CN107312839A - A kind of material, kit and method detected for systemic infection - Google Patents
A kind of material, kit and method detected for systemic infection Download PDFInfo
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Abstract
The present invention relates to a kind of material, kit and detection method detected for systemic infection.The material refers to the Circulating DNA for being disintegrated release in vivo from pathogenic bacteria.Detection of the invention based on human peripheral blood pathogenic bacteria degradation of dna, objectively responds and is invaded with the presence or absence of pathogenic bacteria into blood circulation, can preferably distinguish systemic infection and noninfectious disease.The present invention is identified independent of the separation of viable bacteria thalline, is not influenceed, is not also influenceed by polluted bacteria during collect specimen by viable bacteria content in blood specimen, with higher detection sensitivity and specificity.The detection kit that the present invention is provided includes 662 kinds of pathogenic bacteria 16S rDNA capture probe, can preferably differentiate the category kind of pathogenic bacteria.The detection method that the present invention is provided can preferably be reflected the order of severity of infection, the course of disease and be lapsed to by the semi-quantitative analysis of bacterial origin Circulating DNA in human peripheral blood, and then guiding treatment, assess prognosis.
Description
Technical field
The present invention relates to a kind of material, kit and method detected for systemic infection.
Background technology
Systemic infection is clinically very common complication, with the incidence of disease is high, the death rate is high, medical expense is high
The features such as.Clear and definite etiological diagnosis has vital effect to instructing anti-infective therapy, improving patient's prognosis.At present,
Clinically, what etiological diagnosis relied primarily on pathogenic bacteria is separately cultured identification.Positive blood cultivation results are still whole body at present
The goldstandard of property Infect And Diagnose.However, the maximum shortcoming of blood culture detection is that sensitiveness is too low, positive rate is less than 30%.In addition,
By the factor polluted during collection of specimens, also there is certain false positive rate in the detection method.
The positive rate of blood culture is mainly influenceed by following factor：First, the ex vivo growth capability of pathogenic bacteria；Second,
The service condition of antibiotic before blood drawing；3rd, be also most important influence factor --- the content of viable bacteria in blood specimen.And live
Except the order of severity with infection mutually outside the Pass, the time also with blood sampling is closely related for the content of bacterium, therefore with very big not true
It is qualitative.
16srDNA is the encoding gene of prokaryotes rRNA, total length about 1.5Kb, by multiple variable regions and conserved region
Train interval is rearranged.Conserved region is common to all bacteriums；Variable region sequences have species specificity, between different strain
There is difference to a certain degree.People can design PCR primer according to conserved region consensus sequence, and variable region sequences are expanded with this and are surveyed
Sequence is compared, and the identification that this method is used for bacterium well at present is classified.There is researcher to attempt this method being used for entirely in recent years
The detection of body sexuality dye.But the amplification substrate of these researchs has relied in body fluid the separation and concentration for the thalline that causes a disease.So, together
Blood culture is the same, and the positive rate of the detection method can equally be influenceed by viable bacteria content in blood when taking a blood sample.In addition, sample is dirty
Dye also results in this method and produces a certain proportion of false positive results.
Circulating DNA refers to be present in the dissociative DNA in blood circulation system.Existing research thinks that Circulating DNA mainly comes
Come from the degraded release of nucleus DNA after body cell apoptosis.Recently studies have reported that, by genome sequencing find sense
There is the Circulating DNA in pathogen source in the peripheral blood for contaminating patient, still, due to sequencing depth not enough, these researchs do not have
Individual features to pathogen source Circulating DNA are illustrated.We have found that systemic infection patient's is outer in early-stage Study
The Circulating DNA proportion of bacterial origin is extremely low in all blood, directly carries out the costly of genome sequencing analysis, faces
Bed application feasibility is little.However, for pathogen originate Circulating DNA detection other methods there is not been reported so far.
The content of the invention
Preferable systemic infection Testing index should have following condition：1) systemic infection can be distinguished and non-infectious
Disease；2) there is good specificity and sensitiveness；3) the category kind of pathogenic bacteria can be specified；4) susceptibility can be provided and resistance to
Medicine information；5) order of severity of infection can be reflected；6) course of disease of infection can be reflected and lapsed to；7) detection method is easy, examine
Degree of testing the speed is fast and cheap.The pathogeny detection method of the infection of the past is all based on the separation identification of viable bacteria thalline, and it is examined
Disconnected Sensitivity and Specificity can all be influenceed by more multifactor.And the detection of bacterial origin Circulating DNA can overcome above-mentioned inspection
The deficiency of survey method, provides the relevant information of pathogenic bacteria comprehensively on the basis of high detection efficiency is ensured
It is an object of the invention to provide the feature of systemic infection patient's Circulating DNA, and root comprehensively by deep sequencing
A kind of kit detected for systemic infection and method are provided according to its feature.
To achieve the above object, the technical scheme taken：A kind of material detected for systemic infection, the material
It is derived from the Circulating DNA that pathogenic bacteria are disintegrated release in vivo.The material is to be present in live body blood circulation, from pathogenic bacteria
Dissociative DNA, not including DNA contained by viable bacteria thalline in blood circulation；The ratio that the material accounts for human circulation DNA total amounts is extremely low, should
The Cmax fragment length of material is fluctuated in 100~200bp.
The invention provides a kind of kit detected for systemic infection, the kit includes pathogenic for being enriched with
The capturing liquid chip of the Circulating DNA in bacterium source.
Preferably, the kit includes the capturing liquid probe for being used to be enriched with common pathogen 16S rDNA sequences.Mesh
The common pathogen of preceding discovery is 662 kinds, and the present invention is 8021 for the capture probe sum of this 662 kinds of common pathogen designs
Bar, overall length is 919.445Kbp.
The invention provides a kind of method that systemic infection is detected using kit described above, methods described includes
Following steps：
(1) peripheral blood, centrifugal separation plasma are gathered；
(2) plasma circulation DNA is extracted；
(3) Circulating DNA concentration is detected；
(4) expanded before end reparation plus A tails, jointing, capture are carried out to Circulating DNA；
(5) hybrid capture is carried out with the capturing liquid probe, target dna is enriched with；
(6) expand and tag after capturing, carrying out sequencing using two generation sequencing technologies obtains its sequence；
(7) by comparing analysis with existing bacterial 16 S rDNA reference sequences, bacterial species and half in peripheral blood are determined
It is quantitative.
