CN110468187A - The method and kit be enriched with before high-flux sequence to the pathogenic microorganism infected in host - Google Patents
The method and kit be enriched with before high-flux sequence to the pathogenic microorganism infected in host Download PDFInfo
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- CN110468187A CN110468187A CN201910652240.2A CN201910652240A CN110468187A CN 110468187 A CN110468187 A CN 110468187A CN 201910652240 A CN201910652240 A CN 201910652240A CN 110468187 A CN110468187 A CN 110468187A
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Abstract
The present invention provides the method that the pathogenic microorganism infected in a kind of couple of host is enriched with before high-flux sequence and the kit using this method, the method includes first sufficiently being combined host cell with immunomagnetic beads, then the immunomagnetic beads for being combined with host cell are separated from sample to be tested, remaining sample to be tested is finally carried out to extraction and the high-flux sequence of nucleic acid.The method of the present invention combines the host cell achieved that in expeditiously removal human or animal infection sample simply by simple antigen-antibody, so that the content ratio of the pathogenic microorganism in infection sample is improved.
Description
Technical field
The present invention relates to high-flux sequence samples to be enriched with field, in particular to the pathogenic microorganism infected in host in height
The method and kit being enriched with before flux sequencing detection.
Background technique
Nearly ten years in Molecular Detection field, high throughput sequencing technologies development is swift and violent, can once arrive to millions of
1000000000 DNA moleculars are sequenced parallel, this make its can transcript profile to species and genome carry out it is deep, careful,
The analysis of overall picture, so high-flux sequence is that scientific research is made that tremendous contribution, such as the sequencing of species gene group/transcript profile and
Identification, research of apparent gene group etc..The unique advantage of high throughput sequencing technologies not only advances the progress of scientific research, and
And very big application value is also shown in clinical medicine domain.Deliver an article within 2014 " New England Journal of Medicine ":
It is hospitalized to one and does not make a definite diagnosis within 6 weeks 14 years old boy of the cause of disease, by carrying out the high-flux sequence of CSF sample, be finally diagnosed as hook
Leptospiral infection, then suits the remedy to the case, boy is discharged from hospital after recovery soon.Difficult and complicated cases are carried out in clinical infection field from this
Diagnosis when, high throughput sequencing technologies, which start to obtain, to be paid close attention to and applies widely.
However when the pathogenic microorganism that low amounts in people or animal specimen infects is sequenced, it is faced with such a ask
Topic: since infection sample itself contains the genome of a large amount of human or animal hosts itself, and the carrying capacity of pathogenic microorganism is very
Few, so needing to carry out infection sample the sequencing of superelevation depth, the pathogenic microorganism of low carrying capacity infected could be obtained
Information.Superelevation deep sequencing itself, will lead to that sequencing data amount is big, sequencing cost is high, and even deposit to the analysis of data
Storage all brings very big inconvenience.This problem again limits application of the high-flux sequence in clinical infection diagnosis in turn.So
When the microorganism infected in human or animal's sample is sequenced, need to be enriched with the microorganism in sample, to remove host
The influence of genome is very crucial.
The technology for reaching nucleic acid of pathogenic microorganism enrichment by capturing and removing host genome at present, there is DNA methylation
Technology, differential lysis discharge the technology etc. of different nucleic acid.
DNA methylation technical principle is according to most of 5 '-methylcystein in people or other mammals
(5mC) occurs in the form of dinucleotide CpG, and distribution of the CpG dinucleotide in human genome is very uneven, wherein density
Higher region is known as the island CpG, and the gene of mankind 50%-60% has the island CpG, and be nearly always located at gene promoter and
(or) around exon, the CpGs on the island CpG is always non-methylation under normal circumstances.Methyl is carried out by the CpGs to non-first
Change processing, then realizes the separation of methylated genes and non-methylated genes with multiple technologies means, so that realizing will be in sample
The purpose that is separated with pathogenic microorganism genome of most of host genome, to be enriched with the nucleic acid of pathogenic microorganism.
The method is only suitable for the enrichment of the pathogenic microorganism of most of this type of bacterium, and the pathogenic microorganism of this viral type because
The duplication of itself is carried out using the raw material of host for it, so also there is similar CpGs phenomenon in its gene, meeting when DNA methylation
The portion gene group of virus is also methylated and is separated, missing inspection is be easy to cause.So DNA methylation beneficiation technologies are to infection sample
Do not have universality when host genome in this is removed.
