CN110438199A - A kind of method of novel the pathogenic microorganism examination - Google Patents
A kind of method of novel the pathogenic microorganism examination Download PDFInfo
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Abstract
The invention discloses a kind of methods of novel the pathogenic microorganism examination, comprising: cerebrospinal fluid, bronchoalveolar lavage fluid class sample collection step；Peripheral blood sample acquisition step；DNA extraction step takes 400ul sample for detecting, and reaction reagent buffer GB, Lysis Enzyme K, RNA Carrier are added in sample, 10min is reacted at 56 DEG C, obtains reaction solution；Reaction solution adsorbs column purification through pellosil, removes salt ion and organic reagent, obtains the satisfactory DNA of total amount, purity；Library construction step, the DNA are repaired by end, connector connection, amplified library enrichment, are purified, Quality Control step process, complete library construction；Sequencing steps, library are integrated on sequence testing chip after NaOH denaturation is single-stranded, and the enrichment of each single strand dna is become a cluster through bridge amplification, sequence is read using Illumina SBS sequencing approach, obtains sequencing data；Analytical procedure, the sequencing data are compared through data filtering, removal source of people and joint sequence, remaining data with microorganism reference database.
Description
Technical field
The present invention relates to molecular biology and molecular genetics field, more particularly to one kind based on new-generation sequencing technology
The method of novel the pathogenic microorganism examination.
Background technique
Traditional Pathogenic Microorganisms On Tropical technology is broadly divided into two classes: such as morphology of the method based on cell culture is seen
It examines, cell physiological biochemical character, Bacteria Culture parting, genetic chip, automation microbiological analysis system etc., and based on specifically drawing
Object/probe/antibody method such as antigen-antibody reaction, PCR reaction detection and each species specific pathogenic microorganism is quick
Detection system etc..These technologies have played important function in daily pathogenic microorganism confirmation, but there is also certain deficiencies, such as
The former needs to rely on culture, period length, identifies that precision is low, and the latter needs certain microorganism sequence priori knowledge, can not cope with
Unknown or mutation pathogen etc..
Therefore, pathogenic microorganism gene order-checking is increasingly becoming the important tool and means of Clinical microorganism detection field,
But currently for the Comparison between detecting methods of pathogenic microorganism scarcity, it is difficult to the type of microorganism in accurate detection sample.
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 a kind of methods of novel the pathogenic microorganism examination, it is intended to overcome above-mentioned
Problem detects blood, cerebrospinal fluid, sputum, bronchoalveolar lavage fluid equal samples, can accurately detect the type of microorganism in sample.
Summary of the invention
In order to meet above-mentioned requirements, of the invention is to provide a kind of method of novel the pathogenic microorganism examination.
To achieve the goals above, the invention adopts the following technical scheme:
A kind of method of novel the pathogenic microorganism examination, comprising:
Cerebrospinal fluid, bronchoalveolar lavage fluid class sample collection step collect sample using the sterile cryopreservation tube of 2ml or 5ml, and dry ice is protected
It deposits, the sample size is greater than 1ml；
Peripheral blood sample acquisition step collects peripheral blood 5ml, biological ice bag using EDTA anticoagulant tube or STRECK heparin tube
Or separation process is starched in 4 DEG C or less preservations, separated plasma in 8h, examinations advance promoting circulation of blood；
DNA extraction step takes 400ul sample for detecting, and reaction reagent buffer GB, Lysis are added in sample
Enzyme K, RNA Carrier react 10min at 56 DEG C, obtain reaction solution；Reaction solution adsorbs column purification through pellosil, removal
Salt ion and organic reagent obtain the satisfactory DNA of total amount, purity；
Library construction step, the DNA are repaired by end, connector connection, amplified library enrichment, are purified, Quality Control step
Reason completes library construction；
Sequencing steps, library are integrated on sequence testing chip after NaOH denaturation is single-stranded, will be each single-stranded through bridge amplification
DNA molecular enrichment becomes a cluster, reads sequence using Illumina SBS sequencing approach, obtains sequencing data；
Analytical procedure, the sequencing data are joined through data filtering, removal source of people and joint sequence, remaining data and microorganism
Database comparison is examined, bacterium, the fungi, virus, parasitic microorganisms in sample are identified.
Detection method of the invention is mainly used for extracting cerebrospinal fluid, sputum, bronchoalveolar lavage fluid, blood plasma, Pleural effusions equal samples
Middle circulation dissociative DNA, and directly the DNA of extraction is carried out to build library, analyze wherein possible pathogenic microorganism.