Preferably, in the step (1), peripheral blood collection uses cfDNA special blood-drawing pipes；
The separated plasma comprises the following steps：By blood plasma 1000g~2500g low-speed centrifugals 10 minutes, purified the blood in extraction
Slurry, 20000g high speed centrifugations 10 minutes extract supernatant blood plasma.
Preferably, in the step (2), using the kit QIAamp Circulating of Qiagen companies
Nucleic Acid Kit extract Circulating DNA on request；
In the step (3), DNA concentration is detected using Qubit.
Preferably, in the step (4), (5), (6), entered on request using kit KAPA Hyper Prep Kit
Row builds storehouse.
Preferably, the two generations sequencing technologies are illumina Hiseq.
Preferably, the analysis of the sequencing data obtained in the step (6) comprises the following steps：
S1, lower machine data Quality Control and go sequence measuring joints：Original lower machine sequence needs to remove the sequence for containing " N " and removal connects
Header sequence pollutes, while being used as base quality control standard using Q20；
S2, the DNA sequence dna for filtering Hosts：It is soft by bwa using UCSC hg19 as host genome reference sequences
Part carries out sequence alignment analysis；In paired-end sequencings, either end can compare the sequence antithetical phrase meeting of host genome
It is removed；
S3, De novo assemble microorganism contig and scaffold fragment：Genome is carried out using metaSpades softwares
De novo are assembled, and select the candidate microbial fragment after assembling, it is desirable to：A, more than sequence length 800bp；B, compare through blast
The known strain 16s rDNA sequence libraries that NCBI is included can obtain consistent comparison result with full length sequence database；C, ratio
To concordance rate >=95%, fragment >=85% is covered；Reject and compare to host genome or the biological fragment of its nearly edge；
S4, candidate segment Quality Control：Using the candidate segment of identification as reference sequences, sequence is carried out using bwa softwares
Compare；It is required that：A, non-duplicate aligned sequences (non-duplicated the read) >=10X coverings after comparison per bar segment
(coverage) base accounting >=80%, to ensure the reliability of De novo processes；B, the covering requirement per bar segment are preferable
Homogeneity (uniformly coverage)；
S5, identification pathogenic microorganism and report：The identification precision of 16s rDNA sequences for bacteria, which can reach, " plants
(species) rank ", therefore the probation report first choice of pathogenic microorganism is " strain ", when candidate segment while pointing to same
During the not same species of microorganism of subordinate, then flagship report " Pseudomonas ", and " strain " of mark identification respectively.
It is disintegrated the Circulating DNA of release in vivo the invention provides pathogenic bacteria and is examined for systemic infection screening or prepare
Purposes in disconnected reagent or kit.
Compared with prior art, advantage of the invention：
(1) detection based on human peripheral blood pathogenic bacteria degradation of dna of the invention, objectively respond with the presence or absence of pathogenic bacteria invade into
Blood circulation, can preferably distinguish systemic infection and noninfectious disease.
(2) present invention is identified independent of the separation of viable bacteria thalline, is not influenceed, is not also adopted by viable bacteria content in blood specimen
The influence of polluted bacteria during collection sample, with higher detection sensitivity and specificity.
(3) detection kit that the present invention is provided includes 662 kinds of pathogenic bacteria 16SrDNA capture probe, can be more preferable
Differentiate the category kind of pathogenic bacteria.
(4) detection method that the present invention is provided, can by the semi-quantitative analysis of bacterial origin Circulating DNA in human peripheral blood
Preferably reflect the order of severity infected, the course of disease and lapse to, and then guiding treatment, assess prognosis.
Brief description of the drawings
Fig. 1 is 16SrDNA sequence alignment analysis results；
Fig. 2 is from the beginning splice segment full-length genome comparison result.
Embodiment
To better illustrate the object, technical solutions and advantages of the present invention, below in conjunction with specific embodiment to the present invention
It is described further.
Embodiment 1
The middle-aged male patient of samples sources one, clinical diagnosis is postoperative laryngocarcinoma, severe pneumonia, septic shock.Sputum culturing
And blood culture result is EHEC.
First, peripheral blood, centrifugal separation plasma are gathered
Preserved on request using cfDNA special blood-drawing pipe conventional Christmas peripheral bloods 6ml；2500g is centrifuged 10 minutes, rear to extract
Supernatant blood plasma 20000g is centrifuged 10 minutes, extracts supernatant blood plasma 3ml.
2nd, plasma circulation DNA is extracted using kit QIAamp Circulating Nucleic Acid Kit on request
1st, 3ml blood plasma, 300 μ l Proteinase K, 2.4ml Buffer ACL are added in 50ml centrifuge tubes
(the μ g carrier RNA of containing 1.0), vortex 30s puts 60 DEG C of incubation 30min.
2nd, centrifuge tube is taken out, 5.4ml Buffer ACB, vortex 30s is added, puts and be incubated 5min on ice.
3rd, connection negative pressure extraction device QIAvac 24Plus, QIAamp Mini column and 20ml tube extender,
Negative pressure -900mbar is adjusted, slow to cross mixture in the above-mentioned centrifuge tube of post, rear order adds 600 μ l Buffer ACW1,750 μ l
Buffer ACW2,750 μ l 100%ethanol cross post elution.
4th, QIAamp Mini column are taken out and are put into 2ml EP pipes, 20000g centrifugations 3min.
5th, QIAamp Mini column are taken out and are put into lid in new 2ml EP pipes, opening, 56 DEG C of drying 10min.
6th, QIAamp Mini column are taken out and are put into new 1.5ml EP pipes, to QIAamp Mini column filtration membranes
75 μ l of Buffer AVE of upper addition, close the lid, and normal temperature is incubated 3min.
7th, 20000g centrifuges 3min, takes filtered solution to load new 1.5ml EP pipes and preserves.
8th, Qubit detects sample concentration, 2100 detection clip sizes.
