CN109082469A - The infected's peripheral blood detection method of Integration in hepatitis B liver - Google Patents
The infected's peripheral blood detection method of Integration in hepatitis B liver Download PDFInfo
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Abstract
The present invention provides the detection method of hepatitis B Integration, this method is detected using the dissociative DNA in peripheral blood as sample.The method can be avoided the complication risk of the conventional methods such as open surgical biopsy, aspiration biopsy, improves safety, the numerous periodic monitoring of higher frequency may be implemented, and avoid the heterogeneous sexual deviation of sample of tissue.Method specificity of the invention is good, high sensitivity, can detect in the peripheral blood sample of liver cancer patient after 100% HBV infection, minimum to detect segment in whole cfDNA that 5ml peripheral blood blood plasma extracts.
Description
Technical field
The present invention relates to the detection of hepatitis B Integration, in particular to peripheral blood dissociative DNA is integrated in hepatitis B
Application in condition detection.
Background technique
Hepatitis B (Hepatitis B Virus, HBV) is used as retrovirus, has and is integrated into host genome
Ability is most just reported early in the eighties early stage in 20th century, and the research in hepatocarcinoma gene group also has many accumulation1-13。HBV
Integration ability be not its history of life (Life Cycle) and infection host institute it is necessary, do not recognize at present in liver virus gene group
For there are gene codings the active virus protein of integrase14, it is in full-length genome that the research of early stage, which is thought that HBV integrates event,
The rare events of random distribution15.The research of HBV integrated mechanism thinks that principal mode when viral integrase enters host gene is line
Property double-stranded DNA (double strand linear DNA, dsL DNA)16, it is the intermediate in cccDNA forming process, and
CccDNA is a ring of most critical in HBV infection chronicity17, thus infer that integration event can be along with HBV chronic infection
Overall process.Although researcher continuously attempt to building can be used for cell, the animal model that the HBV history of life is studied, always with
Come that there has been no mature systems18, duck hepatitis B virus (DHBV) is based on to the relevant biological process research of cccDNA, including mesh
The 10 of preceding estimation3~104It can occur once to integrate in the cell of a infection and be also based on DHBV infection experiment16.Earliest researcher
It is thought that the viral fragment being integrated into host genome is in inactivation (Defective) state and can not generate albumen, it is subsequent to grind
Studying carefully the prompt highest HBV gene group segment of integrating frequency is the region DR2-DR1 and preS/S gene region19, can translate respectively
Generate X protein, the surface protein of truncated-type20, and liver cell is induced by respective specific mechanism and is changed to tumour cell21,22, because
And such phenomenon is recognized as trans- (Trans) effect for belonging to integration event in liver disease progression.Current research report,
The segment of integration and neighbouring gene can produce chimeric transcription sheet (Chimeric Transcripts), such as HBV-MLL423、HBV-
cyclin A224；Chiu YT et al. proposes that allowing to translate can not be by host there are pre-mRNA splicing mechanism
The virus of cell degradation-people's chimeric protein, causes cell cycle progression to be accelerated24.It is in recent years even more to think to integrate in chronic infection
Viral dna fragment also will continue in people's liver cell of involvement a series of cells of protein influence that translation generates virus
The immune response of functional activity and host25,26。
Other than there is certain heterogeneous (Heterogeneity) in HBV integration, between different the infecteds in same patient's body
It can also inside observe multiple integration event.2012, the WGS result of Sung WK et al. liver cancer after to 81 HBV infections
The middle nearly 400 integration event of discovery, at most there are 16 different HBV integration in single tumour, wherein young liver cancer patient tumour
More viral integrases can be observed in tissue19.In addition, the research of Yan H in 2015 et al. is it has also been found that after the virus infection
There are difference in integration mode and evening hair liver cancer (>70 years old) patient in early hair liver cancer (<30 years old) patient, in the former liver neoplasm
The viral integrase event that may more influence liver cell is had found in tissue27.Thus, above-mentioned discovery also supports that HBV integration may
Assign the viewpoint of hepatic cell growth advantage, the process that integration can accelerate hepatic disease to develop.Meanwhile in the prevalent clone of tumour
HBV can be analyzed by tumour sequencing data of whole genome based on this Miao R et al. and be incorporated into containing same specific HBV integration
Difference in 2 livers in multiple tumour, successfully identifies the Clonal Origin of tumour: having more tumor focus of identical HBV integration
Belong to monoclonality, it is then polyclonal origin that HBV, which integrates event difference, and the two there can be different clinical prognosis28.2008
Stream survey data show, Chinese there are about 93,000,000 people of Chronic HBV carriers, wherein 19,000,000 people of chronic hepatitis B patient, though
The extensive use of right hepatitis B vaccine so that infection has obtained very effective control, but chronic infection crowd still have it is sizable
Radix, the assessment of hepatic disease development and the early warning of liver cancer are always the emphasis in viral hepatitis treatment.