Differential lysis discharges the technology of different nucleic acid, and the shell by people or animal cell membrane and pathogenic microorganism is (thin
Cell wall perhaps protein coat) component it is different using the lysate of specific cleavage people or zooblast by people or animal
Genome releases, and is then fallen with the absorption of silicon substrate magnetic bead or falls the genomic DNA of people or animal with enzymic digestion, then made
The silicon substrate magnetic bead of absorption someone or Animal genome DNA are separated from sample, finally extract the nucleic acid of pathogenic microorganism,
To achieve the purpose that be enriched with nucleic acid of pathogenic microorganism.But because of lysate it is difficult to ensure that not cracking pathogenic microorganism completely, institute
It will cause the loss of nucleic acid of pathogenic microorganism in this approach, and then also will affect subsequent Molecular Detection quality.
Summary of the invention
To solve the above problems, the present invention provides the pathogenic microorganisms infected in a kind of couple of host before high-flux sequence
Then the method being enriched with will be combined with host the method includes first sufficiently being combined host cell with immunomagnetic beads
The immunomagnetic beads of cell are separated from sample to be tested, and remaining sample to be tested is finally carried out to the extraction and high throughput of nucleic acid
Sequencing.
In one embodiment, the antibody of specifical and efficient combination host cell surface antigen is selected, it then will be described anti-
Body forms immunomagnetic beads together with magnetic bead direct coated, and the immunomagnetic beads are added in sample to be measured and are incubated for,
Combine the host cell in sample to be tested sufficiently with immunomagnetic beads；Or be first incubated for the antibody with sample to be tested,
It combines the host cell in sample to be tested sufficiently with antibody, magnetic bead incubation is then added, so that being combined with host cell
Antibody is sufficiently combined with magnetic bead.
In one embodiment, the antibody of the specifical and efficient combination host cell surface antigen is specific bond host
The antibody for the antigen that all cell surfaces all have.
In one embodiment, the antibody of the specifical and efficient combination host cell surface antigen is specific bond host
Main Types cell surface antigen is anti-in the antibody for the particular category cell surface antigen having, or combination host's sample
Body.
In one embodiment, the antibody of the magnetic bead and specifical and efficient combination host cell surface antigen is before combination
It need to be modified, moditied processing mode is one of the two receptor that coupling can be combined with each other respectively and aglucon.
In one embodiment, the host is human or animal.
In one embodiment, the antibody of specific bond human archeocyte, it is therefore preferable to specific bond human leukocyte table
The antibody for the HLA- class Ⅰ antigens that the antibody of face antigen and specific bond source of people karyocyte surface all have.
In one embodiment, the sample to be tested is blood, Pleural effusions, respiratory tract throat swab or cerebrospinal fluid.
In one embodiment, the present invention provides the kit used in the above-mentioned methods, the kit includes
The immunomagnetic beads being combined with source of people host cell.
In one embodiment, the immunomagnetic beads are the immune magnetic of specific bond human leukocyte surface C D45 antigen
Pearl and specific bond source of people karyocyte surface HLA-A B C antigen immunomagnetic beads mixed immunity magnetic bead
High efficiency is achieved that the advantages and positive effects of the present invention are: combining simply by simple antigen-antibody
Ground removes the host cell in human or animal's infection sample, so that the content ratio of the pathogenic microorganism in infection sample is mentioned
It is high.In high-flux sequence, the sample after pathogenic microorganism enrichment is compared with the sample before enrichment, in the total Reads of sequencing data
In the identical situation of number, the Reads number of the pathogenic microorganism infected can obtain the promotion of hundred times of grades.The foundation of this method is advantageous
The certain infectious diseases of pathogenic microorganism or monitoring in some low infective dose samples of Yu doctor's Accurate Diagnosis are over the course for the treatment of
The variation of pathogenic microorganism quantity.This method is easy to operate and low in cost, is a kind of cost-effectively pathogenic microorganism enrichment
Mode, convenient for promoting the use of.
Specific embodiment
In order to make those skilled in the art more fully understand the technical solution in the application, below in conjunction with embodiment to this
Invention is described further, it is clear that described embodiments are only a part of embodiments of the present application, rather than whole implementation
Example.Based on the embodiment in the application, obtained by those of ordinary skill in the art without making creative efforts
All other embodiment, shall fall within the protection scope of the present application.
