CN110343754A - A method of it is quickly detected for hematopoietic stem cell transplantation donor pathogenic microorganism - Google Patents
A method of it is quickly detected for hematopoietic stem cell transplantation donor pathogenic microorganism Download PDFInfo
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Abstract
The invention discloses a kind of methods quickly detected for hematopoietic stem cell transplantation donor pathogenic microorganism, comprising the following steps: sample collection procedure；Reagent preparation process；CfDNA is extracted and sequencing library construction step；RNA is extracted and sequencing library construction step；Genome DNA extraction and amplicon sequencing library construction step；The variable region 16S rRNA amplification step；ITS amplification step；Sequencing steps；Analytical procedure.Detection method of the invention combines macro genome, macro transcript profile and amplicon sequencing approach, detects the suspicious pathogenic microorganism of hematopoietic stem cell transplantation donor, provides diagnosis and treatment foundation for transplanting safety, reduces transplant infection risk.
Description
Technical field
The present invention relates to molecular biology and molecular genetics field, more particularly to a kind of hematopoietic stem cell transplantation that is used for supply
The method that body pathogenic microorganism quickly detects.
Background technique
For leukaemia, lymthoma, etc. poor prognosis Malignancy patient (hereinafter referred to as leukaemia suffer from
Person), after marrow and hematopoietic stem cell transplantation, in inhibiting drug-induced immune function to be suppressed under state, occurs to be originated from and contribute
" the field planting microorganism " of contributor, " opportunist ", " sepsis caused by outburst infection after caused leukaemic's transplanting
Disease ", " hemorrhagic enteritis ", " hemorrhagic cystitis ", " pneumonia ", etc., this Infective morbidity clinical symptoms are usually very dangerous,
With huge medical treatment, spirit, economy and society burden.
Conventional donor's physical examination is whether body temperature, blood routine, routine urinalysis, biochemical investigation confirm donor " health ",
Whether donor can not be accurately confirmed without " pathogenic microorganisms ", this is so that leukaemic is thin in marrow and Hematopoietic Stem
Infected during hematopoietic reconstitution (12 months) and immunologic reconstitution (24-36 month) after born of the same parents' transplanting occurrence probability close to 80% it is most heavy
Want negative factor.
Traditional Pathogenic Microorganisms On Tropical technology has played important function in daily pathogenic microorganism confirmation, but there is also
Certain deficiency: it needs to rely on culture, period length, identify that precision is low；Need certain microorganism sequence priori knowledge, Wu Faying
To unknown or mutation pathogen etc..Therefore, it is necessary to propose a kind of for being applied to currently used pathogen detection method.
The information disclosed in the background technology section is intended only to deepen understanding of the general background technology to the present invention, and
It is not construed as recognizing or implying in any form that the information constitutes the prior art known to those skilled in the art.
For these reasons, present applicant has proposed one kind quickly examines for hematopoietic stem cell transplantation donor pathogenic microorganism
The method of survey, it is intended to which satisfaction solves the above problems.
Summary of the invention
In order to meet above-mentioned requirements, it is micro- for hematopoietic stem cell transplantation donor cause of disease that the purpose of the present invention is to provide one kind
The method that biology quickly detects can detect suspicious pathogenic microorganism in its blood for clinical hematopoietic stem cell transplantation donor,
Transplant infection risk is effectively reduced in realization, it is therefore intended that is currently known with technological means detection simple, fast, accurately and comprehensively
Include the hundreds of thousands of kinds of microorganisms such as bacterium, fungi, DNA virus, RNA virus, helminth.
To achieve the goals above, the invention adopts the following technical scheme:
A method of it is quickly detected for hematopoietic stem cell transplantation donor pathogenic microorganism, comprising the following steps:
Sample collection procedure collects peripheral blood 5ml using STRECK heparin tube, and 6-30 DEG C saves, separated plasma in 8h, entirely
Blood plasma separating experiment is carried out first before blood sample detection；
After the completion of blood plasma separation test, takes 400 μ l blood plasma to extract for cfDNA and RNA respectively, separately take 200 μ of haemocyte
L is used for Genome DNA extraction；
Reagent preparation process；
CfDNA is extracted and sequencing library construction step, takes 400 μ l blood plasma, and reaction reagent buffer GB, Lysis is added
Enzyme K, RNA Carrier react 10min at 56 DEG C；Reaction solution adsorbs column purification through pellosil, uses cleaning buffer solution
GD, PW remove isolating protein, salt ion and organic reagent, obtain the satisfactory DNA of total amount, purity；The DNA is repaired through end
Multiple, connector connection, amplified library enrichment, purifying, Quality Control step process, complete the library cfDNA that building is completed, and utilize
Illumina sequenator matches the library cfDNA, and sequence is read on sequenator；
RNA is extracted and sequencing library construction step, takes 400 μ l blood plasma, and reaction reagent lysate RZ is added and releases sample
In RNA, chloroform is added, dehydrated alcohol removes protein-based impurity；Reaction solution adsorbs column purification through pellosil, slow using cleaning
Fliud flushing RD, RW removes isolating protein, salt ion and organic reagent, obtains the satisfactory RNA of total amount, purity；The RNA is through segment
Change, reverse transcription and the synthesis of bis- chain of cDNA, end are repaired, connector connection, amplified library enrichment, are purified, Quality Control step process, are completed
The library RNA completed is constructed, the library RNA is matched using Illumina sequenator, sequence is read on sequenator；
Genome DNA extraction and amplicon sequencing library construction step take 200 μ l haemocytes, extract according to spectral element standard nucleic acid
Process extracts total DNA, wherein microbial cell breakage uses bead grinding and combines chemical cracking and enzyme solution, is used for
Extract the DNA of all microorganisms in sample；
The variable region 16S rRNA amplification step, taking total DNA is template, using bacterial universal primers, HiFi PCR enzyme,
DdNTP effect is lower to realize the amplification of the variable region bacterial 16 S rRNA, and gained amplified production is purified using Agencourt AMPure XP
Afterwards and detect its clip size and mass concentration；
ITS amplification step, taking total DNA is template, real under HiFi PCR enzyme, ddNTP effect using fungi universal primer
Existing fungi ITS fragment amplification, gained amplified production using Agencourt AMPure XP after purification and detect its clip size and
Mass concentration；
Sequencing steps, the library cfDNA or the library RNA after NaOH denaturation is single-stranded, are integrated on sequence testing chip,
Each single strand dna or RNA molecule enrichment are become into a cluster through bridge amplification, then are sequenced with Illumina SBS
Method reads sequence, obtains sequencing data；
Analytical procedure, the sequencing data is through data Quality Control, filtering source of people sequence and joint sequence and microorganism reference number
Step is compared according to library, identifies the suspicious pathogenic microorganism in sample.