3rd, set up genomic library and (build storehouse kit：KAPA Hyper Prep Kit KK850496libraries；
SureSelect XT Library Prep Kit ILM cat#5500-0133；SureSelect Target Enrichment
System 1-800-227-9770；Herculase II Fusion Enzyme with dNTP Combo Cat#600679；
SureSelect Target Enrichment Kit ILM Indexing Hyb Module Box 2Cat#5190-4455)
1st, DNA fragmentation end is repaired and end " A " tail
1) mixed liquor of configuration reaction, such as following table：
| Reagent | Consumption |
| End Repair&A-Tailing Buffer | 7μl |
| End Repair&A-Tailing Enzyme Mix | 3μl |
| Amount to | 10μl |
After being mixed with 50 μ l 100ngDNA samples, reaction condition in couveuse is put into as follows：
| Temperature | Time |
| 20℃ | 30min |
| 65℃ | 30min |
| 4℃ | Hold |
2nd, double end adjunction heads
1) reaction mixture, such as following table are configured：
| Reagent | Consumption |
| Nuclease-free water | 5μl |
| Adapter stock | 5μl |
| Ligation Buffer | 30μl |
| DNA Ligase | 10μl |
| Amount to | 50μl |
After being mixed with the product of 60 μ l previous steps, reaction condition in couveuse is put into as follows：
| Temperature | Time |
| 20℃ | 15min |
| 4℃ | Hold |
2) magnetic beads for purifying is carried out：
1st, AMPure XP magnetic beads are taken out into placement 30min from 4 degree of refrigerators, recovers room temperature；The alcohol of configuration 70%.
2nd, be fully vortexed magnetic bead, and it is in uniform state to make it, and the homogeneous magnetic bead that 88 μ l are added into 110 μ l DNA sample is molten
Liquid, is vortexed and mixes, and stands 5min, and magnetic bead is sunken to ttom of pipe by low-speed centrifugal.
3rd, put magnetic frame and stand 2min, wait liquid to become after clarification, discard supernatant, be careful not to be drawn onto magnetic bead.
4th, 500 μ l 70% alcohol is added into pipe, turne tube 1 is enclosed on magnetic frame, about 30s；Suck after alcohol,
A magnetic bead is washed again；The pipe low-speed centrifugal of alcohol will be sucked, put magnetic frame and stand the alcohol for blotting net remaining, airing magnetic bead.
5th, plus magnetic bead is resuspended without enzyme water in 25 μ l, stand after 2min, low-speed centrifugal after vortex, put after magnetic frame 2min, will
Supernatant is sucked in new EP pipes.
3rd, PCR before hybridizing
1) reaction mixture, such as following table are configured：
| Reagent | Consumption |
| 2X KAPA HiFi HotStart ReadyMix | 25μl |
| 10X KAPA Library Amplification Primer Mix | 5μl |
| Amount to | 30μl |
After being mixed with the purified product of 20 μ l previous steps, reaction condition in PCR instrument is put into as follows：
2) magnetic beads for purifying is carried out：Method is the same, plus 90 μ l magnetic beads, and magnetic bead finally is resuspended with 30 μ l water, clear 30 μ l are sucted, point
1 μ l sample detections clip sizes are not taken and detect sample concentration with Qubit.
4th, hybridize with probe：
The pathogenic bacteria captured in the present embodiment are ETEC, and its 16S rDNA total length reference sequences is：
AGAGTTTGATCATGGCTCAGATTGAACGCTGGCGGCAGGCCTAACACATGCAAGTCGAACGGTAACAGGAAGCAGCT
TGCTGCTTTGCTGACGAGTGGCGGACGGGTGAGTAATGTCTGGGAAACTGCCTGATGGAGGGGGATAACTACTGGAA
ACGGTAGCTAATACCGCATAACGTCGCAAGACCAAAGAGGGGGACCTTCGGGCCTCTTGCCATCGGATGTGCCCAGA
TGGGATTAGCTTGTTGGTGGGGTAACGGCTCACCAAGGCGACGATCCCTAGCTGGTCTGAGAGGATGACCAGCCACA
CTGGAACTGAGACACGGTCCAGACTCCTACGGGAGGCAGCAGTGGGGAATATTGCACAATGGGCGCAAGCCTGATGC
AGCCATGCCGCGTGTATGAAGAAGGCCTTCGGGTTGTAAAGTACTTTCAGCGGGGAGGAAGGGAGTAAAGTTAATAC
CTTTGCTCATTGACGTTACCCGCAGAAGAAGCACCGGCTAACTCCGTGCCAGCAGCCGCGGTAATACGGAGGGTGCA
AGCGTTAATCGGAATTACTGGGCGTAAAGCGCACGCAGGCGGTTTGTTAAGTCAGATGTGAAATCCCCGGGCTCAAC
CTGGGAACTGCATCTGATACTGGCAAGCTTGAGTCTCGTAGAGGGGGGTAGAATTCCAGGTGTAGCGGTGAAATGCG
TAGAGATCTGGAGGAATACCGGTGGCGAAGGCGGCCCCCTGGACGAAGACTGACGCTCAGGTGCGAAAGCGTGGGGA
GCAAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGATGTCGACTTGGAGGTTGTGCCCTTGAGGCGTGGCT
TCCGGAGCTAACGCGTTAAGTCGACCGCCTGGGGAGTACGGCCGCAAGGTTAAAACTCAAATGAATTGACGGGGGCC
CGCACAAGCGGTGGAGCATGTGGTTTAATTCGATGCAACGCGAAGAACCTTACCTGGTCTTGACATCCACAGAACTT
TCCAGAGATGGATTGGTGCCTTCGGGAACTGTGAGACAGGTGCTGCATGGCTGTCGTCAGCTCGTGTTGTGAAATGT
TGGGTTAAGTCCCGCAACGAGCGCAACCCTTATCCTTTGTTGCCAGCGGTCCGGCCGGGAACTCAAAGGAGACTGCC
AGTGATAAACTGGAGGAAGGTGGGGATGACGTCAAGTCATCATGGCCCTTACGACCAGGGCTACACACGTGCTACAA
TGGCGCATACAAAGAGAAGCGACCTCGCGAGAGCAAGCGGACCTCATAAAGTGCGTCGTAGTCCGGATTGGAGTCTG
CAACTCGACTCCATGAAGTCGGAATCGCTAGTAATCGTGGATCAGAATGCCACGGTGAATACGTTCCCGGGCCTTGT
ACACACCGCCCGTCACACCATGGGAGTGGGTTGCAAAAGAAGTAGGTAGCTTAACCTTCGGGAGGGCGCTTACCACT
TTGTGATTCATGACTGGGGTGAAGTCGTAACAAGGTAACCGTAGGGGAACCTGCGGTTG。
Probe used in the present embodiment, for common pathogen 16S rDNA sequences Designs, is that liquid of the present invention is caught
Probe is obtained, design method is ordinary skill in the art means.
