CN106893680A - A kind of method that filamentous fungi is enriched with from blood sample - Google Patents
A kind of method that filamentous fungi is enriched with from blood sample Download PDFInfo
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Abstract
The present invention relates to a kind of method that filamentous fungi is enriched with from blood sample, including：(1) 1 2ml blood samples are extracted from Positive Blood Culture blake bottle, centrifuge tube is injected；(2) to 50 150mg quartz sands and 10 20ml enrichment dilutions is added in centrifuge tube, mix, stand, centrifugation is precipitated；(3) precipitation is shaken, centrifugation is subsequently added lauryl sodium sulfate SDS solution and sterile purified water, shakes, and removes quartz sand；Centrifugation, washing, obtains final product.Present invention enrichment hypha,hyphae efficiency high, the fungi impurity being enriched to is few, can be used for fungal nucleic acid extraction, also can be used for mass-spectrometric technique Direct Identification fungi, with wide application space.
Description
Technical field
The invention belongs to blood sample process field, more particularly to a kind of side that filamentous fungi is enriched with from blood sample
Method.
Background technology
With social population's aging progress faster, increase the crowd for suffering from chronic senile disease, meanwhile, wide spectrum resists
Raw element is widely used, and the invasive medical care precess such as intubation intervention treatment, tissue biopsy increases, and tumour, AIDS, white blood
The quantity of the immunocompromised patients such as disease, organ transplant constantly expands.Therefore, in recent years, the incidence of disease of deep fungal infection is big
Width increases, and particularly the incidence of disease of deep filamentous fungi infection just substantially increases year by year, its incidence of disease in some specific crowds
Even above yeast infection rate.Lack the clinical symptoms of characteristic due to deep fungal infection early stage, it is difficult in morbidity early stage
Etiological diagnosis are obtained, when patient occurs continuing hyperpyrexia and invalid antibiotic therapy a few days, often fungal infection has been pointed out
Several organs are diffused into, the best opportunity of rescue patient is missed；Clinical practice shows, sensitivity of the different fungies to antifungal drug
Property there is larger difference, or even some fungi is to commonly using antifungal drug natural drug resistance or insensitive, causes fungus-caused depth
Portion's infectious age is very high.Therefore, in time, correctly diagnosis deep fungal infection, especially filamentous fungi infect, to successfully robbing
Patient vitals are rescued, with very important realistic meaning.
Existing clinical labororatory's two Methods for Fungi Detection relatively lags behind.The conventional method for diagnosing deep fungal infection has form
The methods such as, histopathology, serology.Morphological examination includes the direct microscopy of fungi and culture, is current filamentous fungi disease
The most common method of diagnosis.But, from the flashing lightning magnetic field detector transferred species flat board of blood culture with positive bacteria to turning out filamentous fungi typical case bacterium
Fall, generally require 3 days to 2 weeks, or even the longer time.
Protocols in Molecular Biology is so that its sensitivity is high, high specificity the advantages of, developed rapidly in recent years, existing detection is true
The molecular biology method of bacterium infection is also increasingly mature, such as nucleic acid sequencing, genetic chip, and uses mass-spectrometric technique Direct Identification
Fungi.Therefore, directly it is enriched with from blood sample and separates filamentous fungi, direct detection is carried out using molecular biology method,
And be no longer dependent on the husky weak flat board of guarantor of transferred species and obtain fungi pure culture, it is the focus of current Clinical microbiology experiment room research.
However, filamentous fungi forms very thin mycelia in blood, and content is seldom, slow-growing in patient's blood circulation.Mycelia
It is different from bacterium or fungal spore, using same experiment condition, it is difficult to be effectively efficiently enriched with from blood, isolate this
Class mycelia.