Further technical solution is that the cerebrospinal fluid, bronchoalveolar lavage fluid class sample collection step and peripheral blood sample acquire
Step further includes following sample preservation:
Step a. is in addition to RNA Carrier, remaining reagent is under 15-25 DEG C of drying condition；
Or, being stored under the conditions of 4-8 DEG C, it is being placed at room temperature for using preceding to dissolve precipitating；
Step b.RNA Carrier, concentration 1ug/ul are handled using packing, and storage temperature is -20 DEG C, multigelation
Number is no more than three times；
Buffer GD needed for step c. reagent, rinsing liquid PW use preceding addition dehydrated alcohol；
Step d. avoids the multigelation of reagent；
Step e. is isolated by PCR reaction system preparation area with PCR product zone purification, and uses pipettor timing experiment region
It is cleaned；
During blood plasma separation process described in step f., be added after sample is vortexed and of short duration centrifugation carried out to sample, with from
Residual liquid on heart tube wall；
Step g.PCR reaction tube in PCR instrument after taking down, of short duration centrifugal treating, the liquid for covering tube wall and pipe
Body is centrifuged to tube bottom.
Further technical solution is that the blood plasma separation process includes:
High speed freezing centrifuge is cooled to 4 DEG C in advance；
By the 2ml centrifuge tube containing whole blood sample, pre-cooling centrifuge 1600g centrifugation 10min is set；
The supernatant in the centrifuge tube is transferred in new sterile 2ml centrifuge tube after centrifugation, is carried out using centrifuge
16000g is centrifuged 10min；
The supernatant is transferred in new sterile 2ml centrifuge tube after centrifugation, takes 400ul for detecting, remaining sample
This -80 DEG C freeze.
The DNA is extracted using chemical cracking and enzymatic isolation method, original sample such as cerebrospinal fluid etc. through buffer GB and
After Lysis Enzyme K cracking digestion, column purification is adsorbed through pellosil, obtains the satisfactory DNA of total amount, purity.
In the library construction step, library construction is specifically divided into the following steps again:
Repair end: obtained DNA, need to be through end due to the case where there may be end is uneven and chain damages after extraction
After reparation by double-stranded DNA end-filling and repair chain damage.Prevent from influencing the success rate of subsequent reactions；Examination needed for this reaction
Agent is 5ul EndPrep Enzyme and 5ul EndPrep Buffer, and enzyme action time and enzyme deactivation time are 30min；
Connector connection: 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: adapter dosage 2ul；Ligation Mix is used
Measure 30ul；The reaction time is 15min after Ligation Enhancer dosage 1ul, reaction reagent and DNA mixing；
Purify after connection: connection product need to carry out after purification PCR enrichment through AMPure XP beads；Specific purification process
Step are as follows: after connection product is mixed with 50ul magnetic bead, magnetic bead can adsorb the DNA after connection under room temperature, and magnetic bead is through magnetic force
Remaining ingredient can be removed after frame absorption, non junction, enzyme, other salt can be afforded twice by being washed using 80% dehydrated alcohol
Ion and the higher connection product of purity of organic reagent pollution.
Library enrichment: connection product can carry out under primer, DNA (ddNTP), HiFi amplification enzyme effect
PCR amplification obtains the higher library of total amount；
Library purifying and Quality Control: library can be detected after purification through AMPure XP beads after PCR enrichment.1ul is taken to be used for
The measurement of Qubit nucleic acid concentration；It takes 2ul to detect for agarose gel electrophoresis and determines library fragments size；
Sequencing and analysis: library is integrated on sequence testing chip after NaOH denaturation is single-stranded, through bridge amplification by each list
Ssdna molecule enrichment becomes a cluster, then reads sequence with Illumina SBS sequencing approach.Sequencing data is through data
Filtering, removal source of people and joint sequence, remaining data are compared with microorganism reference database, can be identified thin in sample
The microorganisms such as bacterium, fungi, virus, helminth.
Compared with the prior art, the beneficial effects of the present invention are: using the detection method of this programme, can accurately detect sample
What is be currently known in this includes the hundreds of thousands of kinds of microorganisms such as bacterium, fungi, virus, helminth in addition, the method for Sample preservation is protected
The reliability for having demonstrate,proved sample carries out sample the process of separating treatment, it is ensured that the accuracy of pattern detection realizes and reaches
The purpose accurately detected.
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 library construction flow diagram of the method for novel the pathogenic microorganism examination of the present invention；
Fig. 2 is one plasma sample of embodiment in a kind of specific embodiment of the method for novel the pathogenic microorganism examination of the present invention
The library construction agarose gel electrophoresis results of B1；
Fig. 3 is one plasma sample of embodiment in a kind of specific embodiment of the method for novel the pathogenic microorganism examination of the present invention
The pathogenic microorganism examination result of B1；
Fig. 4 is the cerebrospinal fluid of embodiment two in a kind of specific embodiment of the method for novel the pathogenic microorganism examination of the present invention
Sample CF1 and plasma sample B2 library construction agarose gel electrophoresis results；
Fig. 5 is two cerebrospinal fluid sample of embodiment in a kind of specific embodiment of the method for novel the pathogenic microorganism examination of the present invention
The pathogenic microorganism examination result of this CF1；
Fig. 6 is two plasma sample of embodiment in a kind of specific embodiment of the method for novel the pathogenic microorganism examination of the present invention
The pathogenic microorganism examination result of B2.