1) DNA is prepared:According to the DNA concentration above measured, take 750ng DNA to be concentrated at a temperature of 45 DEG C, concentrate
Being resuspended without enzyme water with 3.4 μ l afterwards.
2) hybridization system is prepared：
Preparing hybrid buffer A
| Reagent | Consumption |
| SureSelect Hyb1 | 6.63μl |
| SureSelect Hyb2 | 0.27μl |
| SureSelect Hyb3 | 2.65μl |
| SureSelect Hyb4 | 3.45μl |
| Amount to | 13μl |
Preparing hybrid blocks liquid B
| Reagent | Consumption |
| SureSelect indexing Block 1 | 2.5μl |
| SureSelect Block 2 | 2.5μl |
| SureSelect ILM Indexing Block 3 | 0.6μl |
| Amount to | 5.6μl |
To the sample (3.4 μ l) being resuspended, 5.6 μ l blocking liquid is added, after mixing, PCR instrument is put into
| Temperature | Time |
| 95℃ | 5min |
| 65℃ | Hold (at least 5min) |
Prepare RNase and block the μ l of liquid C 2 (with water 1：9 mix).
C liquid adds 5 μ l probe, and be fully vortexed 5s, and centrifugation is gently got rid of, 65 DEG C, 2min.
3) 13 μ l A liquid are added in the C liquid of mixed probe, piping and druming for several times, is kept for 65 DEG C.
4) 9 μ l B liquid is added in C mixed liquors, blown and beaten 10 times, keep 65 DEG C of 16h.
5th, the DNA on non-hybridized is eluted, target DNA fragments are reclaimed
1) DynabeadsMyOne Streptavidin T1 magnetic beads are taken out from 4 degree of refrigerators in advance, it is extensive
Multiple room temperature.SureSelect Wash Buffer 2 are put in 65 degree of water-baths and preheated.
2) each sample draws the DynabeadsMyOne Streptavidin T1 magnetic beads mixed in advance
50 μ l are added in EP pipes.
3) 200 μ l SureSelect Binding Buffer are added to it again, are fully mixed.Put magnetic frame standing
1min, becomes after clarification, abandons liquid, be careful not to encounter magnetic bead.Wash altogether 3 times, finally with 200 μ l SureSelect Binding
Magnetic bead is resuspended in Buffer.
4) by the DNA sample after hybridization with 3. in magnetic bead blow and beat mix, put rotation vortex mixer above mix at a slow speed
30min。
5) magnetic frame will be put and stood to clarification, liquid is abandoned and be careful not to encounter magnetic bead after 4. middle mixed liquor gently got rid of.
6) magnetic bead is resuspended with SureSelect Wash Buffer1, after mixing, is stored at room temperature 15min, be vortexed per 3min mixed
It is even once, pipe is put into magnetic frame afterwards and stood to clarification, liquid is abandoned and is careful not to encounter magnetic bead.
7) magnetic frame will be put and stood to clarification, liquid is abandoned and be careful not to encounter magnetic bead after 6. middle mixed liquor gently got rid of.
8) with (65 DEG C) resuspension magnetic beads of SureSelect Wash Buffer2, after mixing, 65 DEG C of incubation 10min, per 3min
It is vortexed and mixes once, pipe is put into magnetic frame afterwards and stood to clarification, liquid is abandoned and is careful not to encounter magnetic bead.Repetition is washed 3 times.
9) add 30 μ l that magnetic bead is resuspended without enzyme water
6th, PCR after hybridizing
1) reaction mixture, such as following table are configured：
Each sample adds 1 μ l Tag primer；After being mixed with the product of 14 μ l previous steps, it is put into PCR instrument.Reaction condition
It is as follows：
2) magnetic beads for purifying is carried out：Method is the same, plus 90 μ l magnetic beads, and magnetic bead finally is resuspended with 30 μ l water, clear 28 μ l are sucted most
Take 1 μ l library detections clip sizes respectively afterwards and detect sample concentration with Qubit HS.
4th, sequencing reaction
WithHiseq1500 high-flux sequences instrument carries out sequencing reaction, and data predicting output is 3G.
5th, data analysis
Sequencing data is analyzed and Pathogenic Microorganisms On Tropical scheme is divided into following steps in principle：
(1) Quality Control of machine data and sequence measuring joints are gone under.Original lower machine sequence needs to remove the sequence for containing " N " and removal connects
Header sequence pollutes.We are used as base quality control standard using Q20 simultaneously.
(2) DNA sequence dna of Hosts is filtered.It is soft by bwa using UCSC hg19 as host genome reference sequences
Part carries out sequence alignment analysis.In paired-end sequencings, either end can compare the sequence antithetical phrase meeting of host genome
It is removed.
(3) De novo assemble microorganism contig and scaffold fragment.Genome is carried out using metaSpades softwares
De novo are assembled.Select the candidate microbial fragment after assembling, it is desirable to：A. more than sequence length 800bp；B. compared through blast
The known strain 16s sequence libraries that NCBI is included can obtain consistent comparison result with full length sequence database；C. one is compared
Cause rate>=95%, cover fragment>=85%.Reject and compare to host genome or the biological fragment of its nearly edge.
(4) candidate segment Quality Control.The candidate segment of identification is carried out as reference sequences using bwa softwares using in (3)
Sequence alignment.It is required that：A. per bar segment non-duplicate aligned sequences (non-duplicated read) after comparison>=10X is covered
(coverage) base accounting>=80%, to ensure the reliability of De novo processes in (3)；B. the covering per bar segment will
Seek preferable homogeneity (uniformly coverage).
(5) identification pathogenic microorganism and report.The identification precision of 16s sequences for bacteria can reach " planting (species) "
Rank.Therefore the probation report first choice of pathogenic microorganism is " strain ".When candidate segment points to the not of the same race of same subordinate simultaneously
During microorganism, then flagship report " Pseudomonas ", and " strain " of mark identification respectively.