In conventional blood sample digestion process liquid clinical at present, hemolytic agent is generally that ammonium chloride, ammonium acetate etc. are poisonous or micro-
Poisonous substance product, such as stimulate skin, mucous membrane, eyes, nasal cavity, throat, damage eyes；High concentration stimulates lung, can cause wet lung；And
And, there is higher concentration protein in blood sample, or even have lymphocyte continued growth, breeding, chlorine in flashing lightning magnetic field detector
Change ammonium, ammonium acetate and easily cause protein precipitation, make solution muddy, viscosity increases, and is unfavorable for the separation of filamentous fungi.
The content of the invention
The technical problems to be solved by the invention are to provide a kind of method that filamentous fungi is enriched with from blood sample, the party
Method is enriched with hypha,hyphae efficiency high, and the fungi impurity being enriched to is few, can be used for fungal nucleic acid extraction, also can be used for mass-spectrometric technique straight
Identification fungi is connect, with wide application space.
A kind of method that filamentous fungi is enriched with from blood sample of the invention, including：
(1) 1-2ml blood samples are extracted from Positive Blood Culture blake bottle, centrifuge tube is injected；
(2) to 50-150mg quartz sands (AR grades, 25-50 mesh) and 10-20ml enrichment dilutions are added in centrifuge tube, mix
It is even, stand, centrifugation is precipitated；Wherein, enrichment dilution is 10-30 by volume ratio:1 80-200g/L urea liquids (are used
Preceding preparation) and 500-600mM EDTA (pH 8.0) compositions；
(3) precipitation is shaken, centrifugation is subsequently added lauryl sodium sulfate SDS solution and sterile purified water, shakes, and removes
Remove quartz sand；Centrifugation, washing, obtains final product the filamentous fungi of enrichment.
Time of repose in the step (2) is 5-10min.
The concentration of the lauryl sodium sulfate SDS solution in the step (3) is 0.5%-10%, the volume ratio with precipitation
It is 1:10-15.
Sterile purified water and the volume ratio of precipitation in the step (3) are 1:0.5-1.
Washing in the step (3) is specially and uses 1.5ml aseptic distillations water washing 3 times.
Centrifugal speed in the step (2) and (3) is 12000r/min, and centrifugation time is 1-2min.
The method have the characteristics that：
High concentration urea solution can not only the blood such as lysed erythrocyte, leucocyte have type composition, and can make red thin
The hemoglobin discharged after cellular lysis is denatured rapidly, is dissolved together with cell fragment without forming precipitation, makes solution more limpid,
Viscosity is small, is conducive to the precipitation of mycelia to be enriched with.A small amount of quartz sand is added, is conducive to further mechanicalness to crush the white thin of remaining
Born of the same parents, blood platelet fragment, remove its dissolving, and impurity is less in making centrifugation；Meanwhile, it is big in supernatant is centrifuged off first
During amount impurity, quartz sand more attaching surfaces for mycelia provides reduce the loss of mycelia during centrifuge washing.And add low dense
The SDS of degree, is anion surfactant, with strong scale removal effect, contributes to filamentous fungi mycelia to be washed from quartz sand
It is de-, it helps to remove the urea of possible remaining in precipitation.Experiment shows that the cell membrane of fungi is very tough and tensile, is adding quartz
Under conditions of sand, the spore of yeast-like fungi needs to connect in the continuous concussion about 10min of high speed oscillator needs, filamentous fungi mycelia
Continuous concussion 20-25min ability effectively broken walls disengage nucleic acid.Therefore, under experiment condition of the invention, hypha,hyphae will not be caused
Or spore cracking.
Particularly, for fungi severe bloodstream infection patient, using the method for the present invention, by (the 3-5 enrichment of many tube method
Pipe), to increase for the blood preparation amount for being enriched with fungi to 6-10ml, the fungi that will be enriched to merges, without blood culture,
But fungi is directly enriched with from patient peripheral's blood, then, using molecular biology method, it is also possible to in blood sample
Fungi is detected.The fungi impurity of present invention enrichment is few, is used directly for fungal nucleic acid extraction.