Integration viral fragment analysis in last century the eighties liver cancer is mostly miscellaneous based on restricted digestion or Southern
Friendship technology, the target fragment of acquisition are sequenced progress subsequent authentication by a generation and determine1-9.Experiment flow is cumbersome, analysis throughput is low,
Thus can not from the overall situation analytical integration event biological impact.Nineteen ninety-five, Minami et al. develop ALU PCR strategy and exist
The flux that screening integration site analyzes viral integrase in genome is promoted29,30.However the premise of the strategy is virus
Integration needs to occur near ALU segment, and this hypotheses destroy the unbiasedness of analysis, are not at ALU segment area
Integration can not be captured by the technology.In addition, round pcr is in the amplification of segment, meeting is because of its intrinsic Template
The problem of Switching cause false positive as a result, liver cancer group genome and f-v-DNA are connected to one in amplification
It rises31,32。
In recent years with the extensive use of high throughput sequencing technologies, researcher carries out full genome in a large amount of liver cancer samples
Group sequencing (Whole Genome Sequencing, WGS) or the screening based on Alu-PCR to integration site19, wherein
Li X in 2014 et al. is by assembling reported 1115 integration events, to occurrence frequency of the HBV in hepatocarcinoma gene group, whole
Close rule have deeper into portray33.It is now recognized that integration event mostly occurs in the region of the transcriptionally active of chromosome, it may
The expression of gene is closed on by the influence of cis- (Cis) regulating and controlling effect17.Although the variation of this expression activity is in tumor development
In effect still need to further investigated, but there are high-frequency integration target genes by the common report of multiple researchs, and be related to cell more
The biological processes such as survival, proliferation and immortalization, such as telomerase reverse transcriptase gene (TERT), histone methyltransferase
(MLL4) and Cyclin E1 (CCNE1) etc.19,28,34.In addition, there is also typical sequence spies in the region of integration generation
Sign, it is easier to appear in the region of genomic instability such as repetitive sequence, long interspersed repeated sequences, short interspersed repeated sequence, micro-
Satellite, telomere, centriolar region35,36Or even there are the structure variations of genome for integration site itself37, some chromosomes (5,
16,17, No. 19) it is determined that HBV viral integrase more easily occurs38-43.However the cost of full genome sequencing technologies is still very high
It is expensive, particular, it is important that the integration event of virus can be found after 80-90%HBV infection in liver cancer tissue, thus it sends out in liver cancer
Raw developing effect can not be ignored44。
Liquid biopsy (Liquid biopsy) technology rapidly develops in recent years, and dissociative DNA (cell is detected in peripheral blood
Free DNA, cfDNA) become the important means for monitoring patient disease disease progression45.Hepar damnification degree caused by HBV infection
When different, there are difference for cfDNA level in peripheral blood in patients, such as after HBV infection in acute hepatic failure patient, cfDNA water
Flat raising can help to judge the prognosis of patient46；It can be in addition to the variation of cfDNA quantity, on the cfDNA of some chronic carriers
The mutation for detecting TP53 gene prompts such patient's liver neoplasm occurrence risk that may improve47；Have trial and passes through monitoring
The methylation of HBV infection different phase blood plasma cfDNA changes, it is found that NF300, SLC22A20 and SHISA are isogenic in cfDNA
Methylation level has differences in slow liver, cirrhosis and liver cancer patient, as the mark of screening tumour High risk group
Object48.However, the information that situations such as detecting mutation and methylation using cfDNA can be provided is still extremely limited, it is necessary to explore
The means of more information amount are provided.Viral integrase is domestic at present as the characteristic event after HBV infection in hepatocytic genes group
There has been no the DNA fragmentations of research and probe integration site whether can be released into peripheral blood outside, and there are no research and probe, whether it has foot
Enough specificity and sensitivity are for testing and analyzing.
Summary of the invention
The present inventor has found that the DNA fragmentation of HBV integration site can be released into peripheral blood for the first time, and can
For testing and analyzing.It is an object of the present invention to provide it is a kind of detect subject hepatitis B Integration method,
It can be realized by non-intruding modes such as liquid biopsies, to avoid present in the tissue biopsy mode such as open surgical biopsy and aspiration biopsy
The defects of security risks and sampling difficulty, sample heterogeneity.
In order to solve the above-mentioned technical problems, the present invention provides a kind of sides of hepatitis B Integration for detecting subject
Method, this method are detected using the dissociative DNA in peripheral blood as sample.
Preferably, the hepatitis B that the hepatitis B Integration is subject's hepatic tissue or liver cell integrates shape
Condition integrates event by the hepatitis B in detection peripheral blood dissociative DNA, to reflect the second of subject's hepatic tissue or liver cell
Hepatovirus Integration；Preferably, the hepatitis B Integration is that the hepatitis B in hepatic tissue or liver cell integrates thing
Part spectrum.
Preferably, the method for the hepatitis B Integration of the detection subject includes the following steps: (1) from tested
Person obtains peripheral blood sample；(2) hepatitis B detected in peripheral blood dissociative DNA integrates event.
In some embodiments, the detection of hepatitis B integration event includes the following steps: in peripheral blood dissociative DNA
(a) peripheral blood dissociative DNA is extracted；
(b) end reparation is carried out to the peripheral blood dissociative DNA that step (a) is extracted, is connect with connector, purify connection product；
(c) optionally, the DNA obtained to step (b) is expanded in advance, purifies amplified production；
(d) DNA obtained using the probe that can hybridize with Hepatitis B virus-DNA sequence in step (b) or step (c)
It is captured in product；
(e) the capture product obtained to step (d) is sequenced；
(f) determine that hepatitis B integrates event by bioinformatic analysis.
In other embodiments, the detection of hepatitis B integration event includes the following steps: in peripheral blood dissociative DNA
(a) peripheral blood dissociative DNA is extracted；
(b) single-molecule sequencing or nano-pore sequencing are carried out to the peripheral blood dissociative DNA that step (a) is extracted；
(c) determine that hepatitis B integrates event by bioinformatic analysis.
