CN111334867A - Method for constructing virus nucleic acid library - Google Patents
Method for constructing virus nucleic acid library Download PDFInfo
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- CN111334867A CN111334867A CN202010124210.7A CN202010124210A CN111334867A CN 111334867 A CN111334867 A CN 111334867A CN 202010124210 A CN202010124210 A CN 202010124210A CN 111334867 A CN111334867 A CN 111334867A
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- 150000007523 nucleic acids Chemical class 0.000 title claims abstract description 66
- 108020004707 nucleic acids Proteins 0.000 title claims abstract description 62
- 102000039446 nucleic acids Human genes 0.000 title claims abstract description 62
- 241000700605 Viruses Species 0.000 title claims abstract description 42
- 238000000034 method Methods 0.000 title claims abstract description 25
- 230000003321 amplification Effects 0.000 claims abstract description 19
- 238000003199 nucleic acid amplification method Methods 0.000 claims abstract description 19
- 230000003612 virological effect Effects 0.000 claims abstract description 13
- 241000894006 Bacteria Species 0.000 claims abstract description 10
- 108091092584 GDNA Proteins 0.000 claims abstract description 10
- 239000007788 liquid Substances 0.000 claims abstract description 8
- 244000052616 bacterial pathogen Species 0.000 claims abstract description 7
- 238000012163 sequencing technique Methods 0.000 claims abstract description 7
- 238000010276 construction Methods 0.000 claims abstract description 6
- 108090000623 proteins and genes Proteins 0.000 claims abstract description 5
- 210000004369 blood Anatomy 0.000 claims abstract description 4
- 239000008280 blood Substances 0.000 claims abstract description 4
- 210000001175 cerebrospinal fluid Anatomy 0.000 claims abstract description 4
- 244000005700 microbiome Species 0.000 claims abstract description 4
- 210000000582 semen Anatomy 0.000 claims abstract description 4
- 210000002966 serum Anatomy 0.000 claims abstract description 4
- 210000004027 cell Anatomy 0.000 claims description 15
- 239000002245 particle Substances 0.000 claims description 12
- 101710163270 Nuclease Proteins 0.000 claims description 7
- 239000000463 material Substances 0.000 claims description 7
- 230000003071 parasitic effect Effects 0.000 claims description 4
- 210000001519 tissue Anatomy 0.000 claims description 4
- 102000007260 Deoxyribonuclease I Human genes 0.000 claims description 3
- 108010008532 Deoxyribonuclease I Proteins 0.000 claims description 3
- 108091028043 Nucleic acid sequence Proteins 0.000 claims description 3
- 238000004458 analytical method Methods 0.000 claims description 3
- 210000000436 anus Anatomy 0.000 claims description 3
- 239000011324 bead Substances 0.000 claims description 3
- 238000005119 centrifugation Methods 0.000 claims description 3
- 239000012141 concentrate Substances 0.000 claims description 3
- 238000000605 extraction Methods 0.000 claims description 3
- 238000001914 filtration Methods 0.000 claims description 3
- 210000002216 heart Anatomy 0.000 claims description 3
- 238000012165 high-throughput sequencing Methods 0.000 claims description 3
- 210000004185 liver Anatomy 0.000 claims description 3
- 210000004072 lung Anatomy 0.000 claims description 3
- 239000012528 membrane Substances 0.000 claims description 3
- 230000001717 pathogenic effect Effects 0.000 claims description 3
- 238000000053 physical method Methods 0.000 claims description 3
- 238000000108 ultra-filtration Methods 0.000 claims description 3
- 238000005199 ultracentrifugation Methods 0.000 claims description 3
- 238000005516 engineering process Methods 0.000 claims description 2
- 244000052769 pathogen Species 0.000 claims description 2
- 238000006073 displacement reaction Methods 0.000 abstract description 4
- 230000000694 effects Effects 0.000 abstract description 4
- 239000000126 substance Substances 0.000 abstract description 2
- 238000001514 detection method Methods 0.000 description 2
- 108020004414 DNA Proteins 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000002068 genetic effect Effects 0.000 description 1
- 230000002503 metabolic effect Effects 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
- 230000003362 replicative effect Effects 0.000 description 1
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- C—CHEMISTRY; METALLURGY
- C40—COMBINATORIAL TECHNOLOGY
- C40B—COMBINATORIAL CHEMISTRY; LIBRARIES, e.g. CHEMICAL LIBRARIES
- C40B50/00—Methods of creating libraries, e.g. combinatorial synthesis
- C40B50/06—Biochemical methods, e.g. using enzymes or whole viable microorganisms
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- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Medicinal Chemistry (AREA)
- Molecular Biology (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention discloses a method for constructing a library of virus nucleic acid, which comprises the following steps: sample treatment, wherein clinical samples are complex and diverse, contain a large amount of host genes and are also mixed with other microorganisms such as bacteria and the like, measures are necessary to reduce the interference of irrelevant sequences and further enrich viruses, sample type liquid samples comprise blood, serum, cerebrospinal fluid, liquid, semen and other pathogenic bacteria under normal conditions, the content of fresh sample host gDNA, rRNA and other background nucleic acids is low, and the sample can be directly used for library construction and sequencing; and (4) removing substances. According to the invention, viral nucleic acid is amplified by a multiple displacement amplification method, the multiple displacement amplification operation is simple, the requirement on experimental instruments is low, and a good effect can be obtained when a non-pre-denatured template is used, so that the problems of low efficiency and high cost in the existing virus nucleic acid library building are solved.