Wherein, the cfDNA extract and sequencing library construction step in cfDNA extract using chemical cracking and enzymatic hydrolysis
Method, primitive plasma sample adsorb column purification through pellosil, obtain after buffer GB and Lysis Enzyme K cracking digestion
The satisfactory DNA of total amount, purity；
In the RNA extraction and sequencing library construction step, RNA is extracted using chemical cleavage method, original blood
Sample is starched after lysate RZ cracking, makes protein denaturation in sample using chloroform, RNA is precipitated using dehydrated alcohol, through silica gel
Film adsorption column obtains the satisfactory RNA of total amount, purity after purification.
The Genome DNA extraction and amplicon sequencing library construction step further include Total DNA extraction method, specifically: blood is thin
Born of the same parents' sample releases total DNA through chemical reagent cracking, Mechanical Crushing, enzymatic hydrolysis etc., then through Buffer BB precipitated impurities, supernatant
Column purification is adsorbed using pellosil, obtains total DNA.
The cfDNA library construction is specifically divided into the following steps again:
1. end is repaired: obtained DNA, need to be through end due to the case where there may be end is uneven and chain damages after extraction
End repair after by double-stranded DNA end-filling and repair chain damage.Prevent from influencing the success rate of subsequent reactions；
This reaction needed for reagent be 5 μ l EndPrep Enzyme and 5 μ l EndPrep Buffer, enzyme action time and
Enzyme deactivation time is 30min；
2. connector connects: the DNA after end is repaired can be with connector (Adapter) in Ligation Mix and Ligation
Connection reaction occurs under Enhancer collective effect；
Each detailed dosage of reagent are as follows: 2 μ l of adapter dosage；30 μ l of Ligation Mix dosage；Ligation
1 μ l of Enhancer dosage.The reaction time is 15min after reaction reagent and DNA mixing；
3. purifying after connection: connection product need to carry out after purification PCR enrichment through AMPure XP beads；
Specific purification procedures are as follows: after connection product is mixed with 50 μ l magnetic beads, magnetic bead can will be after connection under room temperature
DNA absorption, magnetic bead can remove remaining ingredient after magnetic frame adsorbs, and being washed using 80% dehydrated alcohol can elute twice
Obtain non junction, enzyme, other salt ions and the higher connection product of purity of organic reagent pollution；
4. library is enriched with: connection product can carry out under primer, DNA (ddNTP), HiFi amplification enzyme effect
PCR amplification obtains the higher library of total amount；
5. library purifying and Quality Control: library can be detected after purification through AMPure XP beads after PCR enrichment；1 μ l is taken to use
It is measured in Qubit nucleic acid concentration；It takes 2 μ l to detect for agarose gel electrophoresis and determines library fragments size.
The RNA library construction is specifically divided into the following steps:
1. RNA fragmentation: extracting gained total serum IgE, appropriate RNA Fragmentation Buffer and Nuclease- is added
Free Water reacts 4min under the conditions of 94 DEG C, the small fragment that can be about 450bp by long-chain RNA random shearing；
2. RNA reverse transcription: clipped small fragment RNA is added gDNA digestive ferment, cDNA synthetic agent in system, 42 DEG C
20min is reacted, so that the gDNA polluted in sample is degraded and makes RNA reverse transcription cDNA；
3. bis- chain of cDNA synthesizes: dNTPs, archaeal dna polymerase, RNAseH etc. being added in previous step reaction system, with single-stranded
CDNA is templated synthesis double-stranded DNA.Reaction system AMPure XP beads is used to react in next step after purification.Concrete operations step
Suddenly it is mixed for reaction system with magnetic bead 1:1, magnetic bead adsorbs double-stranded DNA under room temperature, is washed twice using 80% dehydrated alcohol
The double-stranded DNA polluted without other albumen, inorganic salts, organic reagent can be afforded；
4. end is repaired: the case where double-stranded DNA is damaged there may be end and chain, it need to be after end be repaired by double-stranded DNA
End-filling and repair chain damage.Reagent needed for this reaction is 5 μ l EndPrep Enzyme and 5 μ l EndPrep
Buffer, enzyme action time and enzyme deactivation time are 30min；
5. connector connects: the DNA after end is repaired can be with connector (Adapter) in Ligation Mix and Ligation
Connection reaction occurs under Enhancer collective effect.Each detailed dosage of reagent are as follows: 2 μ l of adapter dosage；Ligation Mix is used
Measure 30 μ l；1 μ l of Ligation Enhancer dosage.The reaction time is 15min after reaction reagent and DNA mixing；
6. purifying after connection: connection product need to carry out after purification PCR enrichment through AMPure XP beads.Specific purifying behaviour
Make step are as follows: after connection product is mixed with magnetic bead, magnetic bead can adsorb the DNA after connection under room temperature, and magnetic bead is through magnetic frame
Remaining ingredient can be removed after absorption, washed using 80% dehydrated alcohol can afford twice non junction, enzyme, other salt from
Son and the higher connection product of purity of organic reagent pollution；
7. library is enriched with: connection product can carry out under primer, DNA (dNTPs), HiFi amplification enzyme effect
PCR amplification obtains the higher library of total amount；
8. library purifying and Quality Control: library can be detected after purification through AMPure XP beads after PCR enrichment；1 μ l is taken to use
It is measured in Qubit nucleic acid concentration；It takes 2 μ l to detect for agarose gel electrophoresis and determines library fragments size.
The variable region 16S rRNA amplification step further includes the variable region bacterial 16 S rRNA amplification method, specifically: with
Total DNA is template, be added in amplification system the variable region 16S rRNA universal primer, dNTPs, HiFi amplification enzyme, DMSO,
Nuclease-Free Water, amplification program are as follows:
95 DEG C, 2min；98 DEG C, 30s denaturation, 58 DEG C, 20s annealing, 72 DEG C, 30s extends；After circulation terminates, 72 DEG C of extensions
5min, 4 DEG C of constant temperature save.Wherein, " recurring number " is 30,99 DEG C of hot lid temperature, 25 μ l of system.
The ITS amplification step further includes fungi ITS amplification method, the ITS amplification method specifically: be with total DNA
ITS universal primer, dNTPs, HiFi amplification enzyme, Nuclease-Free Water, amplification program are added in amplification system for template
It is as follows:
95 DEG C, 3min；98 DEG C, 30s denaturation, 50 DEG C, 30s annealing, 72 DEG C, 40s extends；After circulation terminates, 72 DEG C of extensions
5min, 4 DEG C of constant temperature save.