Specific embodiment
In order to make the objectives, technical solutions, and advantages of the present invention clearer, with reference to the accompanying drawing and specific implementation
Invention is further described in detail for mode.
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 plasma sample B1 the pathogenic microorganism examination of embodiment
Using detection method of the invention, its plasma sample B1 is detected, and parallel Quality Control is added.Specific implementation step
It is rapid as follows:
1DNA is extracted: dehydrated alcohol is added in buffer GD and rinsing liquid PW using preceding elder generation.
1.1 take 400 μ l samples into the centrifuge tube of 2ml.
1.2 are added 40 μ l Lysis Enzyme K solution, are vortexed uniform.
1.3 are added buffer GB and 5ul the Carrier RNA of 400 μ l, are gently mixed by inversion, 56 DEG C of incubation 10min, and
Sample is shaken frequently.It is of short duration to be centrifuged to remove the drop of cap wall.
Wherein, white precipitate may be generated when buffer GB is added, and whens general 56 DEG C of placements can disappear, after will not influence
Continuous experiment.If solution is unchanged limpid, illustrate that cracking is not thorough, may cause and extract that amount of DNA is few and the DNA that extracts is impure；
1.4 are added pre-cooled ethanol (96-100%) 400 μ l, are gently mixed by inversion sample, are placed at room temperature for 5min, of short duration centrifugation
To remove the drop of cap wall；
1.5 are added to previous step acquired solution in one adsorption column CR2 (adsorption column is put into collecting pipe), 12000rpm
It is centrifuged 30sec, waste liquid is abandoned, adsorption column CR2 is put back in collecting pipe, it is primary to repeat this step；
1.6 500 μ l buffer GD (please first check whether before use and dehydrated alcohol has been added) are added into adsorption column CR2,
12,000rpm centrifugation 30sec, abandon waste liquid, adsorption column CR2 are put back in collecting pipe.
1.7 600 μ l rinsing liquid PW (please first check whether before use and dehydrated alcohol has been added) are added into adsorption column CR2,
12,000rpm centrifugation 30sec, abandon waste liquid, adsorption column CR2 are put back in collecting pipe.
1.8 repetitive operation steps 7.
1.9 12,000rpm are centrifuged 2min, and adsorption column CR2 will be transferred in a clean centrifuge tube, be placed at room temperature for
5min.54 μ l elution buffer TB are vacantly added dropwise to adsorbed film middle position, are placed at room temperature for 5min, 12,000rpm centrifugation 2min,
Solution is collected into centrifuge tube；The purpose of this step is to remove rinsing liquid remaining in adsorption column, ethyl alcohol in rinsing liquid
Residual will affect subsequent enzyme reaction experiment.For the yield for increasing DNA, adsorption column can be added in the solution that centrifugation obtains again
In CR2, it is placed at room temperature for 2min, 12,000rpm centrifugation 2min.Extraction obtains DNA product and is such as not required to carry out subsequent experimental immediately, answers
- 20 DEG C are stored in, to prevent DNA degradation.
2 library constructions: shown in library construction flow chart 1,
It repairs 2.1 ends
2.1.1 reagent shown in table 1 is added into 200ul PCR pipe；
Table 1
| Reagent name | Volume |
| (green)PM EndPrep Enzyme | 5ul |
| (green)PM EndPrep Buffer | 5ul |
| Cell-Free DNA | 50ul |
| Total volume | 60ul |
2.1.2 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)
2.1.3 above-mentioned PCR pipe is placed in PCR instrument, hot lid temperature setting is at 75 DEG C or more, shown in response procedures table 2；Its
Middle 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 2
| Reaction time | Reaction temperature |
| 30minutes | 20℃ |
| 30minutes | 65℃ |
| Hold | 4℃ |
2.2Adaptor connection
2.2.1 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 3:
Table 3
2.2.2 mentioned reagent pressure-vaccum is mixed with pipette tips, of short duration centrifugation makes solution be collected into tube bottom.
2.2.3 above-mentioned PCR pipe is placed in PCR instrument, hot lid temperature is closed, 20 DEG C of incubation 15min.
It is purified after 2.3 connection adaptor；
2.3.1 ensure magnetic bead AMPure XP beads more than equilibrium at room temperature 30min, using preceding oscillation mixing make its at
For uniform solution.