Description
Technical Field
The invention relates to the technical field of viral nucleic acid, in particular to a method for constructing a library of viral nucleic acid.
Background
A virus is a noncellular organism that is small in size, simple in structure, contains only one nucleic acid (DNA or RNA), and must be parasitic in living cells and proliferated in a replicative manner. The virus is a non-cell life form, which is composed of a long nucleic acid chain and a protein shell, and has no own metabolic mechanism and no enzyme system. Therefore, the virus leaves the host cell and becomes a chemical substance which does not have any vital activity and can not independently propagate. Its ability to replicate, transcribe, and translate is performed in the host cell, and when it enters the host cell, it can use the materials and energy in the cell to perform life activities, generating a new generation of virus as it does according to the genetic information contained in its own nucleic acid.
The existing virus library is not sound because the virus is easy to generate variation, new virus can be found every moment, and the existing virus has high cost and low detection efficiency in nucleic acid detection.
Disclosure of Invention
Based on the technical problems in the background art, the invention provides a method for constructing a virus nucleic acid library.
The invention provides a method for constructing a virus nucleic acid library, which comprises the following steps:
s1: sample treatment, wherein clinical samples are complex and diverse, contain a large amount of host genes and are also mixed with other microorganisms such as bacteria and the like, measures are necessary to reduce the interference of irrelevant sequences and further enrich viruses, sample type liquid samples comprise blood, serum, cerebrospinal fluid, liquid, semen and other pathogenic bacteria under normal conditions, the content of fresh sample host gDNA, rRNA and other background nucleic acids is low, and the sample can be directly used for library construction and sequencing;
s2: the material is removed, and because the virus particles are far smaller than bacteria and other cells, the large-particle materials such as cells, cell debris, bacteria and the like in the sample can be removed by physical methods such as centrifugation, filter filtration and the like;
s3: nucleic acid extraction, wherein virus particles with certain density can be subjected to ultracentrifugation to improve the concentration and purity of viruses and then extract virus nucleic acid, and a concentration membrane or an ultrafiltration tube with the molecular weight cutoff of 100kD is adopted to concentrate the sample;
s4: nucleic acid amplification, namely combining random 6-base primers with a template chain at multiple sites, and then realizing amplification of a whole genome by using strong template combining and replacing capacity of phi29DNA polymerase, wherein the amplification method does not depend on nucleic acid sequence information of viruses and can efficiently amplify low-concentration samples, so that a clinical sample can be subjected to high-throughput sequencing under the condition that the nucleic acid information of the viruses is unknown;
s5: and (3) nucleic acid analysis, wherein the nucleic acid concentration, purity and quality of each sample are analyzed by a bioanalyzer, and viruses are sequenced and compared by respectively utilizing a random primer library building method and an oligo (dT) magnetic bead capture library building method.
Preferably, in S1, background nucleic acids such as gDNA and rRNA of the host need to be removed before sequencing due to the interference of a large amount of host nucleic acids in tissue samples such as heart, liver and lung; swab samples including throat swabs, nose swabs, anus swabs and the like have pathogenic bacteria, a large amount of normal parasitic flora and complex nucleic acid background.