The sequencing steps and analytical procedure further include sequencing and analysis method, specifically: sequencing library is denaturalized through NaOH
After single-stranded, can with the short-chain nucleic acids complementary pairing that is anchored on sequence testing chip, through bridge amplification method by each single strand dna
(or RNA molecule) enrichment becomes a cluster (cluster), then reads sequence with Illumina SBS sequencing approach；Sequencing data
Compared through data Quality Control, filtering source of people sequence and joint sequence and microorganism reference database and etc., know in conjunction with pathogenic microorganism
Know library, identifies the suspicious pathogenic microorganism in sample.
Further technical solution is that the blood plasma separation test includes；
Before the blood plasma separating experiment starts, high speed freezing centrifuge is cooled to 4 DEG C in advance；
The whole blood sample is dispensed into sterile 2ml centrifuge tube, pre-cooling centrifuge 1600g centrifugation 10min is set；
Supernatant is transferred in new sterile 2ml centrifuge tube after centrifugation, is centrifuged 10min using 16000g；
Red blood cell and leucocyte cannot be washed when sample number and transfer supernatant；
Supernatant is transferred in new sterile 2ml centrifuge tube after centrifugation.
Further technical solution is that the reagent preparation process includes:
Enzyme needed for RNA Carrier and library construction is stored in -20 DEG C, and multigelation number is no more than three times；
Lysate RZ, is protected from light and deposits in 4-8 DEG C；
The buffer GD and RD, rinsing liquid PW and RW use the preceding dehydrated alcohol that corresponding volume is added.
Further technical solution is that the bacterial universal primers of the variable region 16S rRNA amplification step are 515F/806R
Primer.
Further technical solution is that the fungi universal primer of the ITS amplification step is ITS1F/ITS2R primer.
Compared with the prior art, the beneficial effects of the present invention are: detection methods of the invention to combine macro genome, macro turn
Record group and amplicon sequencing approach detect the suspicious pathogenic microorganism of hematopoietic stem cell transplantation donor, specifically, advantage table
It is now the following aspects: 1. without assuming: being based on the method, without priori knowledge and it is assumed that help to find new cause of disease
Microorganism improves detection efficiency；2. highly sensitive: the method can detect multiple pathogenic microorganisms mixed infection；3. high-resolution:
It, can be with the kind, subspecies, strain of precise Identification pathogenic microorganism based on the method that macro genome, macro transcript profile combination amplicon are sequenced
System, serotype etc., and data can be used for label and the classification parsing of microbial gene level；4. covering comprehensively: the method can be same
When detect the hundreds of thousands of kinds of microorganisms such as bacterium in sample, fungi, DNA virus, RNA virus, helminth；5. high sample flux:
Based on the method, macro genome, macro transcript profile, amplification sublibrary can mix sequencing, and once sequencing can go up more parts of samples of machine simultaneously
This.
The invention will be further described in the following with reference to the drawings and specific embodiments.
Detailed description of the invention
Fig. 1 is a kind of embodiment of the method quickly detected for hematopoietic stem cell transplantation donor pathogenic microorganism of the present invention
One and embodiment two library construction flow chart schematic diagram；
Fig. 2 is a kind of specific reality of the method quickly detected for hematopoietic stem cell transplantation donor pathogenic microorganism of the present invention
Apply one plasma sample B1 library construction agarose gel electrophoresis results schematic diagram of example；
Fig. 3 is a kind of specific reality of the method quickly detected for hematopoietic stem cell transplantation donor pathogenic microorganism of the present invention
Apply two plasma sample B2, B3 library construction agarose gel electrophoresis results schematic diagram of example.
Specific embodiment
Following will be combined with the drawings in the embodiments of the present invention, and technical solution in the embodiment of the present invention carries out clear, complete
Site preparation description, it is clear that described embodiments are only a part of the embodiments of the present invention, instead of all the embodiments.It is based on
Embodiment in the present invention, those skilled in the art's every other implementation obtained without creative efforts
Example, shall fall within the protection scope of the present invention.
In the description of this specification, reference term " one embodiment ", " some embodiments ", " example ", " specifically show
The description of example " or " some examples " etc. means specific features, structure, material or spy described in conjunction with this embodiment or example
Point is included at least one embodiment or example of the invention.In the present specification, schematic expression of the above terms are not
It is interpreted as that identical embodiment or example must be directed to.Moreover, particular features, structures, materials, or characteristics described
It can be combined in any suitable manner in any one or more of the embodiments or examples.In addition, those skilled in the art can
Different embodiments or examples described in this specification are engaged and be combined.
One: 1 part of blood sample B1 the pathogenic microorganism examination of embodiment
Using detection method of the invention, its plasma sample is detected, and parallel Quality Control is added.Specific implementation step
It is as follows:
1cfDNA is extracted
1.1 take 400 μ l plasma samples into the centrifuge tube of 2ml, be added 40 μ l Lysis Enzyme K, 400 μ l it is slow
Fliud flushing GB and 5 μ l RNA Carrier is vortexed uniformly, is gently mixed by inversion, 56 DEG C of incubation 10min.
1.2 enter 400 μ l of pre-cooled ethanol (96-100%), are gently mixed by inversion sample, are placed at room temperature for 5min, of short duration centrifugation with
Remove the drop of cap wall.
1.3 previous step acquired solutions are transferred in adsorption column (adsorption column is put into collecting pipe), 12,000rpm centrifugations
30sec abandons waste liquid, adsorption column is put back in collecting pipe, and it is primary to repeat this step.
It is added 500 μ l buffer GD (please first check whether before use and dehydrated alcohol has been added) in 1.4 adsorption columns, 12,
000rpm is centrifuged 30sec, abandons waste liquid, adsorption column CR2 is put back in collecting pipe.
It is added 600 μ l rinsing liquid PW (please first check whether before use and dehydrated alcohol has been added) in 1.5 adsorption columns, 12,
000rpm is centrifuged 30sec, abandons waste liquid, adsorption column is put back in collecting pipe, repeats this step 1 time.
1.6 12000rpm are centrifuged 2min, and adsorption column will be transferred in a clean centrifuge tube, be placed at room temperature for 5min.To
54 μ l elution buffer Nuclease-Free Water are vacantly added dropwise in adsorbed film middle position, are placed at room temperature for 5min, and 12,
000rpm is centrifuged 2min, and solution is collected into centrifuge tube i.e. extraction and obtains cfDNA in sample.
2.RNA is extracted and cDNA synthesis
2.1 take 400 μ l plasma samples to 400 μ l lysate RZ into the centrifuge tube of 2ml, are added, and acutely shake in vortex instrument
1min is swung, 5min is placed at room temperature for.
2.2 are added 200 μ l chloroforms, acutely shake 30s in vortex instrument, are placed at room temperature for 3min.
2.3 12000rpm, 10min, take supernatant, are slowly added to 500 μ l dehydrated alcohols, mix.Reaction solution is added and is adsorbed
Column is stored at room temperature 3min, 4 DEG C, 12000rpm, 30s, abandons waste liquid.
