CN112795562A - A high-throughput method for extracting trace nucleic acids for detection of large-volume mixed swab samples - Google Patents
A high-throughput method for extracting trace nucleic acids for detection of large-volume mixed swab samples Download PDFInfo
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Abstract
本发明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，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，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。The present invention relates to a high-throughput method for extracting trace nucleic acids for the detection of large-volume mixed swab samples, comprising the following steps: using a lysing solution to lyse a sample to be tested to release the nucleic acid contained in the sample, and obtain a method that mainly contains the nucleic acid. The lysate containing nucleic acid is extracted by a closed column method positive pressure nucleic acid extraction device to obtain an extract; the test solution for virus detection is prepared from the extract. In the present invention, the column method is used to extract the nasopharyngeal swabs of different individuals from multiple sources, and after the swabs are combined, high-speed centrifugation is added at 2000-4000g, and the steps of 10-20min 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 to general extraction, which improves the efficiency of nucleic acid extraction.
Description
Technical Field
The invention belongs to the fields of life science and biotechnology, and particularly relates to a method for extracting trace nucleic acid in high flux for detecting a large-volume mixed swab sample.
Background
At present, the clinical virus detection methods mainly comprise two methods: nucleic acid detection and antigen-antibody immunological detection. Nucleic acid detection requires extraction of viral nucleic acid RNA from various body fluid samples, and the volume of the body fluid sample to be detected is limited by the conventional commercial extraction device and cannot exceed 200-300. mu.l, so that the detection sensitivity is limited.
For example, the novel coronavirus COVID-19 belongs to RNA virus, which mainly invades the body through the upper respiratory tract and the digestive tract, and virus surface spinous process protein (Spike protein) and ACE2 receptor expressed by the upper respiratory tract and the digestive tract cells are combined with high affinity and high specificity to enter host cells, and viral protein synthesis and virus replication are carried out by utilizing host organelles. Samples for detecting the novel coronavirus generally include blood (for detecting antigen and antibody), nasal swab, and pharyngeal swab (for detecting nucleic acid and RNA). 1. The novel coronavirus COVID-19(SARS-CoV-2) is a single-stranded positive-sense RNA virus encapsulated by protein. The genetic material RNA of the novel coronavirus is a direct evidence of the presence of the virus, and RT-PCR nucleic acid detection technology is used for the differential diagnosis of diseases by specifically detecting the viral RNA. 2. The most widely used diagnostic method for COVID-19 infection is reverse transcription quantitative polymerase chain reaction (RT-qPCR). 3. At present, the RT-qPCR method for detecting the COVID-19 nucleic acid RNA needs to extract RNA from a sample as a PCR template. However, the existing RNA extraction technology and device are the main bottleneck in the RNA template extraction process, resulting in low RNA yield, and are limited by the existing extraction methods, including extraction of samples with insufficient volume, especially samples containing low copy nucleic acids.
In addition, the specimen mainly collected for clinically detecting the infection virus of the patient is nasopharyngeal swabs (napopharyngeal swabs), and the detection positive rate is only 30-50%.
And there are two modes for the "mix check". One is that several persons, such as 3 persons or 5 persons, are respectively sampled and put into the same sampling tube during sampling, and the mode is also called as 'mixed sampling' or 'mixed sampling'; the other is to mix 3 or 5 human samples together in the same volume, also called "sample mix", in laboratory tests. Scientifically speaking, the first "mix-sampling" mode does not affect the sensitivity of nucleic acid detection, and the latter mode of mixing the sample for detection has a certain effect on the detection sensitivity, but the degree of the effect is known. Even so, although the nucleic acid detection capability is improved by the above mixed detection method, the detection sensitivity is low, the working efficiency is still not high enough, and particularly, a false negative detection result may occur on a sample containing low-copy nucleic acid.
In the prior art, the magnetic bead method is mostly used for nucleic acid extraction, but the current magnetic bead method is generally applicable to nucleic acid extraction in a sample with a sample volume of 200-. And importantly, a plurality of samples are mixed and detected, the samples are mutually diluted under the influence of the total volume, the target nucleic acid is inevitably diluted, and the sensitivity is reduced under the condition that the sampling amount is only 200-.
