WO2022160479A1 - 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 - Google Patents

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 Download PDF

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WO2022160479A1
WO2022160479A1 PCT/CN2021/088730 CN2021088730W WO2022160479A1 WO 2022160479 A1 WO2022160479 A1 WO 2022160479A1 CN 2021088730 W CN2021088730 W CN 2021088730W WO 2022160479 A1 WO2022160479 A1 WO 2022160479A1
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nucleic acid
swab
detection
extraction
volume
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李同心
钱明伟
杨凡
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福州艾迪康医学检验实验室有限公司
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    • C12N15/09—Recombinant DNA-technology
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • C12N15/1003—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor
    • C12N15/1006—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor by means of a solid support carrier, e.g. particles, polymers

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  • the invention belongs to the fields of life science and biotechnology, and particularly relates to a high-throughput method for extracting trace nucleic acids for the detection of large-volume mixed swab samples.
  • nucleic acid detection requires the extraction of viral nucleic acid RNA from various body fluid samples, and the volume of body fluid samples usually detected is limited by existing commercial extraction devices and cannot exceed 200-300 ⁇ l, thus limiting the sensitivity of the detection.
  • the new coronavirus COVID-19 is an RNA virus, which mainly invades the body through the upper respiratory tract and digestive tract.
  • the spike protein on the surface of the virus has high affinity and specificity with the ACE2 receptor expressed by cells in the upper respiratory tract and digestive tract. Binds, enters host cells, and utilizes host organelles for viral protein synthesis and viral replication.
  • Samples used for new coronavirus testing usually include blood (detecting antigen and antibody), nasal swabs and throat swabs (detecting nucleic acid RNA).
  • the novel coronavirus COVID-19 (SARS-CoV-2) is a single-stranded positive-sense RNA virus wrapped by a protein.
  • RNA of the new coronavirus is the direct evidence of the existence of the virus, and RT-PCR nucleic acid detection technology is used for the differential diagnosis of the disease by specifically detecting the viral RNA.
  • the most widely used method for diagnosing COVID-19 infection is reverse transcription quantitative polymerase chain reaction (RT-qPCR). 3. Currently, RT-qPCR methods are used to detect COVID-19 nucleic acid RNA, and RNA needs to be extracted from the sample as a template for PCR.
  • RNA extraction technologies and devices are the main bottlenecks in the RNA template extraction process, resulting in a low yield of RNA, which is limited by the existing extraction methods, including that the volume of the extracted samples is not large enough, especially in samples containing low-copy nucleic acids. .
  • the main specimens collected clinically for the detection of patients infected with the virus are nasopharyngeal swabs, and the detection positive rate is only 30%-50%.
  • sample mixing There are two modes for “mixed detection”. One is to sample several people, such as 3 or 5 people, and put them into the same sampling tube. This mode is also called “mixed sampling” or “mixed sampling”; For laboratory testing, the same volume of samples from 3 or 5 people is mixed together, also known as “sample mixing”. From a scientific point of view, the first “mixed sampling” mode will not affect the sensitivity of nucleic acid detection, and the latter mode of mixed detection of samples will have a certain impact on the detection sensitivity, but the degree of impact is known . Even so, although the above mixed detection methods have improved the nucleic acid detection ability, their detection sensitivity is low, and the work efficiency is still not efficient enough, especially for samples containing low-copy nucleic acid, "false negative" test results may occur.
  • the magnetic bead method is mostly used for nucleic acid extraction.
  • the applicable sample volume is usually 200-300ul, which is not suitable for large volume requirements (for example, multiple samples. Mixed detection) nucleic acid detection needs.
  • multiple samples are mixed, affected by the total volume, and the samples are diluted with each other, which inevitably dilutes the target nucleic acid, which reduces the sensitivity when the sampling volume is only 200-300ul.
  • the present invention provides a device and method for high-throughput extraction of trace nucleic acid for the detection of large-volume mixed swab samples.
  • the present invention is different from the traditional mixed detection method, but uses a large-volume column method closed column method positive pressure nucleic acid extraction device, firstly, n samples to be tested (n about 2-100) are mixed, and then subjected to cracking, adsorption, cleaning, Elution, extract n samples at a time.
  • the process has high extraction efficiency and less nucleic acid loss, which ensures the detection sensitivity.
  • a method for high-throughput extraction of trace nucleic acid for detection of large-volume mixed swab samples comprising the following steps: lysing the sample to be tested with a lysing solution to release the nucleic acid contained in the sample, and obtaining a lysing solution mainly containing the nucleic acid ;
  • the method also includes a step selected from at least one of the following:
  • test sample is a nasopharyngeal swab
  • test sample can be a mixture of samples from multiple individuals
  • the extraction is performed using a closed column positive pressure nucleic acid extraction device.
  • the volume of the sample to be tested is controlled to be 2-20 mL.