It is another object of the present invention to provide a kind of sides of hepatitis B integration event in detection subject cfDNA
Method.In order to solve this technical problem, the invention provides the following technical scheme:
A method of hepatitis B integrates event in detection subject cfDNA, described to include the following steps:
(a) peripheral blood dissociative DNA is extracted；
(b) end reparation is carried out to the peripheral blood dissociative DNA that step (a) is extracted, is connect with connector, purify connection product；
(c) optionally, the DNA obtained to step (b) is expanded in advance, purifies amplified production；
(d) DNA obtained using the probe that can hybridize with Hepatitis B virus-DNA sequence in step (b) or step (c)
It is captured in product；
(e) the capture product obtained to step (d) is sequenced；
(f) determine that hepatitis B integrates event by bioinformatic analysis.
A method of hepatitis B integrates event in detection subject cfDNA, described to include the following steps:
(a) peripheral blood dissociative DNA is extracted；
(b) single-molecule sequencing or nano-pore sequencing are carried out to the peripheral blood dissociative DNA that step (a) is extracted；
(c) determine that hepatitis B integrates event by bioinformatic analysis.
Preferably, after extracting peripheral blood dissociative DNA, the segment distribution of dissociative DNA is measured, if segment in step (a)
Distribution does not meet the ideal range of 150-200bp, interrupts segment and complies with ideal range.
Preferably, it is that end is added in the peripheral blood dissociative DNA of extraction to repair that the end, which is repaired, in step (b)
Buffer and end, which are repaired, adds A enzymatic mixture, carries out end and repairs reaction.
Preferably, in step (b), it is described be connected to connector end repair connector is added in product and connect reagent into
Row connection reaction.
Preferably, the purifying connection product is pure to connection product progress using nucleic acid purification microballon in step (b)
Change.
Preferably, the pre- amplification is to carry out PCR amplification to the DNA that step (b) obtains in step (c)；Optionally,
It is dried after purifying extension product.
Preferably, the purifying amplified production is pure to amplified production progress using nucleic acid purification microballon in step (c)
Change；Optionally, it is added in amplified production after purification between capable of blocking or interfering joint sequence and matches the oligonucleotides of combination,
And/or it can be with the oligonucleotides in conjunction with the repetitive sequence on human genome.
Preferably, carrying out capture reaction on magnetic bead in step (d)；Preferably, the magnetic bead is the affine magnetic of streptomysin
Pearl.
Preferably, carrying out PCR amplification and purifying after capture in step (d).
Preferably, sequencing amount is 1G, 1.5G or 2G in step (e)；It is preferred that 2G.
Although the hepatitis B integration event in sequencing data can be identified by conventional bioinformatic analysis means,
But in order to provide better recognition effect, the present invention provides the Bioinformatics methods of innovation, include the following steps:
(a) sequencing reading length is compared, identification is from the sequence of receptor gene's group and from hepatitis B
Sequence；
(b) it removes redundancy and reads length；
(c) according to the sequence from receptor gene's group and the sequence from hepatitis B, subject-HBV is found out
It is chimeric to read long and determine its breakpoint；
(d) integration event is determined according to both ends breakpoint.
It further include that the sequencing data of human peripheral blood dissociative DNA is screened preferably, before determining integration event
Step.Preferably, specifically including: (i) filters out low quality sequencing data；And/or (ii) filters out connector polluted sequence.
The present invention further provides a kind of methods for detecting subject liver cell excessive proliferation, and the method includes walking as follows
It is rapid:
(1) using the hepatitis B Integration of method of the present invention detection subject；Or use institute of the present invention
Hepatitis B integration event in the method detection subject cfDNA stated；
(2) the integration event of instruction liver cell excessive proliferation is found in the testing result of step (1), the instruction liver is thin
The criterion of identification of the integration event of born of the same parents' excessive proliferation are as follows: from sequence ginseng corresponding to its of receptor gene's group in integration event
It is no more than 2 than the nucleotide difference between sequence, the core between sequence reference sequence corresponding to its of hepatitis B
Thuja acid difference is no more than 5, and the breakpoint of same every one end of integration event is all in across the breakpoint reading of at least two nonredundancy is long
In the presence of；
(3) there is the integration event for meeting the criterion of identification in subject, then there are excessive proliferations for its liver cell.
The present invention further provides a kind of diagnosis/screening methods of liver cancer, and described method includes following steps:
(1) using the excessive proliferation of method of the present invention detection subject liver cell；
(2) to there are the subjects of liver cell excessive proliferation to carry out the detection of liver cancer iconography.
The present invention further provides peripheral blood dissociative DNAs to detect or monitor answering in subject's hepatitis B Integration
With.
The present invention further provides application of the peripheral blood dissociative DNA in detection subject's hepatitis B integration event.
The present invention further provides application of the peripheral blood dissociative DNA in detection subject liver cell excessive proliferation.
The present invention further provides application of the peripheral blood dissociative DNA in diagnosis or screening liver cancer.
The present invention further provides dissociative DNA reagent preparations to integrate event for detecting the hepatitis B of subject in preparation
Reagent and/or kit in purposes.
The present invention further provides dissociative DNA reagent preparations to prepare the hepatitis B Integration for detecting subject
Reagent and/or kit in purposes.
The present invention further provides dissociative DNA reagent preparations to prepare the examination for detecting subject's liver cell excessive proliferation
Purposes in agent and/or kit.
The present invention further provides dissociative DNA reagent preparations to prepare the purposes in diagnosing cancer of liver reagent and/or kit.