Disclosure of Invention
In view of the above, the present invention provides a device and method for high throughput extraction of trace amounts of nucleic acids for detection of large volume mixed swab samples, which addresses the deficiencies in the prior art. The 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 mixes n samples to be detected (n is about 2-100), and then extracts n samples at one time through cracking, adsorption, cleaning and elution. The process has high extraction efficiency and less nucleic acid loss, and ensures the detection sensitivity.
Specifically, the above object of the present invention is achieved by the following technical solutions:
a method for high throughput extraction of trace nucleic acids for bulk mixed swab sample detection comprising the steps of: carrying out cracking treatment on a sample to be detected by using a cracking solution so as to release nucleic acid contained in the sample, and obtaining the cracking solution mainly containing the nucleic acid;
extracting the lysate containing nucleic acid by a closed column positive pressure nucleic acid extraction device to obtain an extract;
preparing the test solution for virus detection purposes from the extract;
the method further comprises a step selected from at least one of:
A. the detection sample is a nasopharyngeal swab;
B. the detection sample can be a sample mixture of a plurality of individuals;
C. controlling the volume of the sample to be detected to be 1-20 mL;
D. enriching and concentrating nucleic acid in a sample to be detected;
E. the extraction is carried out by adopting a closed column method positive pressure nucleic acid extraction device.
In some preferred embodiments, the volume of the sample to be tested is controlled to be 2-20 mL.
In some preferred embodiments, the closed column positive pressure nucleic acid extraction device comprises a top cap and an adsorption column connected thereto, wherein the top cap has a luer lock socket, and positive pressure is applied by a syringe to promote liquid flow through the adsorption column.
In some preferred embodiments, the method for high throughput extraction of trace nucleic acids for large volume mixed swab sample detection is performed by the following steps:
(1) collecting 2-20ml of nasopharyngeal swab liquid according to specific requirements;
(2) centrifuging the collected body fluid for 10-20min at centrifugal force of 2000-5000g for 2-20ml, and collecting the supernatant fluid for examination, or freezing and storing the supernatant fluid at-20 ℃ for later use;
(3) absorbing lysate Buffer accounting for one tenth of the volume of the sample to a 50ml Lysis tube, and fully and uniformly mixing the lysate Buffer with the supernatant; adding 50-200 mul of protease K solution, and fully and uniformly mixing again;
(4) covering the cracking tube, and placing in a 60 ℃ water bath box or a dry heat module for heat preservation at 60 ℃ for 10-20 minutes; the cracking tube is removed and cooled to room temperature;
(5) adding equal volume of adsorption solution Binding Buffer, mixing with the cracked mixed solution, performing vortex oscillation for 30 seconds, and standing for 3-5 minutes;
(6) pulling out a 30ml syringe push rod, tightly connecting with a luer locking sleeve opening of the closed column positive pressure nucleic acid extraction device, and vertically placing; transferring the lysate to an injector, inserting an injector push rod, slowly and uniformly applying pressure, and injecting the lysate into a closed column positive pressure nucleic acid extraction device; attention is paid to the fact that when liquid is observed to flow out of an adsorption column in the closed column method positive pressure nucleic acid extraction device, intermittent dropping liquid is needed, and the whole pushing process is controlled to be 30-120 seconds; pushing residual air to remove residual liquid;
(7) placing the adsorption column in a clean 1.5ml centrifuge tube, adding 60-300 μ l of Eluent (EB) on the adsorption film in the adsorption column, covering the adsorption column cover, and standing at room temperature for 2 min;
(8) centrifuging the centrifugal tube containing the adsorption column at high speed for 3 minutes under the centrifugal force of 10000-;
(9) the eluate containing the separated nucleic acids is used directly for RT-PCR or other molecular detection, or stored for later use.
In some preferred embodiments, step (7) is performed by washing the adsorption column with the washing solution (WB) 1-2 times before adding the eluent, and then adding the eluent for elution. For further reduction of impurities.
In the present invention, the supernatant refers to a clear liquid from which a precipitated fraction is removed by centrifugation.