It is ETEC to detect comparing analysis result, as shown in Figure 1.
Embodiment 2
The Elderly male patient of samples sources one, clinical diagnosis is severe pneumonia, and Sputum culturing is Acinetobacter bauamnnii, blood culture
As a result it is negative.
First, peripheral blood, centrifugal separation plasma are gathered
Preserved on request using cfDNA special blood-drawing pipe conventional Christmas peripheral bloods 6ml；2500g is centrifuged 10 minutes, rear to extract
Supernatant blood plasma 20000g is centrifuged 10 minutes, extracts supernatant blood plasma 3ml.
2nd, plasma circulation DNA is extracted using kit QIAamp Circulating Nucleic Acid Kit on request
1st, 3ml blood plasma, 300 μ l Proteinase K, 2.4ml Buffer ACL are added in 50ml centrifuge tubes
(the μ g carrier RNA of containing 1.0), vortex 30s puts 60 DEG C of incubation 30min.
2nd, centrifuge tube is taken out, 5.4ml Buffer ACB, vortex 30s is added, puts and be incubated 5min on ice.
3rd, connection negative pressure extraction device QIAvac 24Plus, QIAamp Mini column and 20ml tube extender,
Negative pressure -900mbar is adjusted, slow to cross mixture in the above-mentioned centrifuge tube of post, rear order adds 600 μ l Buffer ACW1,750 μ l
Buffer ACW2,750 μ l 100%ethanol cross post elution.
4th, QIAamp Mini column are taken out and are put into 2ml EP pipes, 20000g centrifugations 3min.
5th, QIAamp Mini column are taken out and are put into lid in new 2ml EP pipes, opening, 56 DEG C of drying 10min.
6th, QIAamp Mini column are taken out and are put into new 1.5ml EP pipes, to QIAamp Mini column filtration membranes
75 μ l of Buffer AVE of upper addition, close the lid, and normal temperature is incubated 3min.
7th, 20000g centrifuges 3min, takes filtered solution to load new 1.5ml EP pipes and preserves.
8th, Qubit detects sample concentration, 2100 detection clip sizes.
3rd, set up genomic library and (build storehouse kit：KAPA Hyper Prep Kit KK8504 96libraries；
SureSelect XT Library Prep Kit ILM cat#5500-0133；SureSelect Target Enrichment
System 1-800-227-9770；Herculase II Fusion Enzyme with dNTP Combo Cat#600679；
SureSelect Target Enrichment Kit ILM Indexing Hyb Module Box 2 Cat#5190-4455)
1st, DNA fragmentation end is repaired and end " A " tail
1) mixed liquor of configuration reaction, such as following table：
| Reagent | Consumption |
| End Repair&A-Tailing Buffer | 7μl |
| End Repair&A-Tailing Enzyme Mix | 3μl |
| Amount to | 10μl |
After being mixed with 50 μ l 100ngDNA samples, reaction condition in couveuse is put into as follows：
| Temperature | Time |
| 20℃ | 30min |
| 65℃ | 30min |
| 4℃ | Hold |
2nd, double end adjunction heads
1) reaction mixture, such as following table are configured：
| Reagent | Consumption |
| Nuclease-free water | 5μl |
| Adapter stock | 5μl |
| Ligation Buffer | 30μl |
| DNA Ligase | 10μl |
| Amount to | 50μl |
After being mixed with the product of 60 μ l previous steps, reaction condition in couveuse is put into as follows：
| Temperature | Time |
| 20℃ | 15min |
| 4℃ | Hold |
2) magnetic beads for purifying is carried out：
(1) AMPure XP magnetic beads are taken out into placement 30min from 4 degree of refrigerators, recovers room temperature；The alcohol of configuration 70%.
(2) be fully vortexed magnetic bead, and it is in uniform state to make it, and the homogeneous magnetic bead that 88 μ l are added into 110 μ l DNA sample is molten
Liquid, is vortexed and mixes, and stands 5min, and magnetic bead is sunken to ttom of pipe by low-speed centrifugal.
(3) put magnetic frame and stand 2min, wait liquid to become after clarification, discard supernatant, be careful not to be drawn onto magnetic bead.
(4) 500 μ l 70% alcohol is added into pipe, turne tube 1 is enclosed on magnetic frame, about 30s；Suck after alcohol,
A magnetic bead is washed again；The pipe low-speed centrifugal of alcohol will be sucked, put magnetic frame and stand the alcohol for blotting net remaining, airing magnetic bead.
(5) plus magnetic bead is resuspended without enzyme water in 25 μ l, stand after 2min, low-speed centrifugal after vortex, put after magnetic frame 2min, will
Supernatant is sucked in new EP pipes.
3rd, PCR before hybridizing
1) reaction mixture, such as following table are configured：
| Reagent | Consumption |
| 2X KAPA HiFi HotStart ReadyMix | 25μl |
| 10X KAPA Library Amplification Primer Mix | 5μl |
| Amount to | 30μl |
After being mixed with the purified product of 20 μ l previous steps, reaction condition in PCR instrument is put into as follows：
2) magnetic beads for purifying is carried out：Method is the same, plus 90 μ l magnetic beads, and magnetic bead finally is resuspended with 30 μ l water, clear 30 μ l are sucted, point
1 μ l sample detections clip sizes are not taken and detect sample concentration with Qubit.