It is conventional method in that art unless otherwise specified in following embodiments.The human antibody of marked by streptavidin
For oneself preparation, the magnetic bead of biotin labeling is purchased from Thermo Fisher company, the U.S., and fluorescent PCR amplifing reagent is purchased from Shenzhen
Fei Peng Biological Co., Ltd., the primer and probe are synthesized by Shanghai biotechnology Services Co., Ltd.Detection
The total DNA and total RNA extraction reagent box of sample are purchased from Qiagen company, Germany.High-flux sequence is in illumina
It is carried out on Nextseq500 sequenator.
Embodiment carries out high-flux sequence after being enriched with using the method for the present invention to source of people host infection sample
1. the enrichment of pathogenic microorganism
Screening can be with the CD45 antibody of specific bond human leukocyte and can be with all karyocytes of specific bond source of people
Capture antibody of the HLA-ABC antibody of surface antigen as source of people host cell, and each personal strepto- of the antibody filtered out is close
It is coated with, is formed containing there are two types of the mixed immunity magnetic beads of antibody with the magnetic bead of biotin labeling again with after element label.
The source of people anticoagulation cirumferential blood sample for drawing 200 μ L dengue virus infections is mixed with 800 μ L PBS buffer solution, uses liquid relief
The two is blown and beaten and is mixed by rifle；It draws 200 μ L internal reference quality-control products to mix with 800 μ L PBS buffer solution, is blown and beaten the two with liquid-transfering gun
It mixes.Into above two mixing liquid, it is separately added into the mixed immunity magnetic bead of 50 μ L, and is covered in respective pipe and indicates " sample
Originally ", " internal reference ".
1. sample tube and internal reference pipe, which are placed in 4 DEG C of rotations, is incubated for 1h；2. being distinguished at room temperature with magnetic frame after the completion of being incubated for
The immunomagnetic beads in sample tube and internal reference pipe are adsorbed, the remaining liq of sample and internal reference quality-control product is sucked out respectively with liquid-transfering gun to one
Clean EP pipe, then enrichment sample, enrichment internal reference are indicated respectively.
2. the internal reference quality-control product and sample of pair enrichment front and back carry out the extraction of nucleic acid respectively
1) Genome DNA extraction is carried out respectively with 200 μ L internal reference Quality Controls not being enriched with to the liquid in enrichment internal reference pipe.
2) peripheral blood sample of liquid and the 200 μ L Dengues infection not being enriched in enrichment sample tube is carried out respectively
Total RNAs extraction.
3. fluorescent PCR is verified
1) utilize quantitative fluorescent PCR, using source of people β-actin gene DNA detection architecture respectively to after enrichment with enrichment
The nucleic acid that preceding internal reference quality-control product extracts is detected；Using Escherichia coli ITS gene DNA detection architecture respectively to enrichment
The nucleic acid proposed afterwards with the internal reference quality-control product before enrichment is detected.
Testing result is as shown in the table, the amplification Ct value and enrichment of the source of people β-actin gene after the enrichment of internal reference quality-control product
Preceding amplification Ct value difference is 4.37, that is, human source gene group is 1/2 before enrichment in the internal reference quality-control product after being enriched with4.37, thus
The amount of human source gene group reduces 93.75% in internal reference quality-control product after enrichment, and the immunomagnetic beads in this visualizingre agent box are that have
Effect.The amplification Ct value of the ITS gene of Escherichia coli after the enrichment of internal reference quality-control product is differed with the amplification Ct value before enrichment
0.11, that is, genome of E.coli is 1/2 before enrichment in internal reference quality-control product after being enriched with0.11, thus the internal reference Quality Control after enrichment
The amount of the genome of Escherichia coli only reduces 7.41% in product.Escherichia coli (simulation pathogenic microorganism) genome reduction amount with
The reduction amount of host genome is compared, and the genome total amount of Escherichia coli is barely affected, and illustrates that the method in the present invention exists
By removal host cell come when being enriched with pathogenic microorganism, pathogenic microorganism itself will not be lost.
| Internal reference state | Source of people β-actin gene C t | Escherichia coli ITS gene C t |
| Before enrichment | 21.62 | 21.04 |
| After enrichment | 25.99 | 21.15 |
2) utilize quantitative fluorescent PCR, using source of people β-actin gene RNA detection architecture respectively to after enrichment with enrichment
The nucleic acid that preceding sample proposes is detected.