Beneficial effect
The present invention can not only efficiently destroy blood type composition, and can make the blood red egg discharged after erythrocytolysis
White rapid denaturation, dissolving, make that enrichment dilution is more limpid, and viscosity is small, is conducive to the gathering trace from blood sample thread
Hypha,hyphae, separates, detects the positive rate of filamentous fungi so as to significantly improve from blood sample；Present invention enrichment hypha,hyphae
Efficiency high, the fungi impurity being enriched to is few, can be used for fungal nucleic acid extraction, also can be used for mass-spectrometric technique Direct Identification fungi, has
There is wide application space.
Specific embodiment
With reference to specific embodiment, the present invention is expanded on further.It should be understood that these embodiments are merely to illustrate the present invention
Rather than limitation the scope of the present invention.In addition, it is to be understood that after the content for having read instruction of the present invention, people in the art
Member can make various changes or modifications to the present invention, and these equivalent form of values equally fall within the application appended claims and limited
Scope.
Embodiment 1
First, sample prepares：
Because filamentous fungi blood culture with positive bacteria blood sample is difficult to find that sufficient amount in a short time in clinic, and Saving specimen
Time was detected in the same time, on interpretation there may be influence.The present embodiment is using simulation
Clinical samples are tested：
1. selection is accredited as the clinical separation strain of white saccharomycete and black-koji mould as experimental subjects, difference through sequence measurement
Two kinds of fungus colony/mycelia on the husky weak culture medium of guarantor are taken, with the grinding of RPMI1640 culture mediums, dilution, with Marienfeld blood cells
Tally is counted to spore/mycelia therein respectively, is 1000CFU/ with the spore/mycelial concentration for adjusting two kinds of fungies
ml。
2. healthy volunteer's blood about 150ml is taken, is divided to A, B two groups, every group of 50ml blood, to adding spore in A group blood
Concentration is the white μ l of saccharomycete bacterium solution 250 of 1000CFU/ml；It is 1000CFU/ml black-koji moulds to mycelial concentration is added in B group blood
The μ l of liquid 250.Blood sample is fully mixed, now, the concentration of fungal spore/mycelia is about 5CFU/ml in blood sample.
3. every group of blood sample is injected separately into 10 Bact/ALERT Blood culture bottles according to every bottle of 5ml.Meanwhile, take 5ml
The blood of bacterium solution is not added to inject Blood culture bottle as control bottle.Blood culture bottle is put intoThe full-automatic blood culture of 3D
Instrument, takes out after after the instrument prompting culture positive, is taken out together with control bottle after last 1 bottle of test bottle report sun；Blood in blake bottle
After sample is fully mixed (particularly aspergillus niger culture test bottle), every bottle takes 3-5ml as test blood sample, and control bottle continues
Culture was to 14 days.
2nd, fungi enrichment process：
1. to adding in advance, the 25ml of 100mg quartz sands is aseptic to have lid centrifuge tube to add 1ml blood samples, adds enrichment to dilute
(100g/L urea liquids are with 500mM EDTA according to volume ratio 20 for liquid 20ml:1 mixing), mix, stand 10min.
2.12000r/min is centrifuged 2min, and supernatant is carefully removed from top, stays about 1.5ml to precipitate.
3. high-speed oscillator concussion 1min is deposited in, 1min is centrifuged with 20ml sterile purified waters 12000r/min, abandon supernatant
Liquid.
4., to addition 0.1ml 5%SDS solution and 1.4ml sterile purified waters is precipitated in pipe, 0.5min is shaken, by intraluminal fluid
Body is all transferred in 1.5ml pipes, discards quartz sand.
5.12000r/min is centrifuged 1min, abandons supernatant, then 1min is centrifuged with 1.5ml sterile purified waters 12000r/min,
Washing 3 times, abandons supernatant, and precipitation is the filamentous fungi being enriched with from blood sample.
3rd, fungi enrichment result detection method：
1ml sterile purified waters are added in precipitation, is fully mixed, take 0.1ml smears, Grain stain microscopy after drying is counted
Hypha,hyphae/spore quantity in full sheet.