2.3.2 adaptor connection reaction solution is transferred in a new 1.5ml centrifuge tube.
2.3.3 plus 50ul magnetic bead is stored at room temperature 5 minutes after vortex concussion 15 seconds.
2.3.4 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)
2.3.5 centrifuge tube is maintained on magnetic frame, is added 80% ethyl alcohol of 200ul (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.
2.3.6 it is primary to repeat step 2.3.5.
2.3.7 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.
2.3.8 centrifuge tube is removed from magnetic frame, draws 52ul EB eluent 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.
2.3.9 centrifuge tube is placed on magnetic frame, adsorbs 5min.It is careful draw 50ul supernatant be transferred to 1.5ml without
In bacterium centrifuge tube.
2.3.10 plus the AMPure XP beads of 40ul, second of purifying of progress, purification process are for example above-mentioned.Purifying terminates
After take 17 μ l Nuc lease-Free Water elute, draw 15ul be transferred in the sterile PCR pipe of 200ul.
The enrichment of 2.4 libraries
2.4.1 it is added in the sterile PCR pipe of sterile 200ul according to reaction system shown in table 4.
Table 4
Reaction condition is as shown in table 5:
Table 5
| Temperature | Time |
| 98℃ | 30s |
| 98℃ | 10s |
| 65℃ | 75s |
| 65℃ | 5min |
| 4℃ | It saves |
It further include that 98 DEG C of holding 10s are warming up to reagent, are then cooled to 65 DEG C of holding 75s, the process being repeated 15 times.
The purifying of 2.5 library productions
2.5.1PCR 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 35ul magnetic bead, and vortex 15s is stored at room temperature 5min.
2.5.2 5min is placed on magnetic frame, removes supernatant, wash 2 times with 80% dehydrated alcohol 200ul, every time about 1min.
2.5.3 pipe lid is opened, volatilization removal ethyl alcohol is added the sterile water eluted dna of 52ul, stands 5min.
2.5.4 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.
2.5.5 5min is placed on magnetic frame, removes supernatant, wash 2 times with 80% dehydrated alcohol 200ul, every time about 1min.
2.5.6 pipe lid is opened, volatilization removal ethyl alcohol is added the Nuclease-Free Water eluted dna of 28ul, stands
5min.26 μ l are sucked out to save to -20 DEG C.
2.6 result Quality Controls
2.6.1 library production takes 1ul to measure for Qubit nucleic acid concentration after purification, and wherein measurement result is shown in Table 6,
2.6.2 the agarose gel electrophoresis of configuration 2% takes 2ul to detect for agarose gel electrophoresis, wherein detection knot
Fruit sees Fig. 2；
Table 6
| Number | Concentration (ng/ul) | Volume (ul) | Remarks |
| B1 | 25.2 | 30 | DNA library |
| Quality Control 1 | 3.60 | 30 | Quality Control |
3 sequencings and analysis
Library is integrated on sequence testing chip after NaOH denaturation is single-stranded, through bridge amplification that each single strand dna is rich
It is integrated into a cluster, then reads sequence with Illumina SBS sequencing approach.Sequencing data is through data filtering, removal people
Source and joint sequence, remaining data are compared with microorganism reference database, can identify bacterium in sample, fungi, virus,
The microorganisms such as helminth, test result figure as shown in Figure 3 are the pathogenic microorganism examination knot in the plasma sample B1 of embodiment one
Fruit, table 7 are the specific type of microorganism that detects in the plasma sample B1 of embodiment one.
Table 7
Two: 1 parts of cerebrospinal fluid of embodiment and 1 part of blood the pathogenic microorganism examination
Two parts of samples are detected using method of the invention, and parallel Quality Control is added.Specific implementation step is as follows:
1DNA is extracted: dehydrated alcohol first please is added in buffer GD and rinsing liquid PW before.
1.1 take 400ul sample into the centrifuge tube of 2ml.
1.2 are added 40ul Lysis Enzyme K solution, are vortexed uniform.
1.3 are added buffer GB and 5ul the Carrier RNA of 400ul, are gently mixed by inversion, 56 DEG C of incubation 10min, and
Sample is shaken frequently.It is of short duration to be centrifuged to remove the drop of cap wall；Wherein, it is heavy that white may be generated when buffer GB is added
It forms sediment, whens general 56 DEG C of placement can disappear, and will not influence subsequent experimental, if solution is unchanged limpid, illustrates to crack and be not thorough, may lead
The DNA for causing extraction amount of DNA few and extracting is impure.
1.4 are added pre-cooled ethanol (96-100%) 400ul, are gently mixed by inversion sample, are placed at room temperature for 5min, of short duration centrifugation
To remove the drop of cap wall.