In some preferred embodiments, the nasopharyngeal swab used as the test sample is a nasopharyngeal swab from a different individual who has pooled multiple sources.
In some preferred embodiments, the nasopharyngeal swab mix can be further divided into subgroups (subgroups) and each subgroup of the swab mix is separately tested. Then the same method of the invention is used for extracting nucleic acid from a large-volume sample so as to rapidly identify a positive specimen.
In some preferred embodiments, when a sample containing a trace amount of nucleic acid is extracted by the bulk liquid adsorption column method, the extraction efficiency is not affected by the volume of the liquid and the concentration of the nucleic acid contained therein.
In some preferred embodiments, the body fluid or nucleic acid sample is extracted, washed, eluted, and detected, all or most of which are not extracted by the magnetic bead method or not detected. The addition of this step can greatly improve the detection sensitivity.
In some embodiments, the nasopharyngeal swab is a combination of 1-100 different individuals.
In some embodiments, the nasopharyngeal swab is a combination of nasopharyngeal swabs from 2 to 100 different individuals.
In some embodiments, the nasopharyngeal swab is a combination of nasopharyngeal swabs from 2 to 20 different individuals.
In some embodiments, the nasopharyngeal swab pools are combined from 20 to 50 different individuals.
In some embodiments, the nasopharyngeal swab is a combination of 30 different individuals.
In some embodiments, the methods of the invention are broadly applicable to nucleic acid extraction of a variety of viruses.
Advantageous effects
The method extracts nasopharyngeal swab solutions for combining different individuals with multiple sources by a column method, and after the nasopharyngeal swab solutions are combined, the steps of high-speed centrifugation of 2000 and 4000g are added for 10-20min to remove precipitates, so that the resistance during column passing is greatly reduced; and as a pretreatment step, 1-2 times of washing and centrifuging steps are reduced compared with the common extraction, so that the efficiency of extracting nucleic acid is improved. Compared with the commonly used sample amount of 200-; meanwhile, the volume of the cleaning solution is also large, about 6-8 times of that of the cleaning solution prepared by a magnetic bead method, and the impurity content after cleaning is low, so that downstream detection is facilitated. Moreover, the nucleic acid extracted by the method is used for detection, and the sensitivity is high; compared with the conventional magnetic bead extraction method, the method improves the sensitivity, namely the Ct value in the RT-PCR reaction is advanced by 2-4.
Drawings
FIG. 1 is a flow chart of a large volume sample nucleic acid extraction system according to the present invention.
FIG. 2 is a schematic view showing the structure of a closed column positive pressure nucleic acid isolation apparatus used in the present invention.
FIG. 3 is a schematic diagram of the top cover and the adsorption column of the closed column method positive pressure nucleic acid extraction apparatus of FIG. 2.
Fig. 4 is a schematic view of the cap of fig. 3 provided with a luer lock cuff.
Detailed Description
Example 1: materials and specific steps used in the bulk sample extraction method are shown in fig. 1.
Reagent component
1. Collecting and extracting reagent
Lysate Lysis Buffer (LB).
And 1b, adsorbing liquid Binding Buffer (BB).
Eluent Elution Buffer (EB) was eluted.
Proteinase K (PK), stored at-20 ℃.
Cleaning solution (WB).
Secondly, the device is self-provided
1. Cracking tube: 50ml centrifuge tube.
2. An eluent collecting pipe: 1.5ml microcentrifuge tube.
3.30 ml disposable syringe (screw connection).
4.5-10 ml disposable syringes (self-contained).
5. Low speed centrifuge (4000 g or more).
5. High-speed micro-centrifuge (12000 g or more).
A 6.60 ℃ water bath or a dry heat module.
7. Closed column method positive pressure nucleic acid extraction element: the extraction container in chinese granted patent CN 201580073867.2. The closed column positive pressure nucleic acid extraction device comprises a top cover 101, an adsorption column 200 connected with the top cover, and a container 400, wherein the adsorption column is positioned in the container, and the top cover 101 is provided with a luer lock sleeve port 120, as shown in figures 2-4. Among them, the filter column 200 in CN201580073867.2 plays a role of filtration and adsorption (paragraphs [ 0045 ] to [ 0047 ]), so the name of the filter column is modified to the adsorption column 200 herein.