  • the closed column method positive pressure nucleic acid extraction device comprises a top cover and an adsorption column connected thereto, the top cover has a luer locking sleeve, and positive pressure is applied through a syringe to promote the flow of the liquid, through the adsorption column.
  • the specific implementation steps are as follows:
  • the eluate containing the isolated nucleic acid is directly used for RT-PCR or other molecular detection, or stored for later use.
  • step (7) before adding the eluent, the adsorption column is washed with a cleaning solution (WB) for 1-2 times, and then the eluent is added to elute. Used to further reduce impurities.
  • WB cleaning solution
  • the supernatant refers to a clear liquid from which the precipitated part is removed after centrifugation.
  • the nasopharyngeal swab fluid used as the test sample is the nasopharyngeal swab fluid from different individuals combined from multiple sources.
  • the mixed samples of nasopharyngeal swab fluid can be further divided into subgroups, and the mixed swab samples of each subgroup are extracted for detection. Then use the same method of the present invention to extract nucleic acid from large-volume samples, so as to facilitate rapid identification of positive samples.
  • the extraction efficiency is not affected by the volume of the body fluid and the concentration of nucleic acid contained therein.
  • all or most of the body fluid or nucleic acid sample that has not been extracted or detected by magnetic bead method is extracted, washed, eluted, and detected. Adding this step can greatly improve the detection sensitivity.
  • the nasopharyngeal swab is a combination of 1-100 nasopharyngeal swabs from different individuals.
  • the nasopharyngeal swab is a combination of 2-100 nasopharyngeal swabs from different individuals.
  • the nasopharyngeal swab is a combination of 2-20 different individuals.
  • the nasopharyngeal swab is a combination of 20-50 different individuals.
  • the nasopharyngeal swab is a combination of 30 different individuals' nasopharyngeal swabs.
  • the methods of the present invention are broadly applicable to nucleic acid extraction of various viruses.
  • column extraction is used to combine nasopharyngeal swab fluids from different individuals from multiple sources, and after combining the swab fluids, high-speed centrifugation is added at 2000-4000 g for 10-20 minutes to remove the precipitate, which greatly reduces the resistance when passing through the column. ; And as a pre-processing step, 1-2 washing and centrifugation steps are reduced compared with general extraction, which improves the efficiency of nucleic acid extraction.
  • the large-volume column method Compared with the 200-300ul sample volume of nucleic acid extracted by the commonly used magnetic bead method, the large-volume column method has a larger sample volume (2-20ml), and can collect 10-100 times the sample collected by the magnetic bead method, improving the detection efficiency; At the same time, the volume of the cleaning solution is also large, about 6-8 times the volume of the magnetic bead method cleaning solution, and the impurity content after cleaning is less, which is more conducive to downstream detection. Moreover, the nucleic acid extracted by the method of the present invention is used for detection, and its sensitivity is high; compared with the conventional magnetic bead extraction method, the method improves the sensitivity, that is, the Ct value in the RT-PCR reaction is advanced by 2-4.
  • FIG. 1 is a flow chart of the nucleic acid extraction system for large-volume samples of the present invention.
  • FIG. 2 is a schematic structural diagram of a closed-column positive-pressure nucleic acid extraction device used in the present invention.
  • FIG. 3 is a schematic diagram of a top cover and an adsorption column of the closed-column positive pressure nucleic acid extraction device of FIG. 2 .
  • FIG. 4 is a schematic diagram illustrating that the top cover of FIG. 3 is provided with a luer locking socket.
  • Example 1 Materials and specific steps used in the method for extracting large volume samples, as shown in FIG. 1 .
  • Lysis tube 50ml centrifuge tube.
  • Eluent collection tube 1.5ml microcentrifuge tube.
  • the closed-column positive-pressure nucleic acid extraction device includes a top cover 101, an adsorption column 200 connected thereto, and a container 400.
  • the adsorption column is located in the container, and the top cover 101 has a luer lock sleeve 120, as shown in FIG. 2 -4 shown.
  • the function of the filter column 200 in CN201580073867.2 is filtration and adsorption (paragraphs [0045]-[0047] of the specification), so the name of the filter column is changed to the adsorption column 200 in this article.
  • A. Collect 2-20ml of nasopharyngeal swabs for inspection according to specific needs; centrifuge 2-20ml of collected body fluids for 10 minutes (centrifugal force> 2000g, in one embodiment, centrifugal force is 2000g-5000g), absorb supernatant 2 -20ml to be tested, or frozen at -20°C for later use.
  • the eluate containing the isolated nucleic acid can be directly sent to a high-throughput magnetic bead extractor for further extraction and purification, automated extraction and RT-PCR, or directly used for RT-PCR or other molecular detection after cleaning, or Save for later use.