1.5 are added to previous step acquired solution in one adsorption column CR2 (adsorption column is put into collecting pipe), and 12,
000rpm is centrifuged 30sec, abandons waste liquid, adsorption column CR2 is put back in collecting pipe, and it is primary to repeat this step.
1.6 are added 500ul buffer GD (please first check whether before use and dehydrated alcohol has been added) into adsorption column CR2,
12,000rpm centrifugation 30sec, abandon waste liquid, adsorption column CR2 are put back in collecting pipe.
1.7 are added 600ul rinsing liquid PW (please first check whether before use and dehydrated alcohol has been added) into adsorption column CR2,
12,000rpm centrifugation 30sec, abandon waste liquid, adsorption column CR2 are put back in collecting pipe.
1.8 repetitive operation steps 1.7.
1.9 are centrifuged 2min using 12000rpm, and adsorption column CR2 will be transferred in a clean centrifuge tube, be placed at room temperature for
5min.54ul elution buffer TB is vacantly added dropwise to adsorbed film middle position, is placed at room temperature for 5min, 12,000rpm centrifugation 2min,
Solution is collected into centrifuge tube；The purpose of this step is to remove rinsing liquid remaining in adsorption column, ethyl alcohol in rinsing liquid
Residual will affect subsequent enzyme reaction experiment.
For the yield for increasing DNA, the solution that centrifugation obtains can be added again in adsorption column CR2, be placed at room temperature for 2min,
12,000rpm is centrifuged 2min.Extraction obtains DNA product and is such as not required to carry out subsequent experimental immediately, -20 DEG C should be stored in, to prevent DNA
Degradation.
2 library constructions: shown in library construction flow chart 1；
It repairs 2.1 ends
2.1.1 reagent shown in table 8 is added into 200 μ l PCR pipes；
Table 8
| Reagent name | Volume |
| (green)PM EndPrep Enzyme | 5ul |
| (green)PM EndPrep Buffer | 5ul |
| Cell-Free DNA | 50ul |
| Total volume | 60ul |
2.1.2 the concussion of solution shown in table 8 15s is mixed, of short duration centrifugation makes all components be collected into tube bottom.It (note: mixes
Extremely important, a small amount of bubble will not interfere experiment)
2.1.3 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；
Table 9
| Reaction time | Reaction temperature |
| 30minutes | 20℃ |
| 30minutes | 65℃ |
| Hold | 4℃ |
(note: sample should directly carry out after the reaction was completed be added adapter step, sample should not be placed on 4 DEG C it is too long when
Between)
2.2Adaptor connection
2.2.1 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
2.2.2 reagent pressure-vaccum shown in table 10 is mixed with pipette tips, of short duration centrifugation makes solution be collected into tube bottom.
2.2.3 above-mentioned PCR pipe is placed in PCR instrument, hot lid temperature is closed, 20 DEG C of incubation 15min.
It is purified after 2.3 connection adaptor
2.3.1 ensure magnetic bead AMPure XP beads more than equilibrium at room temperature 30min, using preceding oscillation mixing make its at
For uniform solution.
2.3.2 adaptor connection reaction solution is transferred in a new 1.5ml centrifuge tube.
2.3.3 plus 50ul magnetic bead is stored at room temperature 5 minutes after vortex concussion 15 seconds.
2.3.4 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)
2.3.5 centrifuge tube is maintained on magnetic frame, is added 80% ethyl alcohol of 200ul (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.
2.3.6 it is primary to repeat step 2.3.5.
2.3.7 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.
2.3.8 centrifuge tube is removed from magnetic frame, draws 52ul EB eluent 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.
2.3.9 centrifuge tube is placed on magnetic frame, adsorbs 5min.It is careful draw 50ul supernatant be transferred to 1.5ml without
In bacterium centrifuge tube.
2.3.10 plus the AMPure XP beads of 40ul, second of purifying of progress, purification process are for example above-mentioned.Purifying terminates
After take 17ul Nuclease-Free Water elute, draw 15ul be transferred in the sterile PCR pipe of 200ul.
The enrichment of 2.4 libraries
2.4.1 it is added in the sterile PCR pipe of sterile 200 μ l according to 11 reaction system of table.
Table 11
Reaction condition such as table 12；
Table 12
| Temperature | Time |
| 98℃ | 30s |
| 98℃ | 10s |
| 65℃ | 75s |
| 65℃ | 5min |
| 4℃ | It saves |
It wherein, further include that 98 DEG C of holding 10s are warming up to reagent, 65 DEG C of holding 75s is then cooled to, is repeated 15 times
Process.
The purifying of 2.5 library productions
2.5.1PCR 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 35ul magnetic bead, and vortex 15s is stored at room temperature 5min.
2.5.2 5min is placed on magnetic frame, removes supernatant, wash 2 times with 80% dehydrated alcohol 200ul, every time about 1min.