4th, experimental result：
1. in first 3 days after blood sample culture starts, the whole report sun of 20 bottles of test cultures bottle instruments, and control bottle after
Sun was not reported in continuous culture to 14 days yet；
2. hypha,hyphae/spore count result see the table below；
In 10 samples of A groups, detected in 0.1ml precipitations and arrive most 250 of white saccharomycete spore, minimum 136, averagely
182, equivalent to being enriched to 1820 fungal spores in every 1ml blood samples；In 10 samples of B groups, detected in 0.1ml precipitations
Most 264 of aspergillus niger mycelia, minimum 102, average 204, equivalent to being enriched to 2040 fungies in every 1ml blood samples
Mycelia；The fungi of two groups of sample enrichments is satisfied by fungal nucleic acid extraction or mass-spectrometric technique to carry out Fungal identification and the minimum of bacterium amount is wanted
Ask.
Claims (6)
1. it is a kind of from blood sample be enriched with filamentous fungi method, including：
(1) 1-2ml blood samples are extracted from Positive Blood Culture blake bottle, centrifuge tube is injected；
(2) to 50-150mg quartz sands and 10-20ml enrichment dilutions is added in centrifuge tube, mix, stand, centrifugation is sunk
Form sediment；Wherein, enrichment dilution is 10-30 by volume ratio:1 80-200g/L urea liquids and 500-600mM EDTA composition；
(3) precipitation is shaken, centrifugation is subsequently added lauryl sodium sulfate SDS solution and sterile purified water, shakes, and removes stone
Sand；Centrifugation, washing, obtains final product the filamentous fungi of enrichment.
2. it is according to claim 1 it is a kind of from blood sample be enriched with filamentous fungi method, it is characterised in that：The step
Suddenly the time of repose in (2) is 5-10min.
3. it is according to claim 1 it is a kind of from blood sample be enriched with filamentous fungi method, it is characterised in that：The step
Suddenly the concentration of the lauryl sodium sulfate SDS solution in (3) is 0.5%-10%, is 1 with the volume ratio of precipitation:10-15.
4. it is according to claim 1 it is a kind of from blood sample be enriched with filamentous fungi method, it is characterised in that：The step
Suddenly the sterile purified water in (3) and the volume ratio of precipitation are 1:0.5-1.
5. it is according to claim 1 it is a kind of from blood sample be enriched with filamentous fungi method, it is characterised in that：The step
Suddenly the washing in (3) is specially and uses 1.5ml aseptic distillations water washing 3 times.
6. it is according to claim 1 it is a kind of from blood sample be enriched with filamentous fungi method, it is characterised in that：The step
Suddenly the centrifugal speed in (2) and (3) is 12000r/min, and centrifugation time is 1-2min.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111607555A (en) * | 2020-06-05 | 2020-09-01 | 杭州同创医学检验实验室有限公司 | Method for collecting fungal spores in deep fungal infection specimen |
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2017
- 2017-03-03 CN CN201710123599.1A patent/CN106893680B/en active Active
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2008154149A2 (en) * | 2007-06-07 | 2008-12-18 | Zimmer Orthobiologics, Inc. | Tissue processing for nonimmunogenic implants |
| WO2009042457A4 (en) * | 2007-09-21 | 2009-06-11 | Streck Inc | Nucleic acid isolation in preserved whole blood |
| CN102725423A (en) * | 2009-12-08 | 2012-10-10 | 比奥卡尔齐什股份有限公司 | Selective lysis of cells |
| CN102146374A (en) * | 2011-01-27 | 2011-08-10 | 四川农业大学 | Cell lysis solution for extracting animal DNA, kit and method |
| CN103518132A (en) * | 2011-06-06 | 2014-01-15 | 比奥卡尔齐什股份有限公司 | Selective lysis of cells by ionic surfactants |
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| 江宁: "《微生物生物技术》", 31 May 2008 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111607555A (en) * | 2020-06-05 | 2020-09-01 | 杭州同创医学检验实验室有限公司 | Method for collecting fungal spores in deep fungal infection specimen |
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