  • step F first wash the adsorption column with cleaning solution (WB) for 1-2 times, place the adsorption column in a clean 1.5ml centrifuge tube, add 300 ⁇ l eluent (EB) on the adsorption membrane in the adsorption column, Cover the adsorption column and let it stand at room temperature for 2 minutes.
  • WB cleaning solution
  • EB eluent
  • steps A-H are performed again.
  • BDS new coronavirus pseudovirus quality control material S2 standard (see the table below for specific concentrations), Guangzhou Bang Desheng Biotechnology Co., Ltd. (referred to as BDS company)
  • Tube 11 200ul S2 quality control product + 200ul throat swab mixture
  • Tube 12 The dilution method is to take 40ul of No. 1 sample and add 360ul of the mixture, that is, 1:9 dilution.
  • 2Sample tubes named No. 1-6 add 6 ml of throat swab mixture to each tube, add 4 ml of lysis solution provided by OBI company, add PK100ul, and place in a 60C water bath for 30 minutes. Then 10 ml of the binding solution provided by OBI was added, the nucleic acid was extracted using a MOES extraction device, the column was washed with 300 ul of eluate, and centrifuged at 12,000 rpm for 3 minutes. Take 200 ul of the eluted product obtained by centrifugation (300 ul of the eluted product in total) by magnetic bead extractor + RT-PCR (automatic nucleic acid extractor, model: S-96. Manufacturer: Wuhan Nano Magnetic Biotechnology Co., Ltd.).
  • the sample (mixture in tubes 11-16) was extracted by magnetic bead method (200ul) by the existing magnetic bead method, and further RT-qPCR was performed.
  • Kit manufacturer Sun Yat-Sen University Daan Gene Co., Ltd.
  • Operation steps 5ul nucleic acid samples + 20ul PCR reagents are mixed and divided into PCR reaction tubes, and the PCR instrument is ABI7500.
  • the specific operation steps and PCR reaction conditions please refer to the instruction manual of Daan Gene's Novel Coronavirus 2019-nCoV Nucleic Acid Detection Kit (Fluorescent PCR Method).
  • RNA stabilization solution to 20ml mixed swab solution and mix well, divide into 6ml/tube, a total of 3 tubes (ie A, B, C tubes).
  • Tube B was centrifuged at 1500 rpm for 10 minutes after the water bath, and the supernatant was collected and passed through the column.
  • Tube C was not centrifuged throughout the process.
  • the test shows that one lysate (OBI provides MOES for large-volume sample extraction) is slightly better than the two lysates (LGX, LGS) provided by comma.
  • OBI provides MOES supporting the lysate used for large-volume sample extraction, which can expand the sample volume to 20 ml (if the current clinical laboratory usually uses 200ul/person for extraction operations, MOES is equivalent to a test that can detect 100 samples at the same time. individual samples).
  • the extraction efficiency is not affected by the volume of the body fluid; and all or part of all nucleic acids obtained can be selectively used for downstream RT-PCR.
  • the existing automatic nucleic acid extractor usually obtains 50-80ul of the eluate containing nucleic acid, but it is limited by the fact that the eluate may contain a higher concentration of substances that inhibit the PCR reaction. material, the actual RT-PCR reaction uses only 5ul eluate, which accounts for about 10% of the total nucleic acid, which greatly reduces the sensitivity).
  • the large-volume extraction method of the present invention is due to: 1. the sampling volume is large (0.2ml vs 2-20ml), and the collected trace nucleic acids are relatively large; 2. the volume of the cleaning solution is large (0.6ml vs 2 -4ml), so the residual impurity content is small; 3.
  • the large-volume extraction device is mixed (n), in the existing extraction method, each sample has a large amount of unused eluent of about 45-75ul, and each tube only absorbs One-tenth of the eluate (5 microliters) was used for rt-PCR detection.
  • the remaining eluate calculated on an average of 45 microliters, has a total of about 45 microliters x n mixed samples, all of which are recovered through the adsorption column and enter into RT-PCR detection, thus greatly improving the sensitivity.