2.4 be added 500 μ l protein liquid removal RD (please first checked whether before use and dehydrated alcohol has been added), 4 DEG C,
12000rpm, 30s abandon waste liquid.
2.5 are added 500 μ l rinsing liquid RW (please first check whether before use and dehydrated alcohol has been added), are stored at room temperature 2min, and 4
DEG C, 12000rpm, 30s abandon waste liquid.Repeat this step 1 time.
2.6 12000rpm, 4 DEG C, centrifugation 2min removes residul liquid-removing and adsorption column is placed in RNase-Free centrifuge tube, quiet
Set 3min.40 μ l RNase-Free ddH2O are vacantly added dropwise to adsorbed film middle position, stand 3min, 4 DEG C, 14000rpm, from
Heart 3min is extracted and is obtained RNA in sample.
5 μ l RNA Fragmentation Buffer and 5 μ l are added in 2.7 RNA for taking 30 μ l to extract
Nuclease-Free Water reacts 4min under the conditions of 94 DEG C, the small pieces that can be about 450bp by long-chain RNA random shearing
Section.It reacts in PCR instrument and carries out, 2 μ l stop buffers are added in system after reaction.
2.8RNA reverse transcription: 2 μ l gDNA digestive ferments, 10 5 μ l cDNA are added in system in clipped small fragment RNA
Synthesis Mix, 42 DEG C of reaction 20min make the gDNA polluted in sample degrade and make the single-stranded cDNA of RNA reverse transcription.
Bis- chain of 2.9cDNA synthesis: in the above system, be added 10 × cDNA2 Buffer, DNA polymerase i, RNAse H,
dNTP(10mM).Reaction condition are as follows: 16 DEG C, 1h；65 DEG C, 10min.
The purifying of bis- chain of 2.10cDNA: after reaction, 100 μ l AMPure XP beads are added, under room temperature 5min
Magnetic bead adsorbs double-stranded DNA, is washed twice using 80% dehydrated alcohol, and adding 52 μ l Nuclease-Free Water can wash
The de- double-stranded DNA for obtaining polluting without other albumen, inorganic salts, organic reagent.
3. Genome DNA extraction and amplification
3.1 take 200 μ l haemocytes to mix to 800 μ l Lysis buffer I concussion into the centrifuge tube of 2ml, is added.
3.2 samples set liquid nitrogen freeze 2min after at room temperature 3-4s place into 65 DEG C of water-bath 2min, be repeated 3 times.
3.3 are added 20 μ l Lysis Enzyme I, and concussion mixes on postposition buoy plate, 37 DEG C of incubation 15min.
3.4 are added 225 μ l Lysis buffer II and Lysis Enzyme II, 56 DEG C of incubation 15min, during which overturn
It mixes for several times, then 65 DEG C of incubation 15min.
14000rpm is centrifuged 2min after 3.5 incubations, shifts 800 μ l of supernatant into new centrifuge tube, 800 μ l are added
Binding Buffer, 12000rpm is centrifuged 2min after concussion mixes.
3.6 previous step acquired solutions are transferred in adsorption column (adsorption column is put into collecting pipe), 12,000rpm centrifugations
30sec abandons waste liquid, adsorption column is put back in collecting pipe, and it is primary to repeat this step.
It is added 500 μ l buffer GD (please first check whether before use and dehydrated alcohol has been added) in 3.7 adsorption columns, 12,
000rpm is centrifuged 30sec, abandons waste liquid, adsorption column CR2 is put back in collecting pipe.
It is added 600 μ l rinsing liquid PW (please first check whether before use and dehydrated alcohol has been added) in 3.8 adsorption columns, 12,
000rpm is centrifuged 30sec, abandons waste liquid, adsorption column is put back in collecting pipe, repeats this step 1 time.
3.9 are centrifuged 2min using 12000rpm, and adsorption column will be transferred in a clean centrifuge tube, be placed at room temperature for
5min.54 μ l elution buffer Nuclease-Free Water are vacantly added dropwise to adsorbed film middle position, are placed at room temperature for 5min,
Solution is collected into centrifuge tube i.e. extraction and obtains cfDNA in sample by 12,000rpm centrifugation 2min.
3.10 to take 2 μ l total DNAs be template, be added in amplification system the variable region 16S rRNA universal primer PrimerB MiX,
DNTPs, HiFi amplification enzyme, DMSO, Nuclease-Free Water, amplification program are as follows: 95 DEG C, 2min；98 DEG C, 30s becomes
Property, 58 DEG C, 20s annealing, 72 DEG C, 30s extends；After circulation terminates, 72 DEG C of extension 5min, 4 DEG C of constant temperature save." recurring number " is
30,99 DEG C of hot lid temperature, 25 μ l of system.
3.11 take 2 μ l total DNAs for template, and ITS universal primer PrimerF MiX, dNTPs, HiFi are added in amplification system
Amplification enzyme, Nuclease-Free Water, amplification program are as follows: 95 DEG C, 3min；98 DEG C, 30s denaturation, 50 DEG C, 30s annealing,
72 DEG C, 40s extends；After circulation terminates, 72 DEG C of extension 5min, 4 DEG C of constant temperature save.
Amplified production after purification, takes 1 μ l to measure for Qubit nucleic acid concentration using AMPure XP beads；2 μ l are taken to use
It is detected in agarose gel electrophoresis and determines amplified production clip size.
The building of 4 sequencing libraries:
Library construction process is as shown in Figure 1；
It repairs 4.1 ends
4.1.1 reagent shown in table 1 is added into 200 μ l PCR pipes；
Table 1
| Reagent name | Volume |
| (green)PM EndPrep Enzyme | 5μl |
| (green)PM EndPrep Buffer | 5μl |
| Cell-Free DNA | 50μl |
| Total volume | 60μl |
4.1.2 above-mentioned solution concussion 15s is mixed, and of short duration centrifugation makes all components be collected into tube bottom.It (note: mixes very heavy
It wants, a small amount of bubble will not interfere experiment)
4.1.3 above-mentioned PCR pipe is placed in PCR instrument, hot lid temperature setting is at 75 DEG C or more, response procedures such as 2 (sample of table
Should directly carry out that adapter step is added after the reaction was completed, sample should not be placed on 4 DEG C and cross for a long time) shown in；
Table 2
| Reaction time | Reaction temperature |
| 20minutes | 25℃ |
| 30minutes | 65℃ |
| Hold | 4℃ |
4.2Adaptor connection
4.2.1 the above-mentioned PCR pipe that end reparation is completed is taken out, after of short duration centrifugation, is directly added into 3 reagent of table:
Table 3
4.2.2 mentioned reagent pressure-vaccum is mixed with pipette tips, of short duration centrifugation makes solution be collected into tube bottom.