2.5.3 pipe lid is opened, volatilization removal ethyl alcohol is added the sterile water eluted dna of 52ul, stands 5min.
2.5.4 50ul eluent is sucked out and is transferred to new second of purifying of 1.5ml EP pipe progress.It is added into the pipe
40ul magnetic bead, vortex 15s, is stored at room temperature 5min.
2.5.5 5min is placed on magnetic frame, removes supernatant, wash 2 times with 80% dehydrated alcohol 200ul, every time about 1min.
2.5.6 pipe lid is opened, volatilization removal ethyl alcohol is added the Nuclease-Free Water eluted dna of 28ul, stands
5min.26ul is sucked out to save to -20 DEG C.
2.6 result Quality Controls
2.6.1 library production takes 1ul to measure for Qubit nucleic acid concentration after purification, specifically, is shown in Table 13, is cerebrospinal fluid
Sample CF1 and the library plasma sample B2 qubit quantitative result.
2.6.2 the agarose gel electrophoresis of configuration 2% takes 2ul to detect for agarose gel electrophoresis, specifically, sees figure
4, it is CSF sample CF1 and plasma sample B2 library construction agarose gel electrophoresis results.
Table 13
| Number | Concentration | Volume | Remarks |
| CF1 | 32.0 | 30 | DNA library |
| B2 | 29.3 | 30 | DNA library |
| Quality Control 2 | 3.12 | 30 | Quality Control |
3 sequencings and analysis
Library is integrated on sequence testing chip after NaOH denaturation is single-stranded, through bridge amplification that each single strand dna is rich
It is integrated into a cluster, then reads sequence with Illumina SBS sequencing approach.Sequencing data is through data filtering, removal people
Source and joint sequence, remaining data are compared with microorganism reference database, can identify bacterium in sample, fungi, virus,
The microorganisms such as helminth, specifically, as shown in Figure 5, be two CSF sample CF1 of embodiment in the pathogenic microorganism examination as a result,
As shown in fig. 6, be in two blood sample B2 of the present embodiment the pathogenic microorganism examination as a result, table 14 is two cerebrospinal fluid sample of the present embodiment
The microbe species detected in this CF1, table 15 are the microbe species detected in two blood sample B2 of the present embodiment.
Table 14
| Type | Chinese name | Latin name | Coverage % | Reads |
| G- | Klebsiella Pneumoniae | Klebsiella_pneumoniae | 0.635 | 237 |
| G+ | Crowded bar bacterium | Corynebacterium_accolens | 0.218 | 27 |
| G- | Become klebsiella of dwelling | Klebsiella_variicola | 0.057 | 19 |
| G+ | Propionic acid bar bacterium | Corynebacterium_propinquum | 0.027 | 4 |
| G- | Pseudomonas oleovorans | Pseudomonas_oleovorans | 0.013 | 4 |
| G- | Klebsiella Pneumoniae | Klebsiella_pneumoniae | 0.635 | 237 |
| G+ | Crowded bar bacterium | Corynebacterium_accolens | 0.218 | 27 |
| G- | Become klebsiella of dwelling | Klebsiella_variicola | 0.057 | 19 |
| G+ | Propionic acid bar bacterium | Corynebacterium_propinquum | 0.027 | 4 |
| G- | Pseudomonas oleovorans | Pseudomonas_oleovorans | 0.013 | 4 |
| G- | Klebsiella Pneumoniae | Klebsiella_pneumoniae | 0.635 | 237 |
| G+ | Crowded bar bacterium | Corynebacterium_accolens | 0.218 | 27 |
| G- | Become klebsiella of dwelling | Klebsiella_variicola | 0.057 | 19 |
| G+ | Propionic acid bar bacterium | Corynebacterium_propinquum | 0.027 | 4 |
| G- | Pseudomonas oleovorans | Pseudomonas_oleovorans | 0.013 | 4 |
Table 15
Before high-flux sequence (NGS) appearance, pathogenic microorganism genome analysis is mainly by means of mulberry lattice sequencing technologies
(Sanger Sequencing), experimental period is longer, and detection flux is low, and testing cost is also higher.Scientific development and technological progress
It is provided for Causal Agent Identification and analysis and is more concisely and efficiently means, NGS is exactly best example.Nowadays, NGS is rapid
As the important detection platform of clinical labororatory, the detectability of clinical pathogenic microorganism is substantially increased.For microorganism base
Because of the measurement of group sequence, in addition to sequencing equipment, computer and various parsers, software and reference database must not yet
It can lack, they collectively constitute gene order-checking " three-major-items ".