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Abstract

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。

Description

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 技术领域
本发明属生命科学和生物技术领域，特别涉及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。
背景技术
目前临床上病毒检测方法主要有两种：核酸检测和抗原抗体免疫学检测。核酸检测需要从各种体液标本中提取病毒核酸RNA，且通常检测的体液标本体积受现有商业化提取装置限定，不能超过200-300μl，因此限制了检测的灵敏度。
比如，新型冠状病毒COVID-19病毒属于RNA病毒，主要通过上呼吸道和消化道入侵机体，病毒表面棘突蛋白(Spike protein)与上呼吸道，消化道细胞表达的ACE2受体高亲和力，高特异性结合，进入宿主细胞，并利用宿主细胞器进行病毒蛋白合成和病毒复制。用于新型冠状病毒检测的样本通常包括血液(检测抗原抗体)、鼻拭子和咽拭子(检测核酸RNA)等标本。1.新型冠状病毒COVID-19(SARS-CoV-2)是由蛋白包裹的单链正义RNA病毒。新冠病毒的遗传物质RNA是病毒存在的直接证据，RT-PCR核酸检测技术通过特异性地检测病毒RNA被用于疾病的鉴别诊断。2.最广泛使用的COVID-19感染诊断方法是逆转录定量聚合酶链式反应(reverse transcription quantitative polymerase chain reaction，RT-qPCR)。3.目前在用RT-qPCR方法检测COVID-19核酸RNA均需从样本中提取RNA作为PCR的模板。但现有RNA提取技术和装置是RNA模板提取过程中的主要瓶颈，导致RNA的得率较低，受限于现有的提取方法，包括提取样本体积不够大，尤其含低拷贝核酸的样本中。
并且，临床上用于检测患者感染病毒主要采集的标本是鼻咽拭子(nasopharyngeal swabs)，检测阳性率仅为30％-50％。
而关于“混检”有两种模式。一种是在采样时，将几个人如3人或5人分别采样后，放至同一采样管中，这种模式也叫做“混合采样”或称“混采”；另一种则是在实验室检测时，将3人或5人的样本取相同体积混合在一起，也称“样本混合”。从科学角度讲，第一种“混采”模式，不会影响核酸检测的敏感性，后一种将样本混合检测的模式，则对检测敏感性有一定的影响，但影响程度是已知的。即便如此，以上混检方法虽提升了核酸检测能力，但其检测敏感度较低，工作效率仍然不够高效，尤其对含低拷贝核酸的样本可能出现“假阴性”的检测结果。
现有技术中，多采用磁珠法进行核酸提取，但目前磁珠法进行样本中核酸提取时，可适用 的样本量通常为200-300ul，并不能适用于大体积需求(比如，多个样本混检)的核酸检测的需要。并且重要的是，多个样本混检，受总体积影响，样本相互稀释，不可避免的稀释了目标核酸，在取样量仅200-300ul的情况下，降低了灵敏度。
发明内容
鉴于上述的情况，针对现有技术中存在的不足，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装置和方法。本发明不同于传统混检方法，而是利用大体积柱法封闭式柱法正压核酸提取装置，首先将n个待检样品(n大约2-100)混合，然后经过裂解，吸附，清洗，洗脱，一次性提取n个样品。其过程提取效率高，核酸丢失少，保证了检测灵敏度。
具体的，本发明的上述目的通过以下技术方案实现：
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；
所述方法还包括选自如下至少一项的步骤：
A.检测样本为鼻咽拭子；
B.检测样本可以为多个个体的样本混合物；
C.控制所述待测样本的体积为1～20mL；
D.富集、浓缩待测样本中核酸；
E.采用封闭式柱法正压核酸提取装置进行所述提取。
一些优选的实施方式中，控制所述待测样本的体积为2～20mL。
一些优选的实施方式中，封闭式柱法正压核酸提取装置包括顶盖和与之连接的吸附柱，所述顶盖上具有鲁尔锁紧套口，通过注射器施以正压促使液体流动，通过吸附柱。
一些优选的实施方式中，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，其具体实施步骤如下：
(1)根据具体需求收集鼻咽拭子液2-20ml；
(2)将收集的体液2-20ml，离心10-20分钟，离心力2000-5000g，吸取上清液2-20ml待检,或将上清液-20℃冰冻保存备用；
(3)吸取占样本十分之一体积的裂解液Lysis Buffer至50ml裂解管，与上清液充分混匀；加入50-200μl Proteinase K溶液，再次充分混匀；
(4)盖上裂解管，置于60℃水浴箱或干热模块保温60℃，10-20分钟；移出裂解管，冷却至室温；
(5)加入等体积吸附液Binding Buffer与上述已裂解的混合液相混，旋涡震荡30秒，静置3-5分钟；
(6)将30ml注射器推杆拔出后与封闭式柱法正压核酸提取装置的鲁尔锁紧套口紧密连接，垂直放置；转移裂解液至注射器，插入注射器推杆，缓慢均匀施压，将裂解液注入封闭式柱法正压核酸提取装置；注意观察液体流出封闭式柱法正压核酸提取装置内的吸附柱时应成间断滴液，控制整个推送过程在30秒至120秒之间；推入残余空气，除尽残余液体；