4.2.3 above-mentioned PCR pipe is placed in PCR instrument, hot lid temperature is closed, 20 DEG C of incubation 15min.
It is purified after 4.3 connection adaptor
4.3.1 ensure magnetic bead AMPure XP beads more than equilibrium at room temperature 30min, using preceding oscillation mixing make its at
For uniform solution.
4.3.2 adaptor connection reaction solution is transferred in a new 1.5ml centrifuge tube.
4.3.3 plus 50 μ l magnetic beads are stored at room temperature 5 minutes after vortex concussion 15 seconds.
4.3.4 centrifuge tube is put on magnetic frame, separates magnetic bead and supernatant solution, until solution clarification (about 2 minutes),
It is careful to draw supernatant and reject, during which avoid contact with the magnetic bead of combining target DNA.If being drawn onto magnetic bead, liquid should be all beaten
It returns in centrifuge tube and adsorbs again.(note: should not reject magnetic bead)
4.3.5 centrifuge tube is maintained on magnetic frame, is added 200 μ l, 80% ethyl alcohol (ready-to-use), is then revolved centrifuge tube
Turn 180 °, after magnetic bead is adsorbed to another side completely, continuing 180 ° of rotation, (in triplicate, centrifuge tube is sure not to leave magnetic force
Frame), 80% ethyl alcohol is carefully sopped up with pipettor.
4.3.6 it is primary to repeat step 2.3.5.
4.3.7 it elutes: centrifuge tube lid is opened, opening is placed at room temperature for 5min on magnetic frame, so that ethyl alcohol volatilization is dry
Only (it can be slightly tilted magnetic frame, observe magnetic bead surfaces, it is non-volatile clean if reflective；If matt, volatilization is clean；If table
There is slight crack in face, then dry excessive), after magnetic bead is dry, all centrifuge tube lids are covered in time, so as not to it is over-drying.
4.3.8 centrifuge tube is removed from magnetic frame, draws 52 μ l EB eluents or deionized water with pipettor, slowly
It is added dropwise and it is flushed to centrifugation bottom of the tube from tube wall in magnetic bead surfaces, then the mixed liquor of suction foot is added dropwise in residue again
Magnetic bead on continue to rinse, until magnetic bead is flushed to bottom completely, drop cannot be stained on tube wall, blown and beaten repeatedly at least 20 times
Mixed liquor is to mix well.It is placed at room temperature for 5min.
4.3.9 centrifuge tube is placed on magnetic frame, adsorbs 5min.It is careful draw 50 μ l supernatants be transferred to 1.5ml without
In bacterium centrifuge tube.
4.3.10 plus the AMPure XP beads of 40 μ l, second of purifying of progress, purification process are for example above-mentioned.Purifying terminates
After take 22 μ l Nuclease-Free Water elute, draw 20 μ l be transferred in the 200 sterile PCR pipes of μ l.
The enrichment of 4.4 libraries
4.4.1 it is added in the sterile PCR pipe of sterile 200 μ l according to such as 4 reaction system of table.
Table 4
Reaction condition is as shown in table 5；
Table 5
| Temperature | Time |
| 98℃ | 30s |
| 98℃ | 10s |
| 65℃ | 60s |
| 72℃ | 5min |
| 4℃ | It saves |
Wherein, further include being warming up to 98 DEG C of holding 10s, be then cooled to 65 DEG C of holding 60s, repetitive cycling 12 times.
The purifying of 4.5 library productions
4.5.1PCR the product after reacting is transferred to sterile 1.5ml EP pipe, and Detailed operating procedures first ensure that magnetic with 2.3
Pearl equilibrium at room temperature 30min or more, adds 35 μ l magnetic beads, and vortex 15s is stored at room temperature 5min.
4.5.2 5min is placed on magnetic frame, removes supernatant, wash 2 times with 80% dehydrated alcohol, 200 μ l, every time about 1min.
4.5.3 pipe lid is opened, volatilization removal ethyl alcohol is added the sterile water eluted dna of 52 μ l, stands 5min.
4.5.4 50 μ l eluents are sucked out and are transferred to new second of purifying of 1.5ml EP pipe progress.40 μ are added into the pipe
L magnetic bead, vortex 15s, is stored at room temperature 5min.
4.5.5 5min is placed on magnetic frame, removes supernatant, wash 2 times with 80% dehydrated alcohol, 200 μ l, every time about 1min.
4.5.6 pipe lid is opened, volatilization removal ethyl alcohol is added the Nuclease-Free Water eluted dna of 28 μ l, stands
5min.26 μ l are sucked out to save to -20 DEG C.
4.6 result Quality Controls
4.6.1 library production takes 1 μ l to measure for Qubit nucleic acid concentration after purification, wherein table 6 is the present embodiment one
The library plasma sample B1 qubit quantitative result.
Table 6
| Number | Concentration (ng/ μ l) | Volume (μ l) | Remarks |
| B1-cfDNA | 16.8 | 24 | The library cfDNA |
| B1-RNA | 12.4 | 24 | The library RNA |
| B1-16S | 35.2 | 24 | The library 16S |
| B1-ITS | 3.28 | 24 | The library ITS |
| Control | 5.60 | 24 | Quality Control |
4.6.2 the agarose gel electrophoresis of configuration 2% takes 2 μ l to detect for agarose gel electrophoresis, and Fig. 2 is this implementation
The library construction agarose gel electrophoresis results of the plasma sample of example one.
5 sequencings and analysis
After NaOH denaturation is single-stranded, can with the short-chain nucleic acids complementary pairing that is anchored on sequence testing chip, through bridge amplification side
The enrichment of each single strand dna is become a cluster (cluster) by method, then reads sequence with Illumina SBS sequencing approach.
Sequencing data is using the analysis software of spectral element independent research through data Quality Control, filtering source of people sequence and joint sequence and microorganism
Reference database comparison and etc., in conjunction with spectral element pathogenic microorganism knowledge base, the suspicious pathogenic microorganism in sample is identified, is seen
Table 7 is the microbe species detected in one plasma sample B1 of the present embodiment, and combines clinical symptoms, which may be viscosity
Actinomyces (Actinomyces_viscosus) infection.
Table 7
Two: 2 parts of blood the pathogenic microorganism examinations of embodiment
Collect the plasma sample B2 of 1 part of suspected infection patient.
Collect the plasma sample B3 of 1 part of suspected infection patient.
Using detection method of the invention, its plasma sample is detected, and parallel Quality Control is added.
Specific implementation step is as follows:
1cfDNA is extracted；
1.1 take 400 μ l plasma samples into the centrifuge tube of 2ml, be added 40 μ l Lysis Enzyme K, 400 μ l it is slow
Fliud flushing GB and 5 μ l RNA Carrier is vortexed uniformly, is gently mixed by inversion, 56 DEG C of incubation 10min.