Macro genome sequencing method main advantage is expanded without targeting, because not needing precognition pathogenic genes group sequence letter
Breath.But since the sequence of host and cause of disease is measured simultaneously, pathogen sequence accounting is few, so how effectively to remove host's sequence
Column, have higher requirements to sample disposal and information analysis techniques.Meanwhile the detection for clinical sample, how to distinguish field planting with
Infection, it is also desirable to big data quantity sample cumulative and clinical experience comprehensive descision.It is (resistance to if you need to be analysed in depth to pathogen sequence
Medicine gene etc.), then it needs to increase sequencing depth, testing cost also increases.
The above analysis, the basis this invention takes macro genome sequencing method as this method, for realizing to sample
The detection and analysis of microorganism in this.
In conclusion, using the detection method of this programme, can accurately detect mesh in sample using the detection method of this programme
It include the hundreds of thousands of kinds of microorganisms such as bacterium, fungi, virus, helminth known to preceding in addition, the method for Sample preservation ensure that sample
This reliability carries out sample the process of separating treatment, it is ensured that the accuracy of pattern detection realizes and reaches accurate inspection
The purpose of survey.
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 deformation, and all these change and deformation should belong to the claims in the present invention protection scope it
It is interior.
Claims (3)
1. a kind of method of novel the pathogenic microorganism examination characterized by comprising
Cerebrospinal fluid, bronchoalveolar lavage fluid class sample collection step collect sample using the sterile cryopreservation tube of 2ml or 5ml, and dry ice saves,
The sample size is greater than 1ml；
Peripheral blood sample acquisition step collects peripheral blood 5ml, biological ice bag or 4 using EDTA anticoagulant tube or STRECK heparin tube
DEG C or less save, separation process is starched in separated plasma in 8h, examinations advance promoting circulation of blood；
DNA extraction step takes 400ul sample for detecting, and reaction reagent buffer GB, Lysis Enzyme are added in sample
K, RNA Carrier reacts 10min at 56 DEG C, obtains reaction solution；Reaction solution adsorbs column purification through pellosil, removes salt ion
And organic reagent, obtain the satisfactory DNA of total amount, purity；
Library construction step, the DNA is repaired by end, connector connection, amplified library enrichment, is purified, Quality Control step process, complete
At library construction；
Sequencing steps, library is integrated on sequence testing chip after NaOH denaturation is single-stranded, through bridge amplification by each single stranded DNA
Molecule enrichment becomes a cluster, reads sequence using Illumina SBS sequencing approach, obtains sequencing data；
Analytical procedure, the sequencing data is through data filtering, removal source of people and joint sequence, remaining data and microorganism reference number
It is compared according to library, identifies bacterium, the fungi, virus, parasitic microorganisms in sample.
2. a kind of method of novel the pathogenic microorganism examination according to claim 1, which is characterized in that the cerebrospinal fluid,
Bronchoalveolar lavage fluid class sample collection step and peripheral blood sample acquisition step further include following sample preservation:
Step a. is in addition to RNA Carrier, remaining reagent is under 15-25 DEG C of drying condition；
Or, being stored under the conditions of 4-8 DEG C, it is being placed at room temperature for using preceding to dissolve precipitating；
Step b.RNA Carrier, concentration 1ug/ul are handled using packing, and storage temperature is -20 DEG C, multigelation number
No more than three times；
Buffer GD needed for step c. reagent, rinsing liquid PW use preceding addition dehydrated alcohol；
Step d. avoids the multigelation of reagent；
Step e. is isolated by PCR reaction system preparation area with PCR product zone purification, and is carried out using pipettor timing experiment region
Cleaning；
During blood plasma separation process described in step f., it is added after sample is vortexed and of short duration centrifugation is carried out to sample, with centrifuge tube
Residual liquid on wall；
Step g.PCR reaction tube in PCR instrument after taking down, of short duration centrifugal treating, the liquid for covering tube wall and pipe from
The heart is to tube bottom.