(7)将吸附柱置于洁净1.5ml离心管，加60-300μl洗脱液(EB)于吸附柱内吸附膜上，盖上吸附柱盖，室温静置2分钟；
(8)将含有吸附柱的离心管高速离心3分钟，离心力10000-12000g，，收集洗脱液；
(9)含有分离出的核酸的洗脱液，直接用于RT-PCR或其它分子检测，或保存待用。
一些优选的实施方式中，步骤(7)在加入洗脱液之前，用清洗液(WB)清洗吸附柱1-2次后再加入洗脱液洗脱。用于进一步减少杂质。
本发明中，上清液指经离心后除去沉淀部分的清亮液体。
一些优选的实施方式中，作为检测样本的鼻咽拭子液为合并多重来源的不同个体的鼻咽拭子液。
一些优选的实施方式中，鼻咽拭子液的混合样本可进一步分为亚组(subgroup)，分别提取各亚组混合拭子样本检测。再用本发明的同样方法进行大体积样本中核酸提取，以便于快速鉴定阳性标本。
一些优选的实施方式中，大体积体液吸附柱法提取含微量核酸的样本时，提取效率不受体液体积大小和所含核酸浓度的影响的影响。
一些优选的实施方式中，将未经磁珠法提取或未经检测体液或核酸样本，全部或大部分经过提取，清洗，洗脱，进行检测。增加这一步骤可以大大提高检测灵敏度。
一些具体的实施方式中，鼻咽拭子液为合并1-100个不同个体的鼻咽拭子液。
一些具体的实施方式中，鼻咽拭子液为合并2-100个不同个体的鼻咽拭子液。
一些具体的实施方式中，鼻咽拭子液为合并2-20个不同个体的鼻咽拭子液。
一些具体的实施方式中，鼻咽拭子液为合并20-50个不同个体的鼻咽拭子液。
一些具体的实施方式中，鼻咽拭子液为合并30个不同个体的鼻咽拭子液。
一些具体的实施方式中，本发明的方法广泛适用于多种病毒的核酸提取。
有益效果
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，大大减小过柱时的阻力；以及作为前处理步骤，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。与通常使用的磁珠法可提取核酸的样本量200-300ul相比，大体积柱法提取样本体积大(2-20ml)，可收集10-100倍磁珠法收集的样本，提高检测效率；同时，清洗液体积也大，约磁珠法清洗液体积6-8倍，清洗后杂质含量少，更有利于下游检测。并且，本发明的方法提取的核酸用于检测，其灵敏度高；本方法相对常规磁珠提取法提高了灵敏度，即RT-PCR反应中Ct值提前2-4。
附图说明
图1为本发明的大体积样本核酸提取系统流程图。
图2为本发明中使用的一种封闭式柱法正压核酸提取装置的结构示意图。
图3为图2封闭式柱法正压核酸提取装置的顶盖与吸附柱的示意图。
图4为图3的顶盖设有鲁尔锁紧套口的示意图。
具体实施方式
实施例1：大体积样本提取方法所用的材料和具体步骤，如图1。
一.试剂组分
1.收集提取试剂
1a.裂解液Lysis Buffer(LB)。
1b.吸附液Binding Buffer(BB)。
1c.洗脱液Elution Buffer(EB)。
1d.Proteinase K(PK)，-20℃保存。
1e.清洗液(WB)。
二.自备
1.裂解管：50ml离心管。
2.洗脱液收集管：1.5ml微量离心管。
3. 30ml一次性注射器(螺口接头)。
4. 5-10ml一次性注射器(自备)。
5.低速离心机(≥4000g)。
5.高速微量离心机(≥12000g)。
6. 60℃水浴箱或干热模块。
7.封闭式柱法正压核酸提取装置：中国授权专利CN201580073867.2中的提取容器。封闭式柱法正压核酸提取装置包括顶盖101和与之连接的吸附柱200，以及容器400，吸附柱位于容器内，所述顶盖101上具有鲁尔锁紧套口120，如图2-4所示。其中，CN201580073867.2中的过滤柱200其所起的作用为过滤吸附(说明书【0045】-【0047】段)，因此在本文中将过滤柱名称修改为吸附柱200。
三.核酸提取操作步骤
A.根据具体需求收集鼻咽拭子2-20ml送检；将收集的体液2-20ml，离心10分钟(离心力>＝2000g，一个实施例中，离心力为2000g-5000g)，吸取上清液2-20ml待检,或-20℃冰冻保存备用。
B.吸取占样本十分之一体积的LB至50ml离心管(裂解管)，与离心后的上清液充分混匀。加入100-200μl Proteinase K溶液，再次充分混匀。
C.盖上裂解管，置于60℃水浴箱(或干热模块保温60℃)，10-30分钟；移出裂解管，冷却至室温。
D.加入等体积BB与上述已裂解的混合液相混，旋涡震荡30秒，静置3-5分钟。
E.将30ml注射器推杆拔出后与封闭式柱法正压核酸提取装置的Luer(鲁尔)锁紧套口紧密连接，垂直放置；转移裂解液至注射器，插入注射器推杆，缓慢均匀施压，将裂解液注入提取装置；注意观察液体流出吸附柱时应成间断滴液，控制整个推送过程在90秒至120秒之间；推入残余空气，除尽残余液体。
F.将吸附柱置于洁净1.5ml离心管，加300μl洗脱液(EB)于吸附柱内吸附膜上，盖上吸附柱盖，室温静置2分钟。
G.将含有吸附柱的离心管高速离心3分钟(离心力10000-12000g)，收集洗脱液。
H.含有分离出的核酸的洗脱液，可直接送入高通量磁珠提取仪进一步提取纯化，进行自动化提取及RT-PCR，或清洗后直接用于RT-PCR或其它分子检测，或保存待用。
或者，在步骤F为：先用清洗液(WB)清洗吸附柱1-2次后，将吸附柱置于洁净1.5ml离心管，加300μl洗脱液(EB)于吸附柱内吸附膜上，盖上吸附柱盖，室温静置2分钟。