1.2 enter 400 μ l of pre-cooled ethanol (96-100%), are gently mixed by inversion sample, are placed at room temperature for 5min, of short duration centrifugation with
Remove the drop of cap wall.
1.3 previous step acquired solutions are transferred in adsorption column (adsorption column is put into collecting pipe), 12,000rpm centrifugations
30sec abandons waste liquid, adsorption column is put back in collecting pipe, and it is primary to repeat this step.
It is added 500 μ l buffer GD (please first check whether before use and dehydrated alcohol has been added) in 1.4 adsorption columns, 12,
000rpm is centrifuged 30sec, abandons waste liquid, adsorption column CR2 is put back in collecting pipe.
It is added 600 μ l rinsing liquid PW (please first check whether before use and dehydrated alcohol has been added) in 1.5 adsorption columns, 12,
000rpm is centrifuged 30sec, abandons waste liquid, adsorption column is put back in collecting pipe, repeats this step 1 time.
1.6 12000rpm are centrifuged 2min, and adsorption column will be transferred in a clean centrifuge tube, be placed at room temperature for 5min.To
54 μ l elution buffer Nuclease-Free Water are vacantly added dropwise in adsorbed film middle position, are placed at room temperature for 5min, and 12,
000rpm is centrifuged 2min, and solution is collected into centrifuge tube i.e. extraction and obtains cfDNA in sample.
2.RNA is extracted and cDNA synthesis；
2.1 take 400 μ l plasma samples to 400 μ l lysate RZ into the centrifuge tube of 2ml, are added, and acutely shake in vortex instrument
1min is swung, 5min is placed at room temperature for.
2.2 are added 200 μ l chloroforms, acutely shake 30s in vortex instrument, are placed at room temperature for 3min.
2.3 12000rpm, 10min, take supernatant, are slowly added to 500 μ l dehydrated alcohols, mix.Reaction solution is added and is adsorbed
Column is stored at room temperature 3min, 4 DEG C, 12000rpm, 30s, abandons waste liquid.
2.4 be added 500 μ l protein liquid removal RD (please first checked whether before use and dehydrated alcohol has been added), 4 DEG C,
12000rpm, 30s abandon waste liquid.
2.5 are added 500 μ l rinsing liquid RW (please first check whether before use and dehydrated alcohol has been added), are stored at room temperature 2min, and 4
DEG C, 12000rpm, 30s abandon waste liquid.Repeat this step 1 time.
2.6 12000rpm, 4 DEG C, centrifugation 2min removes residul liquid-removing and adsorption column is placed in RNase-Free centrifuge tube, quiet
Set 3min.40 μ l RNase-Free ddH2O are vacantly added dropwise to adsorbed film middle position, stand 3min, 4 DEG C, 14000rpm, from
Heart 3min is extracted and is obtained RNA in sample.
5 μ l RNA Fragmentation Buffer and 5 μ l are added in 2.7 RNA for taking 30 μ l to extract
Nuclease-Free Water reacts 4min under the conditions of 94 DEG C, the small pieces that can be about 450bp by long-chain RNA random shearing
Section.It reacts in PCR instrument and carries out, 2 μ l stop buffers are added in system after reaction.
2.8RNA reverse transcription: 2 μ l gDNA digestive ferments, 10 5 μ l cDNA are added in system in clipped small fragment RNA
Synthesis Mix, 42 DEG C of reaction 20min make the gDNA polluted in sample degrade and make the single-stranded cDNA of RNA reverse transcription.
Bis- chain of 2.9cDNA synthesis: in the above system, be added 10 × cDNA2 Buffer, DNA polymerase i, RNAse H,
dNTP(10mM).Reaction condition are as follows: 16 DEG C, 1h；65 DEG C, 10min.
The purifying of bis- chain of 2.10cDNA: after reaction, 100 μ l AMPure XP beads are added, under room temperature 5min
Magnetic bead adsorbs double-stranded DNA, is washed twice using 80% dehydrated alcohol, and adding 52 μ l Nuclease-Free Water can wash
The de- double-stranded DNA for obtaining polluting without other albumen, inorganic salts, organic reagent.
3. Genome DNA extraction and amplification；
3.1 take 200 μ l haemocytes to mix to 800 μ l Lysis buffer I concussion into the centrifuge tube of 2ml, is added.
3.2 samples set liquid nitrogen freeze 2min after at room temperature 3-4s place into 65 DEG C of water-bath 2min, be repeated 3 times.
3.3 are added 20 μ l Lysis Enzyme I, and concussion mixes on postposition buoy plate, 37 DEG C of incubation 15min.
3.4 are added 225 μ l Lysis buffer II and Lysis Enzyme II, 56 DEG C of incubation 15min, during which overturn
It mixes for several times, then 65 DEG C of incubation 15min.
14000rpm is centrifuged 2min after 3.5 incubations, shifts 800 μ l of supernatant into new centrifuge tube, 800 μ l are added
Binding Buffer, 12000rpm is centrifuged 2min after concussion mixes.
3.6 previous step acquired solutions are transferred in adsorption column (adsorption column is put into collecting pipe), 12,000rpm centrifugations
30sec abandons waste liquid, adsorption column is put back in collecting pipe, and it is primary to repeat this step.
It is added 500 μ l buffer GD (please first check whether before use and dehydrated alcohol has been added) in 3.7 adsorption columns, 12,
000rpm is centrifuged 30sec, abandons waste liquid, adsorption column CR2 is put back in collecting pipe.
It is added 600 μ l rinsing liquid PW (please first check whether before use and dehydrated alcohol has been added) in 3.8 adsorption columns, 12,
000rpm is centrifuged 30sec, abandons waste liquid, adsorption column is put back in collecting pipe, repeats this step 1 time.
3.9 12000rpm are centrifuged 2min, and adsorption column will be transferred in a clean centrifuge tube, be placed at room temperature for 5min.To
54 μ l elution buffer Nuclease-Free Water are vacantly added dropwise in adsorbed film middle position, are placed at room temperature for 5min, and 12,
000rpm is centrifuged 2min, and solution is collected into centrifuge tube i.e. extraction and obtains cfDNA in sample.
3.10 to take 2 μ l total DNAs be template, be added in amplification system the variable region 16S rRNA universal primer PrimerB MiX,
DNTPs, HiFi amplification enzyme, DMSO, Nuclease-Free Water, amplification program are as follows: 95 DEG C, 2min；98 DEG C, 30s becomes
Property, 58 DEG C, 20s annealing, 72 DEG C, 30s extends；After circulation terminates, 72 DEG C of extension 5min, 4 DEG C of constant temperature save." recurring number " is
30,99 DEG C of hot lid temperature, 25 μ l of system.