3. a kind of method of novel the pathogenic microorganism examination according to claim 1, which is characterized in that the blood plasma separation
Process includes:
High speed freezing centrifuge is cooled to 4 DEG C in advance；
By the 2ml centrifuge tube containing whole blood sample, pre-cooling centrifuge 1600g centrifugation 10min is set；
The supernatant in the centrifuge tube is transferred in new sterile 2ml centrifuge tube after centrifugation, carries out 16000g using centrifuge
It is centrifuged 10min；
The supernatant is transferred in new sterile 2ml centrifuge tube after centrifugation, takes 400ul for detecting, remaining sample -80
It DEG C freezes.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113025731A (en) * | 2021-03-04 | 2021-06-25 | 宁夏医科大学总医院 | High-throughput pathogen detection kit based on targeted amplicon enrichment strategy |
| CN113061666A (en) * | 2020-12-30 | 2021-07-02 | 中南大学湘雅二医院 | Detection method, application and system of cytomegalovirus after liver transplantation |
| WO2021164472A1 (en) * | 2020-02-18 | 2021-08-26 | 武汉臻熙医学检验实验室有限公司 | Library construction method based on nanopore sequencing platform, microorganism identification method, and application |
| CN115820402A (en) * | 2022-11-29 | 2023-03-21 | 深圳市国赛生物技术有限公司 | Automatic system for microbial testing and microbial testing method |
Citations (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102628082A (en) * | 2012-04-10 | 2012-08-08 | 凯晶生物科技(苏州)有限公司 | Method for qualitatively and quantitatively detecting nucleic acid based on high-flux sequencing technology |
| CN104946623A (en) * | 2015-04-08 | 2015-09-30 | 浙江圣庭生物科技有限公司 | Construction method of small-fragment DNA (deoxyribonucleic acid) library based on Illumina Hiseq 2500 sequencing platform |
| CN105112569A (en) * | 2015-09-14 | 2015-12-02 | 中国医学科学院病原生物学研究所 | Virus infection detection and identification method based on metagenomics |
| WO2016071369A1 (en) * | 2014-11-04 | 2016-05-12 | Genesupport Sa | Method for determining the presence of a biological condition by determining total and relative amounts of two different nucleic acids |
| CN109402223A (en) * | 2018-11-12 | 2019-03-01 | 河南弘腾农业有限公司 | A kind of method and its application based on animal wastes sample building sequencing library |
| US20190071732A1 (en) * | 2016-04-15 | 2019-03-07 | Predicine, Inc. | Systems and methods for detecting genetic alterations |
| CN109609598A (en) * | 2018-12-29 | 2019-04-12 | 北京优迅医学检验实验室有限公司 | A kind of rapidly and efficiently inexpensive library constructing method for two generation sequencing technologies |
| CN109923217A (en) * | 2016-10-13 | 2019-06-21 | 生物梅里埃公司 | The identification of pathogen and antibiotic characterization in macro genomic samples |
-
2019
- 2019-08-15 CN CN201910756062.8A patent/CN110438199A/en active Pending
Patent Citations (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102628082A (en) * | 2012-04-10 | 2012-08-08 | 凯晶生物科技(苏州)有限公司 | Method for qualitatively and quantitatively detecting nucleic acid based on high-flux sequencing technology |
| WO2016071369A1 (en) * | 2014-11-04 | 2016-05-12 | Genesupport Sa | Method for determining the presence of a biological condition by determining total and relative amounts of two different nucleic acids |
| CN104946623A (en) * | 2015-04-08 | 2015-09-30 | 浙江圣庭生物科技有限公司 | Construction method of small-fragment DNA (deoxyribonucleic acid) library based on Illumina Hiseq 2500 sequencing platform |
| CN105112569A (en) * | 2015-09-14 | 2015-12-02 | 中国医学科学院病原生物学研究所 | Virus infection detection and identification method based on metagenomics |
| US20190071732A1 (en) * | 2016-04-15 | 2019-03-07 | Predicine, Inc. | Systems and methods for detecting genetic alterations |
| CN109923217A (en) * | 2016-10-13 | 2019-06-21 | 生物梅里埃公司 | The identification of pathogen and antibiotic characterization in macro genomic samples |
| CN109402223A (en) * | 2018-11-12 | 2019-03-01 | 河南弘腾农业有限公司 | A kind of method and its application based on animal wastes sample building sequencing library |
| CN109609598A (en) * | 2018-12-29 | 2019-04-12 | 北京优迅医学检验实验室有限公司 | A kind of rapidly and efficiently inexpensive library constructing method for two generation sequencing technologies |
Non-Patent Citations (2)
| Title |
|---|
| XIAO-WEI XING等: "Evaluation of Next-Generation Sequencing for the Diagnosis of Infections of the Central Nervous System Caused by the Neurotropic Herpes Viruses: A Pilot Study", 《EUROPEAN NEUROLOGY》 * |
| 缪青等: "基于宏基因二代测序技术检测呼吸道病毒的临床应用", 《中华医院感染学杂志》 * |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2021164472A1 (en) * | 2020-02-18 | 2021-08-26 | 武汉臻熙医学检验实验室有限公司 | Library construction method based on nanopore sequencing platform, microorganism identification method, and application |
| CN113061666A (en) * | 2020-12-30 | 2021-07-02 | 中南大学湘雅二医院 | Detection method, application and system of cytomegalovirus after liver transplantation |
| CN113025731A (en) * | 2021-03-04 | 2021-06-25 | 宁夏医科大学总医院 | High-throughput pathogen detection kit based on targeted amplicon enrichment strategy |
| CN115820402A (en) * | 2022-11-29 | 2023-03-21 | 深圳市国赛生物技术有限公司 | Automatic system for microbial testing and microbial testing method |
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