Thirdly, the operation steps of nucleic acid extraction
A. Collecting 2-20ml nasopharyngeal swab for inspection according to specific requirements; centrifuging the collected body fluid 2-20ml for 10 min (centrifugal force > 2000g, in one embodiment, centrifugal force 2000g-5000g), and collecting supernatant 2-20ml for testing, or freezing at-20 deg.C for storage.
B. And (3) sucking LB which accounts for one tenth of the volume of the sample into a 50ml centrifuge tube (a lysis tube), and fully and uniformly mixing with the centrifuged supernatant. Add 100 and 200. mu.l of protease K solution and mix well again.
C. Covering the cracking tube, and placing in a 60 deg.C water bath tank (or dry heat module for 60 deg.C) for 10-30 min; the tube was removed and cooled to room temperature.
D. Adding BB with the same volume as the mixed liquid obtained by the cracking, mixing the mixed liquid with the cracked mixed liquid, carrying out vortex oscillation for 30 seconds, and standing for 3-5 minutes.
E. Pulling out a push rod of a 30ml syringe, tightly connecting with a Luer (Ruhr) locking sleeve opening of the closed column positive pressure nucleic acid extraction device, and vertically placing; transferring the lysate to an injector, inserting an injector push rod, slowly and uniformly applying pressure, and injecting the lysate into an extraction device; the liquid is observed to be dropped discontinuously when flowing out of the adsorption column, and the whole pushing process is controlled to be between 90 seconds and 120 seconds; residual air is pushed in to remove residual liquid.
F. The adsorption column was placed in a clean 1.5ml centrifuge tube, 300. mu.l of Eluent (EB) was added to the adsorption membrane in the adsorption column, the cover of the adsorption column was closed, and the column was allowed to stand at room temperature for 2 minutes.
G. The tube containing the adsorption column was centrifuged at high speed for 3 minutes (centrifugal force 10000-.
H. The eluent containing the separated nucleic acid can be directly sent into a high-flux magnetic bead extractor for further extraction and purification, and then automatic extraction and RT-PCR are carried out, or the eluent is directly used for RT-PCR or other molecular detection after being cleaned, or the eluent is stored for standby. Or, in step F: washing the adsorption column with the cleaning solution (WB) for 1-2 times, placing the adsorption column in a clean 1.5ml centrifuge tube, adding 300 μ l of Eluent (EB) on the adsorption film in the adsorption column, covering the adsorption column, and standing at room temperature for 2 minutes. Or, after collecting 2-20ml nasopharyngeal swabs according to specific requirements, further dividing into subgroups, and then carrying out the steps A-H.
Example 2: comparison and verification test
Pre-assay reagents and apparatus:
CommaXPTMviral DNA/RNA extraction kit (column method), CommaxPTMSerum plasma free DNA quantitative extraction kit, CommavacTMLuer interfaceNegative pressure device, Shenzhen comma biotechnology Limited (comma for short)
12-channel negative pressure column type adsorption rack
Portable oil-free vacuum pump
Manual Operated Extraction System (MOES), Occam Biolabs, Inc., DE, USA (OBI corporation for short)
BDS new corona pseudovirus quality control: s2 Standard (specific concentrations are shown in the Table below), Guangzhou Bangdong Biotech limited (BDS for short)
| Concentration number | Average (X) copies/mL | X±2SD(coples/mL) |
| S1 (Low value) | 2.01E+03 | 6.24E+02～6.48E+03 |
| S2 (middle value) | 2.87E+04 | 9.17E+03～8.98E+04 |
Detection of nucleic acid by Xeper Xpress SARS-CoV-2
Preparing: 14ml of the mixed swab stock solution obtained in example 1 (obtained from 20 or more different individuals confirmed to be negative) was taken and mixed.
Experiment 1. taking 250ul of mixed swab liquid, adding 50ul of S2 standard, mixing, keeping at room temperature for 2 hours, injecting into Siberian kit, and analyzing with Siberian fully automatic medical PCR analysis System (Cepheid GeneXpert Dx System).