3.11 take 2 μ l total DNAs for template, and ITS universal primer PrimerF MiX, dNTPs, HiFi are added in amplification system
Amplification enzyme, Nuclease-Free Water, amplification program are as follows: 95 DEG C, 3min；98 DEG C, 30s denaturation, 50 DEG C, 30s annealing,
72 DEG C, 40s extends；After circulation terminates, 72 DEG C of extension 5min, 4 DEG C of constant temperature save.
Amplified production after purification, takes 1 μ l to measure for Qubit nucleic acid concentration using AMPure XP beads；2 μ l are taken to use
It is detected in agarose gel electrophoresis and determines amplified production clip size.
The building of 4 sequencing libraries:
Library construction flow chart is as shown in Figure 1；
It repairs 4.1 ends；
4.1.1 reagent shown in table 8 is added into 200 μ l PCR pipes；
Table 8
| Reagent name | Volume |
| (green)PM EndPrep Enzyme | 5μl |
| (green)PM EndPrep Buffer | 5μl |
| Cell-Free DNA | 50μl |
| Total volume | 60μl |
4.1.2 above-mentioned solution concussion 15s is mixed, and of short duration centrifugation makes all components be collected into tube bottom.It (note: mixes very heavy
It wants, a small amount of bubble will not interfere experiment)
4.1.3 above-mentioned PCR pipe is placed in PCR instrument, for hot lid temperature setting at 75 DEG C or more, response procedures are as shown in table 9；
Wherein, sample should directly carry out that adapter step is added after the reaction was completed, and sample should not be placed on 4 DEG C and cross for a long time；
Table 9
| Reaction time | Reaction temperature |
| 20minutes | 25℃ |
| 30minutes | 65℃ |
| Hold | 4℃ |
4.2Adaptor connection；
4.2.1 the above-mentioned PCR pipe that end reparation is completed is taken out, after of short duration centrifugation, is directly added into reagent shown in table 10:
Table 10
4.2.2 mentioned reagent pressure-vaccum is mixed with pipette tips, of short duration centrifugation makes solution be collected into tube bottom.
4.2.3 above-mentioned PCR pipe is placed in PCR instrument, hot lid temperature is closed, 20 DEG C of incubation 15min.
It is purified after 4.3 connection adaptor
4.3.1 ensure magnetic bead AMPure XP beads more than equilibrium at room temperature 30min, using preceding oscillation mixing make its at
For uniform solution.
4.3.2 adaptor connection reaction solution is transferred in a new 1.5ml centrifuge tube.
4.3.3 plus 50 μ l magnetic beads are stored at room temperature 5 minutes after vortex concussion 15 seconds.
4.3.4 centrifuge tube is put on magnetic frame, separates magnetic bead and supernatant solution, until solution clarification (about 2 minutes),
It is careful to draw supernatant and reject, during which avoid contact with the magnetic bead of combining target DNA.If being drawn onto magnetic bead, liquid should be all beaten
It returns in centrifuge tube and adsorbs again.(note: should not reject magnetic bead)
4.3.5 centrifuge tube is maintained on magnetic frame, is added 200 μ l, 80% ethyl alcohol (ready-to-use), is then revolved centrifuge tube
Turn 180 °, after magnetic bead is adsorbed to another side completely, continuing 180 ° of rotation, (in triplicate, centrifuge tube is sure not to leave magnetic force
Frame), 80% ethyl alcohol is carefully sopped up with pipettor.
4.3.6 it is primary to repeat step 2.3.5.
4.3.7 it elutes: centrifuge tube lid is opened, opening is placed at room temperature for 5min on magnetic frame, so that ethyl alcohol volatilization is dry
Only (it can be slightly tilted magnetic frame, observe magnetic bead surfaces, it is non-volatile clean if reflective；If matt, volatilization is clean；If table
There is slight crack in face, then dry excessive), after magnetic bead is dry, all centrifuge tube lids are covered in time, so as not to it is over-drying.
4.3.8 centrifuge tube is removed from magnetic frame, draws 52 μ l EB eluents or deionized water with pipettor, slowly
It is added dropwise and it is flushed to centrifugation bottom of the tube from tube wall in magnetic bead surfaces, then the mixed liquor of suction foot is added dropwise in residue again
Magnetic bead on continue to rinse, until magnetic bead is flushed to bottom completely, drop cannot be stained on tube wall, blown and beaten repeatedly at least 20 times
Mixed liquor is to mix well.It is placed at room temperature for 5min.
4.3.9 centrifuge tube is placed on magnetic frame, adsorbs 5min.It is careful draw 50 μ l supernatants be transferred to 1.5ml without
In bacterium centrifuge tube.
4.3.10 plus the AMPure XP beads of 40 μ l, second of purifying of progress, purification process are for example above-mentioned.Purifying terminates
After take 22 μ l Nuclease-Free Water elute, draw 20 μ l be transferred in the 200 sterile PCR pipes of μ l.
The enrichment of 4.4 libraries；
4.4.1 it is added in the sterile PCR pipe of sterile 200 μ l according to 11 reaction system of table.
Table 11
Reaction condition is as shown in table 12；
Table 12
| Temperature | Time |
| 98℃ | 30s |
| 98℃ | 10s |
| 65℃ | 60s |
| 72℃ | 5min |
| 4℃ | It saves |
Wherein, further include being warming up to 98 DEG C of holding 10s, be then cooled to 65 DEG C of holding 60s, repetitive cycling 12 times.
The purifying of 4.5 library productions
4.5.1PCR the product after reacting is transferred to sterile 1.5ml EP pipe, and Detailed operating procedures first ensure that magnetic with 2.3
Pearl equilibrium at room temperature 30min or more, adds 35 μ l magnetic beads, and vortex 15s is stored at room temperature 5min.
4.5.2 5min is placed on magnetic frame, removes supernatant, wash 2 times with 80% dehydrated alcohol, 200 μ l, every time about 1min.
4.5.3 pipe lid is opened, volatilization removal ethyl alcohol is added the sterile water eluted dna of 52 μ l, stands 5min.
4.5.4 50 μ l eluents are sucked out and are transferred to new second of purifying of 1.5ml EP pipe progress.40 μ are added into the pipe
L magnetic bead, vortex 15s, is stored at room temperature 5min.
4.5.5 5min is placed on magnetic frame, removes supernatant, wash 2 times with 80% dehydrated alcohol, 200 μ l, every time about 1min.
4.5.6 pipe lid is opened, volatilization removal ethyl alcohol is added the Nuclease-Free Water eluted dna of 28 μ l, stands
5min.26 μ l are sucked out to save to -20 DEG C.