Method of the invention can be applied during diagnosis or screening, can be used for non-diagnostic purpose.
Of the invention so-called " integration " refer in the genome of the DNA insertion host from virus, described from virus
DNA can be segment, virus genomic most of region or the entire viral genome of viral gene.
Of the invention so-called " breakpoint " refer to viral genome or genetic fragment insertion host genome restrovirus sequence and
The point of interface of host sequences；
The present invention so-called " subject-HBV is chimeric to read length ", " across breakpoint reading length " refer to that the sequence of a sequencing reading length is same
When covering breakpoint around virus gene sequence and host gene sequence；
So-called " subject " of the invention is mammal, preferably the mankind；The preferred liver cancer patient of the human experimenter, second
Hepatopath, hepatitis carrier or normal person.
What is provided in the public database that so-called " reference sequence " of the invention refers to has determined what base sequence formed
Genomic information is resurveyed the sequencing reading length in sequence used in genome and is compared, and determines the gene order of sequencing reading length covering in the base
Because of the position in group.Receptor gene's group and the reference sequence (or reference sequences) of hepatitis B can be from existing technical literatures
Or obtained in online sequence database, such as US National Biotechnology Information center website (https: //
www.ncbi.nlm.nih.gov/)。
It is substantially noninvasive to patient that the present invention has the following advantages and beneficial effects: method of the invention compared with prior art
Wound may replace tradition tissue biopsy.The hepatitis B of subject is detected and monitored using the dissociative DNA in peripheral blood as sample
Integration, Normal practice compared with the prior art are surveyed after taking tissue sample by open surgical biopsy or aspiration biopsy
Examination can be avoided the risk cases such as complication, improves safety, the numerous periodic monitoring of higher frequency may be implemented, and avoid
The heterogeneous sexual deviation of sample of tissue.And of the invention method specificity is good, high sensitivity, and it at present statistics indicate that can be
It is detected in the peripheral blood sample of liver cancer patient after 100% HBV infection, it is minimum to be extracted in 5ml peripheral blood blood plasma
Segment is detected in whole cfDNA.
Detailed description of the invention
Fig. 1 is liver cancer group figure compared with integrating event level in chronic hepatitis B group cfDNA.
Fig. 2 is that event figure compared with the abundance for integrating event in liver cancer tissue is integrated in liver cancer group cfDNA.
Fig. 3 is the comparison figure of cfDNA and liver cancer tissue point of interruption abundance under best screening criteria.
Fig. 4 is segment insertion/fracture mode figure of liver cancer group Yu chronic hepatitis B group cfDNA.
Specific embodiment
In the present invention, unless specifically stated otherwise, used reagent, equipment etc. are commonly used in the art, or can be led to
Market purchase is crossed to obtain.The present invention is described in detail combined with specific embodiments below, it should be appreciated that these embodiments are only used for
The illustration present invention, the present invention are not limited merely to these embodiments, those skilled in the art's announcement according to the present invention, in this hair
Any modification, equivalent replacement, improvement and so within bright spirit and principle, be all contained in protection scope of the present invention it
It is interior.
Embodiment 1, sample acquire and the extraction of peripheral blood dissociative DNA
In Beijing, You An hospital has collected the peripheral blood sample of liver cancer patient after 7 HBV infections.For each liver cancer patient,
In addition to having collected peripheral blood sample before surgery, 2 parts of liver cancer tissue sample and Para-cancerous tissue sample 2 are also had collected after surgery
Part, for compareing the ability of discovery of the viral integrase event in peripheral blood.Above-mentioned patient includes 3 people of women, and 4 people of male is average
Age 51 years old.
It is extracted the cfDNA of the peripheral blood sample and DNA of liver cancer tissue sample and Para-cancerous tissue sample.DNA's mentions
It takes using QIAGEN DNeasy Blood&Tissue Kits kit, is extracted according to the standardization process of kit.cfDNA
Extraction use following steps, and carry out quantitative and quality evaluation:
1) dissociative DNA heparin tube (Streck Cell-Free DNA is usedTubes) collection 10ml periphery whole blood, 4
It send in hour to laboratory.
2) upper plasma, centrifugal condition are as follows: 4 DEG C, 3000 × g, 15 minutes are centrifugated.5ml blood plasma utilizes free nucleic acid
Extracts kit QIAamp Circulating Nucleic Acid Kit (Qiagen, Valencia, CA) extracts peripheral blood trip
From DNA, elution stage uses ultrapure water of the 20ul without DNASE enzyme.
3) fluorescent quantitation instrument (Thermo Fisher) measurement cfDNA concentration.
4) 2100 analyzing biochips system instrument of Agilent (2100 Bioanalyzer instrument, DNA are used
1000 assay) analysis cfDNA segment distribution.If segment is distributed the ideal range for not meeting 150-200bp, use as one sees fit
Ultrasonic (Covaris Focused-ultrasonicators, Covaris, Inc.MA) interrupts segment, complies with ideal model
It encloses.Product after interrupting repeats implementation steps 3) -4), it is anti-whether analysis assessment concentration of specimens and clip size reach subsequent experimental
The requirement answered.
The results show that the extracted cfDNA of most of patients sample has the short-movie section distribution for concentrating on 167bp peak value, always
About 600ng is measured, subsequent capture experiment is completely used for.