再或者，根据具体需求收集鼻咽拭子2-20ml后，进一步分为亚组(subgroup)后，再进行步 骤A-H。
实施例2：比较和验证试验
预实验试剂与装置:
Figure PCTCN2021088730-appb-000001
Xpress SARS-CoV-2，
Figure PCTCN2021088730-appb-000002
Cepheid，904 Caribbean Drive，Sunnyvale,CA 94089 USA
CommaXP TM病毒DNA/RNA提取试剂盒(柱式法)，CommaXP TM血清血浆游离DNA中量提取试剂盒，CommaVac TM鲁尔接口负压装置，深圳逗点生物技术有限公司(简称：逗点公司)
12通道负压柱式吸附架
手提式无油真空泵
Manually Operated Extraction System(MOES)，Occam Biolabs,Inc.，DE，USA(简称OBI公司)
BDS新冠假病毒质控品：S2标准品(具体浓度见下表)，广州邦德盛生物科技有限公司(简称BDS公司)
浓度编号 平均数(X)coples/mL X±2SD(coples/mL)
S1(低值) 2.01E+03 6.24E+02～6.48E+03
S2(中值) 2.87E+04 9.17E+03～8.98E+04
一.赛沛Xpert Xpress SARS-CoV-2检测大体积提取核酸
准备：取14ml实施例1中获得的混合拭子保存液(取自20个以上确认阴性的不同个体)，混均。
实验1.取混合拭子液250ul，加入S2标准品50ul，混合，室温下保持2小时，注入赛沛试剂盒，利用赛沛全自动医用PCR分析系统(Cepheid GeneXpert Dx System)进行分析。
实验2.混合拭子液1.5ml，加入S2标准品50ul，混合，加入GLX(逗点公司的配套提供的裂解液)，利用CommaVac TM鲁尔接口负压装置按逗点公司负压提取说明书进行提取，干净水300ul洗脱，注入赛沛试剂盒，利用赛沛全自动医用PCR分析系统(Cepheid GeneXpert Dx System进行分析。
实验3.混合拭子液6ml，加入S2标准品50ul，加入GLX(逗点公司的配套提供的裂解液)，利用CommaVac TM鲁尔接口负压装置按逗点公司负压提取说明书进行提取(注：提取一半时负 压消失，改离心)；其余步骤同试验2。
实验4.混合拭子液6ml，加入S2标准品50ul，使用MOES提取装置及配套裂解液(LB)和结合液(BB)，重复实验3。
试验结果：
Figure PCTCN2021088730-appb-000003
该实验结果显示：实验2(大体积)和实验4(大体积)质控品基因片段E和N2Ct值明显低于实验1(小体积)，大体积拭子液柱式提取装置对检测灵敏度影响不大。
二.含不同核酸浓度的样本的实际过柱得率。
①标准品序列稀释方案：
11号管：200ulS2质控品+200ul咽拭子混合液，12号管：稀释方法为取1号样本40ul,再加入360ul混合液，即1：9稀释。13-16号管以此类推，分别取2-5号样本，加入混合液，1：9稀释。
②样本管命名1-6号：每管加入咽拭子混合液6毫升，加入OBI公司提供的裂解液4毫升，加入PK100ul，置60C水浴30min。再加入OBI提供的结合液10毫升，使用MOES提取装置提取核酸，300ul洗脱液洗柱，12000rpm离心3分钟。取200ul离心获得的洗脱产物(洗脱产物共300ul)经磁珠提取仪+RT-PCR(全自动核酸提取仪，型号：S-96。生产企业：武汉纳磁生物科技有限公司)。
③11-16号管各取200ul经磁珠提取仪+RT-PCR。
样品(11-16号管中混合液)经现有磁珠法仪进行磁珠法提取(200ul)，进一步RT-qPCR。试剂盒厂家：中山大学达安基因股份有限公司。操作步骤：5ul核酸样本+20ulPCR试剂混合分置PCR反应管，PCR仪器为ABI7500。具体的操作步骤和PCR反应条件见达安基因公司新型冠状病毒2019-nCoV核酸检测试剂盒(荧光PCR法)说明书)。
RT-PCR结果如下：
Figure PCTCN2021088730-appb-000004
Figure PCTCN2021088730-appb-000005
结论：系列稀释的质控品在6ml体积混合拭子液用柱法提取，和在200ul中用磁珠法提取对比结果。结果表明，柱法提取混合拭子液6ml vs磁珠法提取0.2ml，得到的Ct值近似，表明体积对含有相同量新冠病毒核酸影响甚微。因此，大体积体液滤柱法提取含微量核酸的样本时，提取效率不受体液体积大小和所含核酸浓度的影响。
三.样本裂解前离心对过柱是否堵塞的影响情况。
具体步骤：
1. 20ml混合拭子液加入2ml RNA稳定液混匀，分为6ml/管，共3管(即A,B,C管)。
2.加入标准品S2 50ul/管。
3. A管离心1500rpm，20分钟，收集上清液。
4.分别加入BLX buffer 3ml/管到A,B,C管。
5.加PK100ul/管，置60C水浴30min。
6. B管水浴后离心1500rpm，10分钟，收集上清液过柱。
7. C管全程未离心。
8.过柱(RC2),阻塞情况：
A管      -     正常
B管      +     极微堵
C管      +++  阻塞严重。
9.加300ul洗脱液，高速离心3分钟，收集洗脱液。
10.仪器进一步提取及RT-qPCR。
PCR反应结果(Ct值)：
标本编号 IC N ORF-1ab