Experiment 2. mix swab 1.5ml, add S2 standard 50ul, mix, add GLX (comma' S kit lysate), utilize CommavacTMThe luer negative pressure device was extracted according to the comma corporation negative pressure extraction instructions, 300ul of clean water was eluted, and a sippy kit was injected and analyzed using a sipheid full-automatic medical PCR analysis System (Cepheid GeneXpert Dx System).
Experiment 3. mixing the swab fluid 6ml, adding S2 standard 50ul, adding GLX (comma corporation' S complete kit lysate), using CommavacTMExtracting by a luer negative pressure device according to the negative pressure extraction instruction of comma corporation (note: when half of the extraction is carried out, the negative pressure disappears, and the centrifugation is changed); the remaining steps were as in experiment 2.
Experiment 4. mix swab liquid 6ml, add S2 standard 50ul, use MOES extraction equipment and supporting Lysate (LB) and binding solution (BB), repeat experiment 3.
And (3) test results:
the experimental results show that: the gene fragment E and the Ct value of N2 of the quality control products of experiment 2 (large volume) and experiment 4 (large volume) are obviously lower than those of experiment 1 (small volume), and the large-volume swab liquid column type extraction device has little influence on the detection sensitivity.
Second, actual column yields for samples containing different nucleic acid concentrations.
A standard substance sequence dilution scheme:
tube No. 11: 200ulS2 quality control +200ul throat swab mix, tube 12: the dilution method is to take 40ul of No. 1 sample, and then add 360ul of mixed solution, namely 1: 9, diluting. And (3) performing the same analogy on tubes 13-16, respectively taking samples 2-5, adding the mixed solution, and performing the following steps of 1: 9, diluting.
Sample tube designation No. 1-6: 6ml of throat swab mixture was added to each tube, 4ml of lysate from OBI was added, PK100ul was added, and the mixture was placed in a 60C water bath for 30 min. Then, 10ml of the binding solution supplied by OBI was added, and the nucleic acid was extracted using a MOES extraction apparatus, and the column was washed with 300. mu.l of the eluate and centrifuged at 12000rpm for 3 minutes. 200ul of the eluate (300 ul in total) obtained by centrifugation was subjected to magnetic bead extraction and RT-PCR (full-automatic nucleic acid extractor, model: S-96, manufacturer: Wuhanna magnetic Biotechnology Co., Ltd.).
③ taking 200ul of No. 11-16 tubes respectively, and passing through a magnetic bead extractor and RT-PCR.
The samples (mixed solution in tubes 11-16) were extracted by the paramagnetic particle method (200ul) using the existing paramagnetic particle method apparatus, followed by RT-qPCR. The kit manufacturer: daan Gen-John GmbH, Zhongshan university. The method comprises the following operation steps: 5ul of nucleic acid sample and 20ul of PCR reagent are mixed and separately placed in a PCR reaction tube, and the PCR instrument is ABI 7500. The specific operation steps and PCR reaction conditions are shown in the specification of a novel coronavirus 2019-nCoV nucleic acid detection kit (fluorescent PCR method) of Daan Gene company).
The RT-PCR results were as follows:
and (4) conclusion: the serially diluted quality control was extracted by column method in a 6ml volume of mixed swab, and comparative results were extracted by magnetic bead method in 200 ul. The result shows that the Ct value obtained by extracting 0.2ml of the mixed swab liquid by the column method by the 6ml vs magnetic bead method is approximate, and the volume has little influence on the nucleic acid containing the same amount of the new coronavirus. Therefore, when a sample containing a trace amount of nucleic acid is extracted by the bulk liquid column method, the extraction efficiency is not affected by the volume of the liquid and the concentration of the nucleic acid contained therein.
And thirdly, the influence of centrifugation on whether the column is blocked or not before sample lysis.
The method comprises the following specific steps:
adding 2ml of RNA stabilizing solution into 1.20ml of mixed swab liquid, mixing, and dividing into 3 tubes (i.e. 6 ml/tube)
Tubes a, B, C).