4th, hybridize with probe：
The pathogenic bacteria captured in the present embodiment are Acinetobacter bauamnnii, and its 16S rDNA total length reference sequences is：
CGCACACTACGGCCGGGAGTCCTGTGGGGAGCTATCTCCTACTATTTGTGGCCCACCGCCCACACCCACATCCCAGC
CTTGCATAAGAAGGCGAAGGAGGCAATAAAGGTTTTAAAGCACGTTGAGGGGTGGTCCAGTCTTATTCTACCTAATA
CCTAGGGGATAGTTGCCCGTACTAAGCAGAATAAGCATCTGTTTAACTCCGCGCCAGCAGCCGCGGTAATACAGAGG
GTGCGAGCGTTAATCTGATGTACTGAGCGTAAAGCGTGCGTAGGCGGCTTATTAAGTCGGATGTGAAATCCCCGAGC
TTAACTTGTGAATTGCATTCGATACTGGTGAGCTAGAGTATGGGAGAGGATGGTAGAATTCCAGGTGTAGCGGTGAA
ATGCGTAGAGATCTGGAGGAATACCGATGGCGAAGGCAGCCATCTGGCCTAATACTGACGCTGAGGTACGAAAGCAT
GGGGAGCAAACAGGATTAGATACCCTGGTAGTCCATGCCGTAAACGATGTCTACTAGCCGTTGGGGCCTTTGAGGCT
TTAGTGGCGCAGCTAACGCGATAAGTAGACCGCCTGGGGAGTACGGTCGCAAGACTAAAACTCAAATGAATTGACGG
GGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGATGCAACGCGAAGAACCTTACCTGGCCTTGACATACTAGA
AACTTTCCAGAGATGGATTGGTGCCTTCGGGAATCTAGATACAGGTGCTGCATGGCTGTCGTCAGCTCGTGTCGTGA
GATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTTTCCTTACTTGCCAGCATTTCGGATGGGAACTTTAAGGATA
CTGCCAGTGACAAACTGGAGGAAGGCGGGGACGACGTCAAGTCATCATGGCCCTTACGGCCAGGGCTACACACGTGC
TACAATGGTCGGTACAAAGGGTTGCTACACAGCGATGTGATGCTAATCTCAAAAAGCCGATCGTAGTCCGGATTGGA
GTCTGCAACTCGACTCCATGAAGTCGGAATCGCTAGTAATCGCGGATCAGAATGCCGCGGTGAATACGTTCCCGGGC
CTTGTACACACCGCCCGTCACACCATGGGAGTTTGTTGCACCAGAAGTAGCTAGCCTAACTGCAAAGAGGGCGGTTA
CCACGGTGGGCCGATGACTGGGGTGAAGTCGTATCAAGGACGACC。
Probe used in the present embodiment, for common pathogen 16S rDNA sequences Designs, is that liquid of the present invention is caught
Probe is obtained, design method is ordinary skill in the art means.
1) DNA is prepared:According to the DNA concentration above measured, take 750ng DNA to be concentrated at a temperature of 45 DEG C, concentrate
Being resuspended without enzyme water with 3.4 μ l afterwards.
2) hybridization system is prepared：
Preparing hybrid buffer A
| Reagent | Consumption |
| SureSelect Hyb1 | 6.63μl |
| SureSelect Hyb2 | 0.27μl |
| SureSelect Hyb3 | 2.65μl |
| SureSelect Hyb4 | 3.45μl |
| Amount to | 13μl |
Preparing hybrid blocks liquid B
| Reagent | Consumption |
| SureSelect indexing Block 1 | 2.5μl |
| SureSelect Block 2 | 2.5μl |
| SureSelect ILM Indexing Block 3 | 0.6μl |
| Amount to | 5.6μl |
To the sample (3.4 μ l) being resuspended, 5.6 μ l blocking liquid is added, after mixing, PCR instrument is put into
| Temperature | Time |
| 95℃ | 5min |
| 65℃ | Hold (at least 5min) |
Prepare RNase and block the μ l of liquid C 2 (with water 1：9 mix).
C liquid adds 5 μ l probe, and be fully vortexed 5s, and centrifugation is gently got rid of, 65 DEG C, 2min.
3) 13 μ l A liquid are added in the C liquid of mixed probe, piping and druming for several times, is kept for 65 DEG C.
4) 9 μ l B liquid is added in C mixed liquors, blown and beaten 10 times, keep 65 DEG C of 16h.
5th, the DNA on non-hybridized is eluted, target DNA fragments are reclaimed
1) DynabeadsMyOne Streptavidin T1 magnetic beads are taken out from 4 degree of refrigerators in advance, it is extensive
Multiple room temperature.SureSelect Wash Buffer 2 are put in 65 degree of water-baths and preheated.
2) each sample draws the DynabeadsMyOne Streptavidin T1 magnetic beads mixed in advance
50 μ l are added in EP pipes.
3) 200 μ l SureSelect Binding Buffer are added to it again, are fully mixed.Put magnetic frame standing
1min, becomes after clarification, abandons liquid, be careful not to encounter magnetic bead.Wash altogether 3 times, finally with 200 μ l SureSelect Binding
Magnetic bead is resuspended in Buffer.
4) by the DNA sample after hybridization with 3. in magnetic bead blow and beat mix, put rotation vortex mixer above mix at a slow speed
30min。
5) magnetic frame will be put and stood to clarification, liquid is abandoned and be careful not to encounter magnetic bead after 4. middle mixed liquor gently got rid of.
6) magnetic bead is resuspended with SureSelect Wash Buffer1, after mixing, is stored at room temperature 15min, be vortexed per 3min mixed
It is even once, pipe is put into magnetic frame afterwards and stood to clarification, liquid is abandoned and is careful not to encounter magnetic bead.
7) magnetic frame will be put and stood to clarification, liquid is abandoned and be careful not to encounter magnetic bead after 6. middle mixed liquor gently got rid of.
8) with (65 DEG C) resuspension magnetic beads of SureSelect Wash Buffer2, after mixing, 65 DEG C of incubation 10min, per 3min
It is vortexed and mixes once, pipe is put into magnetic frame afterwards and stood to clarification, liquid is abandoned and is careful not to encounter magnetic bead.Repetition is washed 3 times.
9) add 30 μ l that magnetic bead is resuspended without enzyme water
6th, PCR after hybridizing
1) reaction mixture, such as following table are configured：
Each sample adds 1 μ l Tag primer；After being mixed with the product of 14 μ l previous steps, it is put into PCR instrument.Reaction condition
It is as follows：
2) magnetic beads for purifying is carried out：Method is the same, plus 90 μ l magnetic beads, and magnetic bead finally is resuspended with 30 μ l water, clear 28 μ l are sucted most
Take 1 μ l library detections clip sizes respectively afterwards and detect sample concentration with Qubit HS.
4th, sequencing reaction
WithHiseq1500 high-flux sequences instrument carries out sequencing reaction, and data predicting output is 3G.