A 19.93 29.43 32.68
B 19.64 29.95 32.69
C 22.32 32.13 35.54
D(S2标准品50ul) 24.45 29.17 32.56
试验得知：离心可以提高过柱效率。
四.不同厂家(逗点生物、OBI等公司)的多种裂解液对大体积样本过柱是否堵塞的影响情况。
具体方案：20ml混合拭子液加50ulS2混匀，分为3管，6ml混合拭子液/管。离心3900rpm，20min，加三种不同裂解缓冲液(OBI提供MOES配套使用于大体积样本提取的裂解液，逗点公司提供的两种裂解液即LGX、LGS)，按各自说明书裂解处理，提取过程均使用MOES装置提取，洗脱液进行RT-PCR。
PCR反应结果(Ct值)：
Figure PCTCN2021088730-appb-000006
试验得知：一种裂解液(OBI提供MOES配套使用于大体积样本提取的裂解液)略优于逗点提供的两种裂解液(LGX、LGS)。
五.OBI提供MOES配套使用于大体积样本提取的裂解液可以将样本体积扩大至20毫升(若以现临床实验室通常提取操作使用200ul/人来计算，MOES相当于一个检测可以同时检测100份个体样本)。
具体方案：取60毫升不同个体混合咽拭子样本，A管和B管每管装10毫升，C管和D管每管装20毫升，按照MOES提取说明书并使用MOES抽提装置进行核酸提取。
Figure PCTCN2021088730-appb-000007
PCR反应结果(Ct值)如下：
Figure PCTCN2021088730-appb-000008
Figure PCTCN2021088730-appb-000009
经上述实验发现，本发明的方法用于提取含微量核酸的大体积体液时，提取效率不受体液体积大小的影响；且获取的所有核酸可选择性的全部或部分用于下游RT-PCR，优于现有通过自动核酸提取仪提取核酸的方法(现有自动核酸提取仪通常获取50～80ul含有核酸的洗脱液，但受限于洗脱液中可能含有较高浓度抑制PCR反应的物质，实际RT-PCR反应仅使用了5ul洗脱液，约占全部核酸的10％，大大降低了灵敏度)。在进行混合检测时，我们认为，本发明大体积提取方法由于：1.采样量大(0.2ml vs 2-20ml)，收集的微量核酸相对较多；2.清洗液体积大(0.6ml vs 2-4ml),因此残余杂质含量少；3.在大体积提取装置混检时(n个)，现有提取方法中每个标本较大量未使用洗脱液约45-75ul，每管只吸取了10分之一洗脱液(5微升)进入rt-PCR检测。而本发明的大体积提取方法中，其剩余洗脱液，按平均45微升计算，总共约45微升x n个混合样本，皆通过吸附柱得以回收，进入RT-PCR检测，因此大大提高了灵敏度。

Claims (10)

  1. 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，其特征在于，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；
    由所述提取物制备用于病毒检测目的的测试液；
    所述方法还包括选自如下至少一项：
    A.检测样本为鼻咽拭子；
    B.检测样本可以为多个个体的样本混合物；
    C.控制所述待测样本的体积为2～20mL；
    D.富集、浓缩待测样本中核酸；
    E.采用封闭式柱法正压核酸提取装置进行提取。
  2. 根据权利要求1所述的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，其特征在于，封闭式柱法正压核酸提取装置包括顶盖和与之连接的吸附柱，所述顶盖上具有鲁尔锁紧套口，通过注射器施以正压促使液体流动，通过吸附柱。
  3. 根据权利要求1或2所述的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，其特征在于，具体实施步骤如下：
    (1)根据具体需求收集鼻咽拭子液2-20ml；
    (2)将收集的体液2-20ml，离心10-20分钟，离心力2000-5000g，吸取上清液2-20ml待检,或将上清液-20℃冰冻保存备用；
    (3)吸取占样本十分之一体积的裂解液Lysis Buffer至50ml裂解管，与上清液充分混匀；加入50-200μl Proteinase K溶液，再次充分混匀；
    (4)盖上裂解管，置于60℃水浴箱或干热模块保温60℃，10-20分钟；移出裂解管，冷却至室温；
    (5)加入等体积吸附液Binding Buffer与上述已裂解的混合液相混，旋涡震荡30秒，静置3-5分钟；
    (6)将30ml注射器推杆拔出后与封闭式柱法正压核酸提取装置的鲁尔锁紧套口紧密连接，垂直放置；转移裂解液至注射器，插入注射器推杆，缓慢均匀施压，将裂解液注入封闭式柱法正压核酸提取装置；注意观察液体流出封闭式柱法正压核酸提取装置内的吸附柱时应成间断滴液，控制整个推送过程在30秒至120秒之间；推入残余空气，除尽残余液体；
    (7)将吸附柱置于洁净1.5ml离心管，加60-300μl洗脱液(EB)于吸附柱内吸附膜上，盖上吸附柱盖，室温静置2分钟；
    (8)将含有吸附柱的离心管高速离心3分钟，离心力10000-12000g，，收集洗脱液；
    (9)含有分离出的核酸的洗脱液，直接用于RT-PCR或其它分子检测，或保存待用。