4.6 result Quality Controls
4.6.1 library production takes 1 μ l to measure for Qubit nucleic acid concentration after purification, wherein is this reality as shown in table 13
Apply two library plasma sample B2, B3 qubit quantitative result of example.
Table 13
| Number | Concentration (ng/ μ l) | Volume (μ l) | Remarks |
| B2-cfDNA | 25.7 | 24 | The library cfDNA |
| B3-cfDNA | 20.4 | 24 | The library cfDNA |
| B2-RNA | 10.6 | 24 | The library RNA |
| B3-RNA | 15.3 | 24 | The library RNA |
| B2-16S | 37.2 | 24 | The library 16S |
| B3-16S | 40.4 | 24 | The library 16S |
| B2-ITS | 8.60 | 24 | The library ITS |
| B3-ITS | 6.44 | 24 | The library ITS |
| Control | 4.40 | 24 | Quality Control |
4.6.2 the agarose gel electrophoresis of configuration 2% takes 2 μ l to detect for agarose gel electrophoresis, as shown in figure 3, being
Plasma sample B2, B3 library construction agarose gel electrophoresis results of the present embodiment two.
5 sequencings and analysis
After NaOH denaturation is single-stranded, can with the short-chain nucleic acids complementary pairing that is anchored on sequence testing chip, through bridge amplification side
The enrichment of each single strand dna is become a cluster (cluster) by method, then reads sequence with Illumina SBS sequencing approach.
Sequencing data is using the analysis software of spectral element independent research through data Quality Control, filtering source of people sequence and joint sequence and microorganism
Reference database comparison and etc., in conjunction with spectral element pathogenic microorganism knowledge base, the suspicious pathogenic microorganism in sample is identified,
In, 14 are shown in Table, for the microbe species detected in two plasma sample B2 of the present embodiment.
Table 14
Table 15
It will be apparent to those skilled in the art that it is various that other can be made according to the above description of the technical scheme and ideas
It is corresponding to change, and all these changes should belong within the scope of protection of the claims of the present invention.
Claims (5)
1. a kind of method quickly detected for hematopoietic stem cell transplantation donor pathogenic microorganism, which is characterized in that including following
Step:
Sample collection procedure collects peripheral blood 5ml using STRECK heparin tube, and 6-30 DEG C saves, separated plasma in 8h, whole blood
Blood plasma separating experiment is carried out before this detection first；
After the completion of blood plasma separation test, 400 μ l blood plasma is taken to extract for cfDNA and RNA respectively, 200 μ l of haemocyte is separately taken to use
In Genome DNA extraction；
Reagent preparation process；
CfDNA is extracted and sequencing library construction step, takes 400 μ l blood plasma, and reaction reagent buffer GB, Lysis is added
Enzyme K, RNA Carrier react 10min at 56 DEG C；Reaction solution adsorbs column purification through pellosil, uses cleaning buffer solution
GD, PW remove isolating protein, salt ion and organic reagent, obtain the satisfactory DNA of total amount, purity；The DNA is repaired through end
Multiple, connector connection, amplified library enrichment, purifying, Quality Control step process, obtain the library cfDNA that building is completed, and utilize
Illumina sequenator matches the library cfDNA, and sequence is read on sequenator；
RNA is extracted and sequencing library construction step, takes 400 μ l blood plasma, and reaction reagent lysate RZ is added and releases in sample
Chloroform is added in RNA, dehydrated alcohol removes protein-based impurity；Reaction solution adsorbs column purification through pellosil, uses cleaning buffer solution
RD, RW remove isolating protein, salt ion and organic reagent, obtain the satisfactory RNA of total amount, purity；The RNA through fragmentation,
Reverse transcription and the synthesis of bis- chain of cDNA, end are repaired, connector connection, amplified library enrichment, are purified, Quality Control step process, and structure is obtained
The library RNA for building completion matches the library RNA using Illumina sequenator, sequence is read on sequenator；
Genome DNA extraction and amplicon sequencing library construction step take 200 μ l haemocytes, extract process according to spectral element standard nucleic acid
Extract total DNA, wherein microbial cell breakage uses bead grinding and combines chemical cracking and enzyme solution, for extracting
The DNA of all microorganisms in sample；
The variable region 16S rRNA amplification step, taking total DNA is template, using bacterial universal primers, is made in HiFiPCR enzyme, ddNTP
It is expanded with the lower realization variable region bacterial 16 S rRNA, gained amplified production after purification and is examined using Agencourt AMPure XP
Survey its clip size and mass concentration；
ITS amplification step, taking total DNA is template, using fungi universal primer, is realized under HiFiPCR enzyme, ddNTP effect true
Bacterium ITS fragment amplification, gained amplified production after purification and detect its clip size and quality using Agencourt AMPure XP
Concentration；
Sequencing steps, the library cfDNA or the library RNA after NaOH denaturation is single-stranded, are integrated on sequence testing chip, through bridge
Each single strand dna or RNA molecule enrichment are become a cluster by formula amplification, then with Illumina SBS sequencing approach
Sequence is read, sequencing data is obtained；
Analytical procedure, the sequencing data is through data Quality Control, filtering source of people sequence and joint sequence and microorganism reference database
Step is compared, the suspicious pathogenic microorganism in sample is identified.
2. a kind of method quickly detected for hematopoietic stem cell transplantation donor pathogenic microorganism according to claim 1,
It is characterized in that, the blood plasma separation test includes；
Before the blood plasma separating experiment starts, high speed freezing centrifuge is cooled to 4 DEG C in advance；
The whole blood sample is dispensed into sterile 2ml centrifuge tube, pre-cooling centrifuge 1600g centrifugation 10min is set；
Supernatant is transferred in new sterile 2ml centrifuge tube after centrifugation, is centrifuged 10min using 16000g；
Red blood cell and leucocyte cannot be washed when sample number and transfer supernatant；
Supernatant is transferred in new sterile 2ml centrifuge tube after centrifugation.
3. a kind of method quickly detected for hematopoietic stem cell transplantation donor pathogenic microorganism according to claim 1,
It is characterized in that, the reagent preparation process includes:
Enzyme needed for RNA Carrier and library construction is stored in -20 DEG C, and multigelation number is no more than three times；
Lysate RZ, is protected from light and deposits in 4-8 DEG C；
The buffer GD and RD, rinsing liquid PW and RW use the preceding dehydrated alcohol that corresponding volume is added.
4. a kind of method quickly detected for hematopoietic stem cell transplantation donor pathogenic microorganism according to claim 1,
It is characterized in that, the bacterial universal primers of the variable region 16S rRNA amplification step are 515F/806R primer.
5. a kind of method quickly detected for hematopoietic stem cell transplantation donor pathogenic microorganism according to claim 1,
It is characterized in that, the fungi universal primer of the ITS amplification step is ITS1F/ITS2R primer.
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