5th, data analysis
Sequencing data is analyzed and Pathogenic Microorganisms On Tropical scheme is divided into following steps in principle：
(1) Quality Control of machine data and sequence measuring joints are gone under.Original lower machine sequence needs to remove the sequence for containing " N " and removal connects
Header sequence pollutes.We are used as base quality control standard using Q20 simultaneously.
(2) DNA sequence dna of Hosts is filtered.It is soft by bwa using UCSC hg19 as host genome reference sequences
Part carries out sequence alignment analysis.In paired-end sequencings, either end can compare the sequence antithetical phrase meeting of host genome
It is removed.
(3) De novo assemble microorganism contig and scaffold fragment.Genome is carried out using metaSpades softwares
De novo are assembled.Select the candidate microbial fragment after assembling, it is desirable to：A. more than sequence length 800bp；B. compared through blast
The known strain 16s sequence libraries that NCBI is included can obtain consistent comparison result with full length sequence database；C. one is compared
Cause rate>=95%, cover fragment>=85%.Reject and compare to host genome or the biological fragment of its nearly edge.
(4) candidate segment Quality Control.The candidate segment of identification is carried out as reference sequences using bwa softwares using in (3)
Sequence alignment.It is required that：A. per bar segment non-duplicate aligned sequences (non-duplicated read) after comparison>=10X is covered
(coverage) base accounting>=80%, to ensure the reliability of De novo processes in (3)；B. the covering per bar segment will
Seek preferable homogeneity (uniformly coverage).
(5) identification pathogenic microorganism and report.The identification precision of 16s sequences for bacteria can reach " planting (species) "
Rank.Therefore the probation report first choice of pathogenic microorganism is " strain ".When candidate segment points to the not of the same race of same subordinate simultaneously
During microorganism, then flagship report " Pseudomonas ", and " strain " of mark identification respectively.
Data results：Analyzed through above-mentioned bioinformatics method, identify 2 candidate pathogens antimicrobial moieties,
Originate as Acinetobacter bauamnnii.As a result it is consistent with infection focus body fluid cultivation results, as shown in Figure 2.
Finally, it should be noted that the above embodiments are merely illustrative of the technical solutions of the present invention rather than the present invention is protected
The limitation of scope is protected, although being explained in detail with reference to preferred embodiment to the present invention, one of ordinary skill in the art should
Understand, technical scheme can be modified or equivalent substitution, without departing from the essence of technical solution of the present invention
And scope.
Sequence table
<110>Zhongshan Univ. Cancer Cure Center
<120>A kind of material, kit and method detected for systemic infection
<130> 2017
<160> 2
<170> PatentIn version 3.3
<210> 1
<211> 1522
<212> DNA
<213>ETEC
<400> 1
agagtttgat catggctcag attgaacgct ggcggcaggc ctaacacatg caagtcgaac 60
ggtaacagga agcagcttgc tgctttgctg acgagtggcg gacgggtgag taatgtctgg 120
gaaactgcct gatggagggg gataactact ggaaacggta gctaataccg cataacgtcg 180
caagaccaaa gagggggacc ttcgggcctc ttgccatcgg atgtgcccag atgggattag 240
cttgttggtg gggtaacggc tcaccaaggc gacgatccct agctggtctg agaggatgac 300
cagccacact ggaactgaga cacggtccag actcctacgg gaggcagcag tggggaatat 360
tgcacaatgg gcgcaagcct gatgcagcca tgccgcgtgt atgaagaagg ccttcgggtt 420
gtaaagtact ttcagcgggg aggaagggag taaagttaat acctttgctc attgacgtta 480
cccgcagaag aagcaccggc taactccgtg ccagcagccg cggtaatacg gagggtgcaa 540
gcgttaatcg gaattactgg gcgtaaagcg cacgcaggcg gtttgttaag tcagatgtga 600
aatccccggg ctcaacctgg gaactgcatc tgatactggc aagcttgagt ctcgtagagg 660
ggggtagaat tccaggtgta gcggtgaaat gcgtagagat ctggaggaat accggtggcg 720
aaggcggccc cctggacgaa gactgacgct caggtgcgaa agcgtgggga gcaaacagga 780
ttagataccc tggtagtcca cgccgtaaac gatgtcgact tggaggttgt gcccttgagg 840
cgtggcttcc ggagctaacg cgttaagtcg accgcctggg gagtacggcc gcaaggttaa 900
aactcaaatg aattgacggg ggcccgcaca agcggtggag catgtggttt aattcgatgc 960
aacgcgaaga accttacctg gtcttgacat ccacagaact ttccagagat ggattggtgc 1020
cttcgggaac tgtgagacag gtgctgcatg gctgtcgtca gctcgtgttg tgaaatgttg 1080
ggttaagtcc cgcaacgagc gcaaccctta tcctttgttg ccagcggtcc ggccgggaac 1140
tcaaaggaga ctgccagtga taaactggag gaaggtgggg atgacgtcaa gtcatcatgg 1200
cccttacgac cagggctaca cacgtgctac aatggcgcat acaaagagaa gcgacctcgc 1260
gagagcaagc ggacctcata aagtgcgtcg tagtccggat tggagtctgc aactcgactc 1320
catgaagtcg gaatcgctag taatcgtgga tcagaatgcc acggtgaata cgttcccggg 1380
ccttgtacac accgcccgtc acaccatggg agtgggttgc aaaagaagta ggtagcttaa 1440
ccttcgggag ggcgcttacc actttgtgat tcatgactgg ggtgaagtcg taacaaggta 1500
accgtagggg aacctgcggt tg 1522
<210> 2
<211> 1200
<212> DNA
<213>Acinetobacter bauamnnii
<400> 2
cgcacactac ggccgggagt cctgtgggga gctatctcct actatttgtg gcccaccgcc 60
cacacccaca tcccagcctt gcataagaag gcgaaggagg caataaaggt tttaaagcac 120
gttgaggggt ggtccagtct tattctacct aatacctagg ggatagttgc ccgtactaag 180
cagaataagc atctgtttaa ctccgcgcca gcagccgcgg taatacagag ggtgcgagcg 240
ttaatctgat gtactgagcg taaagcgtgc gtaggcggct tattaagtcg gatgtgaaat 300
ccccgagctt aacttgtgaa ttgcattcga tactggtgag ctagagtatg ggagaggatg 360
gtagaattcc aggtgtagcg gtgaaatgcg tagagatctg gaggaatacc gatggcgaag 420
gcagccatct ggcctaatac tgacgctgag gtacgaaagc atggggagca aacaggatta 480
gataccctgg tagtccatgc cgtaaacgat gtctactagc cgttggggcc tttgaggctt 540
tagtggcgca gctaacgcga taagtagacc gcctggggag tacggtcgca agactaaaac 600
tcaaatgaat tgacgggggc ccgcacaagc ggtggagcat gtggtttaat tcgatgcaac 660
gcgaagaacc ttacctggcc ttgacatact agaaactttc cagagatgga ttggtgcctt 720
cgggaatcta gatacaggtg ctgcatggct gtcgtcagct cgtgtcgtga gatgttgggt 780
taagtcccgc aacgagcgca acccttttcc ttacttgcca gcatttcgga tgggaacttt 840
aaggatactg ccagtgacaa actggaggaa ggcggggacg acgtcaagtc atcatggccc 900
ttacggccag ggctacacac gtgctacaat ggtcggtaca aagggttgct acacagcgat 960
gtgatgctaa tctcaaaaag ccgatcgtag tccggattgg agtctgcaac tcgactccat 1020
gaagtcggaa tcgctagtaa tcgcggatca gaatgccgcg gtgaatacgt tcccgggcct 1080
tgtacacacc gcccgtcaca ccatgggagt ttgttgcacc agaagtagct agcctaactg 1140
caaagagggc ggttaccacg gtgggccgat gactggggtg aagtcgtatc aaggacgacc 1200
Claims (10)
1. a kind of material detected for systemic infection, it is characterised in that the material is derived from pathogenic bacteria and collapsed in vivo
Solve the Circulating DNA of release.