Detection display, the amplification Ct value of the source of people β-actin gene of sample is 24.27 after enrichment, the people of sample before being enriched with
The amplification Ct value of source β-actin gene is 20.38, and the difference of the two is 3.89, that is, human source gene group is in the sample after being enriched with
1/2 before enrichment3.89, so that the amount of human source gene group reduces 93.26% in sample after enrichment, reached removal source of people place
Purpose that is main and improving pathogen content ratio in the sample.
4. high-flux sequence
Requirement by high-flux sequence to Nucleic acid quality and total amount is ready to after being enriched with propose with the sample before enrichment total
RNA carries out building library respectively according to the Library development flow of standard library, high-flux sequence is then carried out, to sequencing after meeting the requirements
As a result it is briefly summarized as follows shown in table:
It can be seen that from upper table, the survey in the total Reads number of same sequencing data, after the enrichment of dengue virus infection sample
The Reads number of ordinal number dengue virus obtained in is 285 times before enrichment, and pathogen Reads number has obtained mentioning for hundred times of grades
It rises；Accounting of the Reads number of dengue virus obtained in the sequencing data of front and back in total data is enriched with from every ten thousand Reads
Improving less than one has ten or so into every ten thousand Reads, it can be seen that this method is micro- to actual clinical sample progress cause of disease
Good concentration effect can be obtained when biological concentration.
It should be understood that the present invention disclosed is not limited only to specific method, scheme and the substance of description, because these
It is alterable.It will also be understood that purpose of the terminology used here just for the sake of the specific embodiment scheme of description, rather than
It is intended to limit the scope of the invention, the scope of the present invention is limited solely by the attached claims.
Those skilled in the art, which will also be appreciated that or be able to confirm that, uses no more than routine experiment, institute herein
The many equivalents for the specific embodiment of the invention stated.These equivalents are also contained in the attached claims.
Claims (10)
1. the method that the pathogenic microorganism infected in a kind of couple of host is enriched with before high-flux sequence, which is characterized in that institute
Stating method includes first sufficiently being combined host cell with immunomagnetic beads, then will be combined with the immunomagnetic beads of host cell to
It is separated in test sample sheet, remaining sample to be tested is finally carried out to extraction and the high-flux sequence of nucleic acid.
2. the method according to claim 1, wherein selecting the anti-of specifical and efficient combination host cell surface antigen
Then the antibody is formed immunomagnetic beads together with magnetic bead direct coated by body, the immunomagnetic beads are added to be measured
It is incubated in sample, combines the host cell in sample to be tested sufficiently with immunomagnetic beads；Or by the antibody first with to
Test sample is originally incubated for, and combines the host cell in sample to be tested sufficiently with antibody, then adds magnetic bead incubation, so that knot
Conjunction has the antibody of host cell sufficiently to be combined with magnetic bead.
3. according to the method described in claim 2, it is characterized in that, the specifical and efficient combination host cell surface antigen resists
Body is the antibody for the antigen that all cell surfaces of specific bond host all have.
4. according to the method described in claim 2, it is characterized in that, the specifical and efficient combination host cell surface antigen resists
Body is the antibody for the particular category cell surface antigen that specific bond host has, or combines main Types in host's sample thin
The antibody of cellular surface antigen.
5. according to the method described in claim 2, it is characterized in that, the magnetic bead and specifical and efficient combination host cell surface are anti-
Both former antibody needs to be modified before combination, and moditied processing mode the is receptor that coupling can be combined with each other respectively with
One of aglucon.
6. -5 any method arrived according to claim 1, which is characterized in that the host is human or animal.
7. according to the method described in claim 6, it is characterized in that, the antibody of specific bond human archeocyte, it is therefore preferable to special
The HLA- class Ⅰ antigens all having in conjunction with the antibody of human leukocyte surface antigen and specific bond source of people karyocyte surface it is anti-
Body.
8. -5 any method arrived according to claim 1, which is characterized in that the sample to be tested is blood, Pleural effusions, exhales
Inhale road throat swab or cerebrospinal fluid.
9. the kit used in any one of claim 1-8 the method, which is characterized in that the kit include with
The immunomagnetic beads that source of people host cell is combined.
10. kit according to claim 9, which is characterized in that the immunomagnetic beads are specific bond human leukocyte
The immunomagnetic beads and specific bond source of people karyocyte surface HLA-A of surface C D45 antigen B C antigen immunomagnetic beads mixing
Immunomagnetic beads.
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Cited By (1)
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| CN113249436A (en) * | 2020-02-12 | 2021-08-13 | 广州微远基因科技有限公司 | Method and application for reducing host nucleic acid in biological sample |
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