WO2022160479 METHOD FOR HIGH-THROUGHPUT EXTRACTION OF TRACE NUCLEIC ACID FOR LARGE-VOLUME MIXED SWAB SAMPLE DETECTION

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Publication Number WO/2022/160479

Publication Date 04.08.2022

International Application No. PCT/CN2021/088730

International Filing Date 21.04.2021

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CPC

Applicants

福州艾迪康医学检验实验室有限公司 FUZHOU ADICON CLINICAL LABORATORIES, INC. [CN]/[CN]

Inventors

李同心 LI, Tongxin
钱明伟 QIAN, Mingwei
杨凡 YANG, Fan

Agents

浙江杭州金通专利事务所有限公司 ZHEJIANG HANGZHOU JINTONG PATENT AGENCY CO., LTD.

Priority Data

202110125242.3

29.01.2021

Publication Language Chinese (zh)

Filing Language Chinese (ZH)

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Title

(EN) METHOD FOR HIGH-THROUGHPUT EXTRACTION OF TRACE NUCLEIC ACID FOR LARGE-VOLUME MIXED SWAB SAMPLE DETECTION

(FR) PROCÉDÉ D'EXTRACTION À HAUT DÉBIT D'ACIDE NUCLÉIQUE À L'ÉTAT DE TRACE POUR LA DÉTECTION D'ÉCHANTILLON D'ÉCOUVILLON MÉLANGÉ À GRAND VOLUME

(ZH) 用于大体积混合拭子样本检测的高通量提取微量核酸的方法

Abstract

(EN) Provided in the present invention is a method for high-throughput extraction of trace nucleic acid for large-volume mixed swab sample detection. The method comprises the following steps: performing lysis treatment on a sample to be detected with a lysis solution to release nucleic acid contained in the sample, thereby obtaining a lysis solution mainly containing the nucleic acid; extracting the lysis solution containing the nucleic acid by means of a closed-column-method positive pressure nucleic acid extraction device, thereby obtaining an extract; and using the extract to prepare a test solution for the purpose of virus detection. According to the present invention, a column extraction method is used for combining nasopharyngeal swab liquids of different individuals from multiple sources, and after the swab liquids are combined, the step of high-speed centrifugation at 2000-4000 g for 10-20 min is added to remove a precipitate, thereby reducing the resistance during column passing. Compared with ordinary extraction, the number of steps of washing and centrifugation is reduced by 1-2, and the efficiency of nucleic acid extraction is improved.

(FR) La présente invention concerne un procédé d'extraction à haut débit d'acide nucléique à l'état de trace pour la détection d'échantillon d'écouvillon mélangé à grand volume. Le procédé comprend les étapes suivantes : réalisation d'un traitement de lyse sur un échantillon à détecter avec une solution de lyse pour libérer l'acide nucléique contenu dans l'échantillon, ce qui permet d'obtenir une solution de lyse contenant principalement l'acide nucléique ; extraction de la solution de lyse contenant l'acide nucléique au moyen d'un dispositif d'extraction d'acide nucléique à pression positive à procédé en colonne fermée, ce qui permet d'obtenir un extrait ; et utilisation de l'extrait pour préparer une solution d'essai à des fins de détection de virus. Selon la présente invention, un procédé d'extraction à colonne est utilisé pour combiner des liquides d'écouvillon nasopharyngiens de différents individus de multiples sources, et une fois que les liquides d'écouvillon sont combinés, l'étape de centrifugation à grande vitesse à 2 000-4 000 g pendant 10-20 min est ajoutée pour éliminer un précipité, ce qui permet de réduire la résistance pendant le passage de la colonne. Par comparaison avec une extraction ordinaire, le nombre d'étapes de lavage et de centrifugation est réduit de 1 à 2, et l'efficacité d'extraction d'acide nucléique est améliorée.

(ZH) 本发明提供用于大体积混合拭子样本检测的高通量提取微量核酸的方法，包括以下步骤：采用裂解液对待测样本进行裂解处理以释放出样本中包含的核酸，获得主要包含所述核酸的裂解液；将所述含有核酸的裂解液通过封闭式柱法正压核酸提取装置进行提取，获得提取物；由所述提取物制备所述用于病毒检测目的的测试液。本发明将柱法提取用于合并多重来源的不同个体的鼻咽拭子液，并且合并拭子液后增加高速离心2000-4000g，10-20min的步骤除去沉淀，减小过柱时的阻力；比一般提取减少1-2次清洗和离心步骤，提高了提取核酸的效率。

C	CHEMISTRY; METALLURGY
12	BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
Q	MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
1	Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
68	involving nucleic acids

C	CHEMISTRY; METALLURGY
12	BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
Q	MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
1	Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
68	involving nucleic acids

C	CHEMISTRY; METALLURGY
12	BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
N	MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
15	Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
09	Recombinant DNA-technology
10	Processes for the isolation, preparation or purification of DNA or RNA
1003	Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor
1006	by means of a solid support carrier, e.g. particles, polymers

C	CHEMISTRY; METALLURGY
12	BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
N	MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
15	Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
09	Recombinant DNA-technology
10	Processes for the isolation, preparation or purification of DNA or RNA
1003	Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor
1006	by means of a solid support carrier, e.g. particles, polymers

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1. WO2022160479 - METHOD FOR HIGH-THROUGHPUT EXTRACTION OF TRACE NUCLEIC ACID FOR LARGE-VOLUME MIXED SWAB SAMPLE DETECTION