Embodiment 2, the end reparation of peripheral blood dissociative DNA, connector connection and pre- amplification
The cfDNA obtained to embodiment 1 is handled in accordance with the following steps:
1) it is quantitative to 50ul volume with ultrapure water that the cfDNA obtained will be extracted, the end 7ul is added and repairs buffer (End
Repair&A-Tailing Buffer) and the end 3ul is repaired plus A enzymatic mixture (End Repair &A-Tailing
Enzyme Mix), to 60ul after mixing, complete end on thermal cycler and repair reaction, reaction condition are as follows: 20 DEG C 30 minutes,
65 DEG C 30 minutes, 4 DEG C preservation, heat lid be maintained at 70 DEG C.
2) by the connector of above-mentioned 60ul product addition 5ul, (connector includes following amplification primer region, individual tag identification
Sequence, corresponding with connector needed for subsequent microarray dataset), it is attached reaction on ice: the connection reaction mixing of 55ul is added
Liquid, including 5ul nuclease-free water, the connection buffer of 30ul, 10ul DNA ligase.20 DEG C of incubations, 15 minutes (Wu Regai).
The sample fewer for amount of DNA can extend to incubation time to improve joint efficiency no more than 4 hours or 4 DEG C
Overnight.
3) connection product of 110ul is taken, 88ul is added and balances to nucleic acid purification microballon (the Agencourt AMPure of room temperature
XP Beads), soft mixing 5-10 times, and bubble is avoided to generate, it is incubated at room temperature 5 minutes.Centrifuge tube is put to magnetic frame, 1
Magnetic bead is agglomerating in minute, and supernatant is limpid, removes supernatant；80% ethyl alcohol of 200ul, room temperature are continuously added in pipe on magnetic frame
It is absorbed after being incubated for 30 seconds；It is repeated 2 times；Drying at room temperature magnetic bead 3-5 minutes, avoid overdrying that Product yields is caused to decline.It takes
22ul nuclease-free water eluted dna.
4) DNA of 20ul elution, is added high-fidelity DNA polymerase premixed liquid 25ul, and each 2.5ul of amplimer is added and prepares
At 50ul reaction system, the amplimer corresponds to the amplification primers region in connector, carries out PCR amplification building 1ug's
Sequencing library.
5) product carries out aforementioned purifying with nucleic acid purification microballon (Agencourt AMPure XP Beads), with 30ul without
The DNA of nuclease water dissolution after purification, obtains product about 28ul.Pass through 2100 analyzing biochips system (2100 of Agilent
Bioanalyzer instrument, DNA 1000assay) fragment length is analyzed, about 275-325bp is left as the result is shown
It is right.
6) concentration for measuring previous step purified product obtained, takes the solution of certain volume, so that wherein containing
750ng purified product is added 1ul and blocks liquid 1,5ul that liquid 2 and 5ul is blocked to block liquid 3, dry powder is converted on freeze dryer.Its
In, it blocks and contains oligonucleotides Block1 and Block2 in liquid 1 and 2 respectively, Block1 and Block2 are designed to block and connect
Pairing between header sequence declines in conjunction with caused capture rate, blocks and contains oligonucleotides Block3 in liquid 3, and Block3 is designed
To combine repetitive sequence on human genome etc. to avoid its interference to experiment.
Embodiment 3, hybrid capture experiment
Human peripheral blood cfDNA and liver cancer tissue and cancer beside organism DNA carry out the capture of integration event, as follows into
Row:
1) design of viral integrase capture probe group: according to the hepatitis B A-H type for arranging acquisition in ncbi database
Sequence, c-type 457, designs the degeneracy probe of Corticovirus full-length genome 3.2K by Type B 212 including CHINESE REGION prevalence,
Each probe length is about 100bp.The design method of probe be it is known in the art that using conventional design method, such as it is common
Software or website can be completed, the probe groups in the present embodiment are particular by common design of primers websitehttps:// design.igenetech.com/Design obtains.
2) prepare reaction solution: it is quantitative that nuclease-free water is added in the peripheral blood cfDNA dry powder sample that Example 2 obtains
It is soft to mix to 10ul, it is transferred in new 500ul PCR pipe (B pipe), is placed on ice；It is added in 200ul PCR pipe (A pipe)
20ul hybridization buffer is preheated to 65 DEG C；5ul ribalgilase is added in 500ul PCR pipe (C pipe) and blocks liquid (RNase
Block) and 2ul probe is placed on ice, and wherein concentration and probe concentration has carried out corresponding adjustment according to the amount of cfDNA product.
The liver cancer tissue DNA and cancer beside organism DNA prepared in embodiment 1 also uses same method to handle.
3) capture reaction system is prepared: 95 DEG C of thermal cycler instrument setting program 5 minutes, 65 DEG C of holdings, and 100 DEG C of hot lid.It
After be put into B pipe startup program.When instrument shows 65 DEG C, A pipe is placed 5 minutes in the thermal cycler, is put into the holding of C pipe later
2 minutes；It takes 13ul hybridization buffer to be put into C pipe from A pipe, and C pipe is also added in the 10ul sample of B pipe, generated during avoiding
Bubble.After C pipe strictly is sealed, 65 DEG C incubation 16-24 hours, 100 DEG C of hot lid on thermal cycler.
4) the affine magnetic bead of streptomysin prepares: Dynabeads MyOne Streptavidin T1 magnetic bead 50ul is put into 500ul
In PCR pipe, pipe is placed in magnetic board (DynaMag-96Side), magnetic bead is agglomerating to be moved back except supernatant, and pipe is taken from shelf
Under, 200ul combination buffer is added, it is soft to mix；Again pipe is placed on the top of the shelf, magnetic bead is agglomerating, removes supernatant, pays attention to
Magnetic bead is not broken up, repeats above-mentioned cleaning step 3 times, magnetic bead is resuspended with 200ul combination buffer later and is immediately available for following catch
Obtain reaction.