2. a kind of kit detected for systemic infection, it is characterised in that the kit includes being used to be enriched with pathogenic bacteria
The capturing liquid chip of the Circulating DNA in source.
3. kit according to claim 2, it is characterised in that the kit includes being used to be enriched with common pathogen
The capturing liquid probe of 16S rDNA sequences.
4. a kind of method that systemic infection is detected using kit described in claim 3, it is characterised in that methods described bag
Include following steps：
(1) peripheral blood, centrifugal separation plasma are gathered；
(2) plasma circulation DNA is extracted；
(3) Circulating DNA concentration is detected；
(4) expanded before end reparation plus A tails, jointing, capture are carried out to Circulating DNA；
(5) hybrid capture is carried out with the capturing liquid probe, target dna is enriched with；
(6) expand and tag after capturing, carrying out sequencing using two generation sequencing technologies obtains its sequence；
(7) by comparing analysis with existing bacterial 16 S rDNA reference sequences, bacterial species and sxemiquantitative in peripheral blood are determined.
5. method according to claim 4, it is characterised in that in the step (1), peripheral blood collection is special using cfDNA
Heparin tube；
The separated plasma comprises the following steps：By blood plasma 1000g~2500g low-speed centrifugals 10 minutes, supernatant blood plasma is extracted,
20000g high speed centrifugations 10 minutes, extract supernatant blood plasma.
6. method according to claim 4, it is characterised in that in the step (2), using the kit of Qiagen companies
QIAamp Circulating Nucleic Acid Kit extract Circulating DNA on request；
In the step (3), DNA concentration is detected using Qubit.
7. method according to claim 4, it is characterised in that in the step (4), (5), (6), using kit KAPA
Hyper Prep Kit carry out building storehouse on request.
8. method according to claim 4, it is characterised in that the two generations sequencing technologies are illumina Hiseq.
9. method according to claim 4, it is characterised in that the analysis of the sequencing data obtained in the step (6) include with
Lower step：
S1, lower machine data Quality Control and go sequence measuring joints：Original lower machine sequence needs to remove the sequence containing " N " and removes joint sequence
Row pollution, while being used as base quality control standard using Q20；
S2, the DNA sequence dna for filtering Hosts：Using UCSC hg19 as host genome reference sequences, entered by bwa softwares
Row sequence alignment analysis；In paired-end sequencings, the sequence antithetical phrase that either end can compare host genome can be picked
Remove；
S3, De novo assemble microorganism contig and scaffold fragment：Genome De is carried out using metaSpades softwares
Novo is assembled, and selects the candidate microbial fragment after assembling, it is desirable to：A, more than sequence length 800bp；B, compare through blast
The known strain 16s rDNA sequence libraries that NCBI is included can obtain consistent comparison result with full length sequence database；C, ratio
To concordance rate >=95%, fragment >=85% is covered；Reject and compare to host genome or the biological fragment of its nearly edge；
S4, candidate segment Quality Control：Using the candidate segment of identification as reference sequences, sequence alignment is carried out using bwa softwares；
It is required that：A, base accounting >=80% that non-duplicate aligned sequences >=10X is covered after comparison per bar segment, to ensure De novo
The reliability of process；B, the covering per bar segment require preferable homogeneity；
S5, identification pathogenic microorganism and report：The identification precision of 16s rDNA sequences for bacteria can reach the rank of " kind ", therefore
The probation report first choice of pathogenic microorganism is " strain ", when candidate segment points to the not same species of microorganism of same subordinate simultaneously,
Then flagship report " Pseudomonas ", and " strain " of mark identification respectively.
10. the Circulating DNA that pathogenic bacteria are disintegrated release in vivo is screening or prepared the reagent diagnosed for systemic infection or examination
Purposes in agent box.
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| CN110343754A (en) * | 2019-08-15 | 2019-10-18 | 深圳谱元科技有限公司 | A method of it is quickly detected for hematopoietic stem cell transplantation donor pathogenic microorganism |
| CN111154905A (en) * | 2019-06-11 | 2020-05-15 | 青海大学附属医院 | Method and kit for detecting hydatid cyst infection |
| CN111187813A (en) * | 2020-02-20 | 2020-05-22 | 予果生物科技（北京）有限公司 | Full-process quality control pathogenic microorganism high-throughput sequencing detection method |
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| CN111411144B (en) * | 2020-04-21 | 2023-12-22 | 深圳华大因源医药科技有限公司 | Plasma free DNA marker for diagnosing blood flow infection pathogen |
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