  4. 根据权利要求3所述的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，其特征在于，步骤(7)中在加入洗脱液之前，可用清洗液(WB)清洗吸附柱1-2次后再加入洗脱液洗脱。
  5. 根据权利要求1或3所述的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，其特征在于，作为检测样本的鼻咽拭子液为合并多重来源的不同个体的鼻咽拭子液。
  6. 根据权利要求1或3所述的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，其特征在于，作为检测样本的鼻咽拭子液为合并1-100个不同个体的鼻咽拭子液。
  7. 根据权利要求1或3所述的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，其特征在于，作为检测样本的鼻咽拭子液为合并2-100个不同个体的鼻咽拭子液。
  8. 根据权利要求5所述的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，其特征在于，鼻咽拭子液的混合样本可进一步分为亚组，分别提取各亚组混合拭子样本检测。
  9. 根据权利要求1或3所述的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，其特征在于，大体积体液吸附柱法提取含微量核酸的样本时，提取效率不受体液体积大小和所含核酸浓度的影响。
  10. 根据权利要求1或3所述的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，其特征在于，将未经磁珠法提取或未经检测体液或核酸样本，全部或大部分经过提取，清洗，洗脱，进行检测。
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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN115161385A (zh) * 2022-08-30 2022-10-11 中国医科大学附属盛京医院 一种多样本快速核酸检测方法及系统

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5958677A (en) * 1997-07-28 1999-09-28 The New York Blood Center, Inc. Method for purifying viral nucleic acids
WO2019174468A1 (zh) * 2018-03-13 2019-09-19 武汉医蒂生物科技有限公司 一种核酸提取纯化正压装置
CN110272808A (zh) * 2018-03-13 2019-09-24 武汉医蒂生物科技有限公司 一种核酸提取系统

Family Cites Families (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
RU2729113C2 (ru) * 2014-11-21 2020-08-04 Оккам Байолэбс, Инк. Система и способ сбора образца нуклеиновой кислоты
CN111534570B (zh) * 2020-07-07 2020-11-13 上海思路迪医学检验所有限公司 一种高通量混样裂解病毒核酸提取方法
CN111850166A (zh) * 2020-07-10 2020-10-30 中国人民解放军总医院第五医学中心 一种大规模筛查新型冠状病毒感染人群的核酸检测方法及应用

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5958677A (en) * 1997-07-28 1999-09-28 The New York Blood Center, Inc. Method for purifying viral nucleic acids
WO2019174468A1 (zh) * 2018-03-13 2019-09-19 武汉医蒂生物科技有限公司 一种核酸提取纯化正压装置
CN110272808A (zh) * 2018-03-13 2019-09-24 武汉医蒂生物科技有限公司 一种核酸提取系统

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
ANONYMOUS: "Technical Specifications on Covid-19 Nucleic Acid 10-in-1 Pool Test", CHINESE JOURNAL OF VIRAL DISEASES, vol. 10, no. 5, 30 September 2020 (2020-09-30), pages 330 - 332, XP055954626, ISSN: 2095-0136, DOI: 10.16505/j.2095-0136.2020.0070 *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN115161385A (zh) * 2022-08-30 2022-10-11 中国医科大学附属盛京医院 一种多样本快速核酸检测方法及系统

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