5) capture reaction of the affine magnetic bead of streptomysin: above-mentioned 200ul magnetic bead is directly added into 65 DEG C of thermal cycler
500ul PCR pipe C is soft to mix；By mixture, the 10rpm on rotation vortex mixer is mixed 30 minutes room temperature；It is placed on magnetic force later
On plate DynaMag-96Side, supernatant is abandoned after magnetic bead is agglomerating, is added 200ul eluent 1 (room temperature less salt liquid eluent), removes magnetic
Room temperature 10rpm is mixed 15 minutes on rotation vortex mixer after power plate.Magnetic board is put back to after concussion pipe, is abandoned after magnetic bead is agglomerating
Clearly；It is added 200ul eluent 2 (high temperature and high salt eluent), is incubated for 10 points at upper 65 DEG C of mixer (ThermoMixer) after mixing
Clock, speed 800rpm；It is put into magnetic board after concussion tube wall, abandons supernatant after magnetic bead is agglomerating, repeats this step 3 time of eluent 2,
Ensure that its complete reject is clean later.Then magnetic bead is resuspended with 30ul nuclease-free water.
6) PCR and purifying after capturing: buffer 18ul is added, corresponding to amplification region in connector in 30ul previous step product
Universal amplification primer (this primer contain sequence label for mark the different libraries of differentiation) each 1ul, DNA high fidelity enzyme 1ul is made into
The reaction system of 50ul, after mixing on thermal cycler PCR amplification, PCR condition are as follows: 100 DEG C of hot lid；95 DEG C entered after 4 minutes
Circulation, cyclic process be 98 DEG C 20 seconds, 60 DEG C 30 seconds, 72 DEG C 30 seconds, totally 10 circulation；72 DEG C 5 minutes later, 12 DEG C of holdings.
Then nucleic acid purification microballon (Agencourt AMPure XP Beads) (at least room temperature 30 minutes) 55ul of balance to room temperature
It is added in pipe, piping and druming 5-10 times (attention avoids generating bubble), is incubated at room temperature 5min.Magnetic bead is in 1 minute on magnetic board
Supernatant is abandoned in agglomeration, is incubated at room temperature to move back for 30 seconds with 80% ethyl alcohol of 200ul and be removed, and 2 times repeatedly.Allow magnetic bead dry at room temperature, from magnetic
Power plate removes pipe, with 25ul nuclease free water elution.Magnetic board is put back to later, by acquisition after 2 minutes magnetic beads are sufficiently agglomerating
23ul product moves on in new pipe.Use 2100 analyzing biochips system (2100 Bioanalyzer of Agilent
1000 assay of instrument, DNA) fragment length is analyzed, about 275-325bp or so as the result is shown.
Embodiment 4, the sequencing for capturing product
In order to determine that best sequencing amount required for the event is integrated in capture, the positive reference system based on HepG2.2.15 is constructed
System.HepG2.2.15 cell line is as cell line the most famous in HBV research, integrated therein with complete HBV full-length gene
Group sequence, has specific integration site.The HepG2.2.15 of culture passes through the removal point of hepatitis B Core antibody after harvesting
The virion for secreting generation purifies to obtain its nucleus using nucleus separating kit, to exclude the cccDNA in cytoplasm
Influence for standard quantitative, the strategy synthesized using excess probe guarantee the sensibility of capture.1G, 1.5G, 2G is respectively adopted
Sequencing amount be sequenced, the microarray dataset used is Illumina X-Ten sequenator, and sequencing steps are using microarray dataset
Standardized method.
As a result show such as following table, tests probe and devise and tests three times, be set separately 1G sequencing amount, 1.5G sequencing amount,
2G sequencing amount, capture the results show that integrated in breakpoint for 5 in HepG2.2.15, can stablize discovery chromosome 1,
7, the breakpoint on 9, but the breakpoint on chromosome 2 cannot be found by 1G sequencing amount, it is promoted in sequencing amount to 2G and is sequenced
It is can be found that when amount.
In fact, best sequencing amount can be adjusted with the variation of used probe combinations, it is contemplated that actual sample
Between the difference that may be present to sequencing depth requirements, such as the sequence of cfDNA amount less, from HBV is limited or patient itself
The HBV for being released into blood integrates situations such as segment is not sufficient enough, in order to guarantee accuracy and sensitivity as far as possible, has finally chosen
Biggish sequencing amount carries out follow-up test, in case the sequencing depth of part sample not enough leads to not find integration event.
According to result above, high-throughput sequencing library building is carried out to the purified capture product obtained in embodiment 3,
It using the commercialization Library development flow of illumina X-ten platform, is then sequenced, each library measures about 2G data volume.
When sequencing amount is 2G, sequencing result meets following quality requirement as the result is shown: Raw bases is not less than 2000M base, after Quality Control
Sequencing amount (Clean bases) is not less than 1600M bases (QC rate > 80%), and average fragment is read long not less than 120bp, reading
Long redundancy rate (Duplication rate) is not more than 1%, and for the coverage of HBV gene group entirety, the region of 4X is not less than
The region of 90%, 50X are not less than 50%.