Preferably, in said S2, the nuclease is added to the sample to degrade not only the background nucleic acids of the host but also the free pathogen nucleic acids. Since most viral nucleic acids are typically encapsidated and present within the viral particle to be resistant to nuclease action ", free host gDNA can be degraded by DNase I and free host rRNA can be degraded by the addition of RNaseA.
Preferably, in S4, the nucleic acid amplification protocol includes sequence-independent single primer amplification, VIDISCA technique, and random PCR. .
The beneficial effects of the invention are as follows:
the virus nucleic acid library building method amplifies virus nucleic acid by a multiple displacement amplification method, has simple multiple displacement amplification operation and low requirement on experimental instruments, can obtain good effect when a non-pre-denatured template is used, and solves the problems of low efficiency and high cost in the existing virus nucleic acid library building.
Drawings
FIG. 1 is a flow chart of a method for library construction of viral nucleic acids according to the present invention.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments.
Referring to FIG. 1, a method for library construction of viral nucleic acid comprising the steps of:
s1: sample treatment, wherein clinical samples are complex and diverse, contain a large amount of host genes and are also mixed with other microorganisms such as bacteria and the like, measures are necessary to reduce the interference of irrelevant sequences and further enrich viruses, sample type liquid samples comprise blood, serum, cerebrospinal fluid, liquid, semen and other pathogenic bacteria under normal conditions, the content of fresh sample host gDNA, rRNA and other background nucleic acids is low, and the sample can be directly used for library construction and sequencing;
s2: the material is removed, and because the virus particles are far smaller than bacteria and other cells, the large-particle materials such as cells, cell debris, bacteria and the like in the sample can be removed by physical methods such as centrifugation, filter filtration and the like;
s3: nucleic acid extraction, wherein virus particles with certain density can be subjected to ultracentrifugation to improve the concentration and purity of viruses and then extract virus nucleic acid, and a concentration membrane or an ultrafiltration tube with the molecular weight cutoff of 100kD is adopted to concentrate the sample;
s4: nucleic acid amplification, namely combining random 6-base primers with a template chain at multiple sites, and then realizing amplification of a whole genome by using strong template combining and replacing capacity of phi29DNA polymerase, wherein the amplification method does not depend on nucleic acid sequence information of viruses and can efficiently amplify low-concentration samples, so that a clinical sample can be subjected to high-throughput sequencing under the condition that the nucleic acid information of the viruses is unknown;
s5: and (3) nucleic acid analysis, wherein the nucleic acid concentration, purity and quality of each sample are analyzed by a bioanalyzer, and viruses are sequenced and compared by respectively utilizing a random primer library building method and an oligo (dT) magnetic bead capture library building method. .
In the invention, in S1, background nucleic acids such as gDNA and rRNA of a host need to be removed before sequencing on tissue samples including heart, liver, lung and other tissues due to the interference of a large amount of host nucleic acids; swab samples including throat swabs, nose swabs, anus swabs and the like have pathogenic bacteria and also have a large number of normal parasitic flora, the background of nucleic acid is complex, and in S2, nuclease is added into the samples, so that the nuclease can degrade background nucleic acid of a host and free pathogenic nucleic acid. Since most viral nucleic acids are typically encapsidated and present within the viral particle to be resistant to nuclease action ", free host gDNA can be degraded by DNase I, free host rRNA can be degraded by the addition of RNaseA, S4, and nucleic acid amplification protocols include sequence-independent single primer amplification, VIDISCA technology and random PCR.
The above description is only for the preferred embodiment of the present invention, but the scope of the present invention is not limited thereto, and any person skilled in the art should be considered to be within the technical scope of the present invention, and the technical solutions and the inventive concepts thereof according to the present invention should be equivalent or changed within the scope of the present invention.
