amount of mucus to cover the surface of the mucous membrane, which can protect the mucous membrane and keep the tracheal mucous membrane moist, so as to adhere to the dust particles and bacteria inhaled into the trachea and bronchi, and block them. It penetrates deep into the lung tissue. When the trachea, bronchi, and lungs are stimulated by harmful factors or inflamed by pathogenic bacteria, the mucous membrane of the respiratory tract is congested, edematous, a large number of inflammatory cells infiltrate, the blood vessels di...

Claims

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Application Information

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IPC IPC(8): C12N15/10
CPCC12N15/1013
Inventor 高利飞李振红王玮于永敏吕萌萌李桂林付光宇吴学炜苗拥军
Owner AUTOBIO DIAGNOSTICS CO LTD
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