Embodiment 5, bioinformatic analysis
Bioinformatic analysis is carried out to the sequencing result that embodiment 4 obtains.First with the removals such as Cutdapt software sequencing
In low quality and remnants joint sequence, sequencing reading length after Quality Control is compared to people and HBV by sequence alignment program BWA and is joined
It examines on genome, determines the coverage for HBV gene group, the redundancy after being compared by Picard software removal reads length, later
Assess the valid data amount and quality of sequencing.It recalls the chimeric reading of people-HBV in data long (junction reads) that is, covering is disconnected
The sequencing reading length of point carries out the Sequence annotation of genome to the end HBV and people end respectively, so that it is determined that the generation of integration event is special
Sign: the position etc. on the region of viral integrase, human genome.
According to the common knowledge of this field, cfDNA greatly mostly from normal tissue rather than tumour, it is generally recognized that tumor patient
CfDNA in the ratio of Circulating tumor DNA (Circulating Tumor DNA, ctDNA) be about to be based between 0.1%-1%
This understanding is it is contemplated that the integration event in cfDNA from non-cancer hepatic tissue should be more than the integration thing from liver cancer tissue
Part.However after peripheral blood, liver cancer tissue and the integration of the cancer beside organism event to 7 liver cancer patients are compared and analyze
(result is as shown in following table), it was unexpectedly found that, the 23 integration events captured in peripheral blood cfDNA all can
It is captured in liver cancer tissue, but 18 therein can only be captured in normal liver tissue.The quantity of cancer cell is far fewer than just
Normal hepatic tissue, the frequency of dead cracking occurs for cancer cell also below normal tissue cell, however experimental result but shows that cancer is thin
Born of the same parents seem that releasing integration events more more than normal tissue enters peripheral blood, this phenomenon causes the curiosity of inventor, mentions
Show that perhaps the HBV integration event in peripheral blood has the potentiality as diagnosing cancer of liver or the marker of early-warning, it is possible to apply in liver
In assessment, hepatocyte growth or the clonal expansion of dirty immune response and the monitoring process of tumour progression.
Embodiment 6, Chronic Hepatitis B cfDNA analysis
With the presence or absence of connection between generation and liver cancer state in order to verify the integration event in peripheral blood cfDNA and liver cancer
System, so that the diagnosis and early warning for liver cancer provide useful reference information, it is thus necessary to determine that in peripheral blood, tumor tissues and normal group
All existing 18 integration events, are derived from tumor tissues actually, are also derived from normal tissue, or source simultaneously in knitting
In the two.For this purpose, be extracted the peripheral blood cfDNA of the Chronic Hepatitis B that 10 are not suffering from liver cancer for verifying, according to it is aforementioned
Identical method is captured and has been sequenced in embodiment, whole in normal liver tissue (i.e. non-tumour hepatic tissue) to determine with this
Whether conjunction event can be discharged into peripheral blood.
This 10 patients's (referring to below table) experienced long-time HBV infection state and (be greater than 25 years, when being birth
Infect), the results show that no matter actively whether the horizontal height of AFP, HBV virus replication, can only detect in peripheral blood cfDNA
To low-level integration event.Event will be integrated in the cfDNA of 7 liver cancer patients in 10 slow hepatopaths and previous embodiment
Level be compared, there are significant difference both as the result is shown (result is as shown in Figure 1).
This is the results show that integration event is difficult to be discharged into peripheral blood, previous in the slow hepatopath for being not suffering from cancer
The integration event captured in peripheral blood cfDNA in embodiment is derived from tumor cell of liver mostly, is because containing integration
Excessive proliferation has occurred in the liver cell of event, and cfDNA therein is caused largely to enter blood and have little time to remove, thus in blood
In detect the integration event of high abundance.
Embodiment 7, integration event enrichment analysis
In order to determine whether to replace hepatic tissue sample to reflect that the hepatitis B of subject integrates shape using peripheral blood cfDNA
Condition, the integration site (Fig. 2, black) in integration site (Fig. 2, white) and liver cancer tissue detected in human peripheral blood compare,
It was found that the two shows more consistent abundance, i.e., the relatively high integration event of abundance, rich in blood plasma in liver cancer tissue
It spends also relatively high, but integrates abundance between liver cancer tissue and normal tissue there are significant difference (p < 0.05), further support
CfDNA is tumor tissues source.Since the integration event in the cell of excessive proliferation can largely enter peripheral blood, and it is common thin
Integration in born of the same parents only enters blood on a small quantity, and integrates that event proliferation degree is higher, and the abundance in cfDNA is also higher, thus
Hepatitis B integration in peripheral blood cfDNA is able to reflect the hepatitis B Integration of subject.
It can best reflect the whole of liver cell excessive proliferation situation to find in the integration event of peripheral blood cfDNA
Conjunction event compares different inclusion criterias, chooses genome sequence matching degree, hepatitis B sequences match degree and breakpoint and occurs
Rate is as index, each selecting index number grade standard, i.e. core in integration event between host genome sequence and reference sequence
No more than 1/2/3/4, the nucleotide difference in integration event between hepatitis B sequence and reference sequence does not surpass thuja acid difference
3/4/5/6 is crossed, the breakpoint of same every one end of integration event is all in across the breakpoint reading length of at least 1/2/3 nonredundancy
(Junction) exist in.After the integration event in above-mentioned standard screening cfDNA, optimal screening conditions have been obtained: whole
The matching degree of genome sequence is no more than 2 nucleotide differences in conjunction event, and the matching degree of hepatitis B sequence is no more than 5 cores
The breakpoint of thuja acid difference, same integration event exists in across the breakpoint reading of at least two nonredundancy is long.As shown in figure 3, at this
It is available with the optimal correlation (R of tumor tissues breakpoint abundance under part2=0.62).