Claims (4)
1. A method for banking viral nucleic acid comprising the steps of:
s1: sample treatment, wherein clinical samples are complex and diverse, contain a large amount of host genes and are also mixed with other microorganisms such as bacteria and the like, measures are necessary to reduce the interference of irrelevant sequences and further enrich viruses, sample type liquid samples comprise blood, serum, cerebrospinal fluid, liquid, semen and other pathogenic bacteria under normal conditions, the content of fresh sample host gDNA, rRNA and other background nucleic acids is low, and the sample can be directly used for library construction and sequencing;
s2: the material is removed, and because the virus particles are far smaller than bacteria and other cells, the large-particle materials such as cells, cell debris, bacteria and the like in the sample can be removed by physical methods such as centrifugation, filter filtration and the like;
s3: nucleic acid extraction, wherein virus particles with certain density can be subjected to ultracentrifugation to improve the concentration and purity of viruses and then extract virus nucleic acid, and a concentration membrane or an ultrafiltration tube with the molecular weight cutoff of 100kD is adopted to concentrate the sample;
s4: nucleic acid amplification, namely combining random 6-base primers with a template chain at multiple sites, and then realizing amplification of a whole genome by using strong template combining and replacing capacity of phi29DNA polymerase, wherein the amplification method does not depend on nucleic acid sequence information of viruses and can efficiently amplify low-concentration samples, so that a clinical sample can be subjected to high-throughput sequencing under the condition that the nucleic acid information of the viruses is unknown;
s5: and (3) nucleic acid analysis, wherein the nucleic acid concentration, purity and quality of each sample are analyzed by a bioanalyzer, and viruses are sequenced and compared by respectively utilizing a random primer library building method and an oligo (dT) magnetic bead capture library building method.
2. The method for creating a library of viral nucleic acids according to claim 1, wherein in S1, background nucleic acids such as gDNA and rRNA of hosts need to be removed before sequencing due to the interference of nucleic acids of a large number of hosts in tissue samples such as heart, liver and lung; swab samples including throat swabs, nose swabs, anus swabs and the like have pathogenic bacteria, a large amount of normal parasitic flora and complex nucleic acid background.
3. The method of claim 1, wherein the step of adding nuclease to the sample degrades not only background host nucleic acids but also free pathogen nucleic acids. Since most viral nucleic acids are typically encapsidated and present within the viral particle to be resistant to nuclease action ", free host gDNA can be degraded by DNase I and free host rRNA can be degraded by the addition of RNaseA.
4. The method of claim 1, wherein in step S4, the nucleic acid amplification protocol comprises sequence-independent single primer amplification, VIDISCA technology and random PCR.
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| CN202010124210.7A CN111334867A (en) | 2020-02-27 | 2020-02-27 | Method for constructing virus nucleic acid library |
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| CN202010124210.7A CN111334867A (en) | 2020-02-27 | 2020-02-27 | Method for constructing virus nucleic acid library |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN116426696A (en) * | 2023-06-14 | 2023-07-14 | 北京大学人民医院 | Plasma virus detection and analysis method based on sequencing technology |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2000063440A2 (en) * | 1999-04-20 | 2000-10-26 | The Government Of The United States Of America, As Represented By The Secretary, Department Of Health & Human Services | Diagnostic standards for virus detection and quantification |
| US20040132132A1 (en) * | 2001-02-22 | 2004-07-08 | Ugur Sahin | Method for identifying biologically active structures of microbial pathogens |
| CN105112569A (en) * | 2015-09-14 | 2015-12-02 | 中国医学科学院病原生物学研究所 | Virus infection detection and identification method based on metagenomics |
| CN110093455A (en) * | 2019-04-27 | 2019-08-06 | 中国医学科学院病原生物学研究所 | A kind of detection method of Respirovirus |
-
2020
- 2020-02-27 CN CN202010124210.7A patent/CN111334867A/en active Pending
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2000063440A2 (en) * | 1999-04-20 | 2000-10-26 | The Government Of The United States Of America, As Represented By The Secretary, Department Of Health & Human Services | Diagnostic standards for virus detection and quantification |
| US20040132132A1 (en) * | 2001-02-22 | 2004-07-08 | Ugur Sahin | Method for identifying biologically active structures of microbial pathogens |
| CN105112569A (en) * | 2015-09-14 | 2015-12-02 | 中国医学科学院病原生物学研究所 | Virus infection detection and identification method based on metagenomics |
| CN110093455A (en) * | 2019-04-27 | 2019-08-06 | 中国医学科学院病原生物学研究所 | A kind of detection method of Respirovirus |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN116426696A (en) * | 2023-06-14 | 2023-07-14 | 北京大学人民医院 | Plasma virus detection and analysis method based on sequencing technology |
| CN116426696B (en) * | 2023-06-14 | 2024-01-26 | 北京大学人民医院 | Plasma virus detection and analysis method based on sequencing technology |
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