2. Add standard S250 ul/tube.
Tube A was centrifuged at 1500rpm for 20 minutes and the supernatant was collected.
4. Add BLX buffer 3 ml/tube to tubes A, B and C, respectively.
5. Adding PK100 ul/tube, and placing in 60C water bath for 30 min.
Centrifuging at 1500rpm for 10 minutes after water bath in the tube B, and collecting supernatant to pass through a column.
Tube C was not centrifuged throughout.
8. Column pass (RC2), blocking condition:
9. adding 300ul of eluent, centrifuging at high speed for 3 minutes, and collecting the eluent.
10. Further extraction and RT-qPCR by the instrument.
PCR reaction results (Ct value):
| specimen number | 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 standard 50ul) | 24.45 | 29.17 | 32.56 |
The experiment shows that: centrifugation can improve column efficiency.
And fourthly, the influence of various lysates of different manufacturers (comma biology, OBI and the like) on whether the large-volume sample column passes through the blockage or not.
The specific scheme is as follows: 20ml of the mixed swab was mixed with 50ulS2 and divided into 3 tubes and 6ml of mixed swab/tube. Centrifuging at 3900rpm for 20min, adding three different lysis buffers (OBI provides lysate for MOES matched with large-volume sample extraction, and comma provides two lysates, namely LGX and LGS), performing lysis treatment according to respective instructions, extracting with MOES device, and performing RT-PCR with eluate.
PCR reaction results (Ct value):
the experiment shows that: one lysate (the OBI provides the lysate used in conjunction with MOES for bulk sample extraction) was slightly superior to the two lysates (LGX, LGS) provided by comma.
The OBI provides a lysate that is used in conjunction with MOES for large volume sample extraction, which can expand the sample volume to 20ml (when calculated using 200 ul/person for current clinical laboratory extraction procedures, MOES corresponds to a test that can simultaneously test 100 individual samples).
The specific scheme is as follows: 60 ml of each individual throat swab sample was mixed, each of tubes A and B contained 10ml, and each of tubes C and D contained 20ml, and nucleic acid extraction was performed according to MOES extraction instructions using a MOES extraction apparatus.
The results of the PCR reaction (Ct values) were as follows:
the experiment shows that when the method is used for extracting the large-volume body fluid containing trace nucleic acid, the extraction efficiency is not influenced by the volume of the body fluid; and all the obtained nucleic acids can be selectively used for downstream RT-PCR (reverse transcription-polymerase chain reaction) completely or partially, and the method is superior to the existing method for extracting nucleic acids by an automatic nucleic acid extractor (the existing automatic nucleic acid extractor usually obtains 50-80 ul of nucleic acid-containing eluent, but is limited by the fact that the eluent possibly contains substances inhibiting PCR reaction at higher concentration, and the actual RT-PCR reaction only uses 5ul of eluent, which accounts for about 10% of all the nucleic acids, and greatly reduces the sensitivity). When mixed detection is carried out, the large-volume extraction method is considered to be due to the following steps: 1. the sampling amount is large (0.2ml vs 2-20ml), and the collected trace nucleic acid is relatively more; 2. the volume of the cleaning solution is large (0.6ml vs 2-4ml), so the content of residual impurities is low; 3. when mixed detection is carried out by a large-volume extraction device (n samples), the larger amount of unused eluent in each sample in the existing extraction method is about 45-75ul, and only 10 times of eluent (5 microliter) is absorbed in each tube to enter rt-PCR detection. In the large-volume extraction method, the residual eluent is calculated according to the average 45 microliters, and x n mixed samples of about 45 microliters are recovered through the adsorption column and enter the RT-PCR detection, so the sensitivity is greatly improved.
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| CN111534570A (en) * | 2020-07-07 | 2020-08-14 | 上海思路迪医学检验所有限公司 | High-throughput mixed sample lytic virus nucleic acid extraction method |
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| CN111534570A (en) * | 2020-07-07 | 2020-08-14 | 上海思路迪医学检验所有限公司 | High-throughput mixed sample lytic virus nucleic acid extraction method |
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