Integration event abundance in peripheral blood is about 10% in tissue, is influenced, cuts by Tumor Heterogeneity in fact
It takes tumor tissues and analyzes the integration of the inside, not necessarily coincide with abundance actual in tumour, under the influence of this condition, periphery
Integration coverage in blood still shows preferable correlation, it is contemplated that sample of tissue itself integrally forms tumour reflection
When bring sampling bias, the detection of peripheral blood possibly even preferably reflects the patient's body tumour clone than sample of tissue
Ratio.
Embodiment 8, integration event insertion/fracture mode analysis
10 slow hepatopaths are compared with the integration event in the peripheral blood cfDNA of 7 liver cancer patients, as the result is shown
The two shows consistent segment insertion/fracture mode (referring to fig. 4), but the breakpoint abundance of slow hepatopath has not yet been reached and can sentence
The required threshold value of the disconnected liver cell abnormality proliferation containing integration.It is also integrated constantly in slow hepatopath's liver, and one
After dividing the cell death with integration, DNA may also be detected after being released into blood, when selecting slow hepatopath, be chosen emphatically
It has selected risk of hepatic cancer high but existing ultrasonic diagnostic technique is not yet diagnosed to be the patient of liver cancer, utilized Acquisition Scheme discovery
Integrate segment candidate region, show with insertion/fracture mode consistent in Peripheral Blood of Patients with Hepatocellular Carcinoma, prompt integration really can when
It carves and occurs, but this abundance for integrating segment outside in all blood is extremely limited, the chimeric reading of only 1 integration is long, it was demonstrated that releases
The abundance for integrating segment put needs to reach certain threshold value, i.e., the liver cell with integration needs to be proliferated to a certain extent,
There can be tumorigenic possibility.By being monitored to the integration event in subject's peripheral blood cfDNA, discovery abundance reaches
After threshold value, diagnosing cancer of liver or regular screening can be carried out, diagnosis and screening can use common means, such as iconography to detect.
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The above is only a preferred embodiment of the present invention, it is not intended to limit the scope of protection of the present invention, should refer to
Out, for those skilled in the art, without departing from the principle of the present invention, can also make several
Improvements and modifications, these modifications and embellishments should also be considered as the scope of protection of the present invention.
Claims (10)
1. a kind of method of the hepatitis B Integration of detection or monitoring subject, which is characterized in that with the trip in peripheral blood
It is detected from DNA as sample.
2. the method according to claim 1, wherein the hepatitis B Integration be subject's hepatic tissue or
The hepatitis B Integration of liver cell, by detection peripheral blood dissociative DNA in hepatitis B integrate event, with reflect by
Examination person's hepatic tissue or the hepatitis B Integration of liver cell；Preferably, the hepatitis B Integration is hepatic tissue or liver
Hepatitis B in cell integrates event tree.
3. a kind of method of hepatitis B integration event in detection subject cfDNA.
4. according to the method described in claim 3, it is characterized in that, described include the following steps:
(a) peripheral blood dissociative DNA is extracted；
(b) end reparation is carried out to the peripheral blood dissociative DNA that step (a) is extracted, is connect with connector, purify connection product；
(c) optionally, the DNA obtained to step (b) is expanded in advance, purifies amplified production；
(d) DNA product obtained using the probe that can hybridize with Hepatitis B virus-DNA sequence in step (b) or step (c)
In captured；
(e) the capture product obtained to step (d) is sequenced；
(f) determine that hepatitis B integrates event by bioinformatic analysis.
5. a kind of for determining the Bioinformatics method of the hepatitis B Integration of subject, which is characterized in that pass through
Hepatitis B in peripheral blood dissociative DNA integrates event, reflects the hepatitis B Integration of subject.
6. a kind of method for detecting subject liver cell excessive proliferation, which comprises the steps of:
(1) using the hepatitis B Integration of the method as described in claim 1 detection subject；Or it is wanted using such as right
Method described in asking 3 detects hepatitis B in subject cfDNA and integrates event；
(2) the integration event of instruction liver cell excessive proliferation, the instruction liver cell mistake are found in the testing result of step (1)
Measure the criterion of identification of the integration event of proliferation are as follows: from sequence reference sequence corresponding to its of receptor gene's group in integration event
Nucleotide difference between column is no more than 2, the nucleotide between sequence reference sequence corresponding to its of hepatitis B
Difference is no more than 5, and the breakpoint of same every one end of integration event is all deposited in across the breakpoint reading of at least two nonredundancy is long
?；
(3) there is the integration event for meeting the criterion of identification in subject, then there are excessive proliferations for its liver cell.
7. a kind of diagnosis/screening method of liver cancer, which comprises the steps of:
(1) using the excessive proliferation for detecting subject liver cell method as claimed in claim 6；
(2) to there are the subjects of liver cell excessive proliferation to carry out the detection of liver cancer iconography.
8. peripheral blood dissociative DNA is detecting or is monitoring subject's hepatitis B Integration/detection subject's hepatitis B integration
Application in event/detection subject liver cell excessive proliferation/diagnosis or screening liver cancer.
9. dissociative DNA reagent preparation preparation for detect subject hepatitis B integrate event/hepatitis B Integration/
The reagent of liver cell excessive proliferation and/or the purposes in kit.
10. dissociative DNA reagent preparation is preparing the purposes in diagnosing cancer of liver reagent and/or kit.
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