CN101851684A - Fungus colony polymerase chain reaction (PCR) method and pathogenic fungus identification method - Google Patents
Fungus colony polymerase chain reaction (PCR) method and pathogenic fungus identification method Download PDFInfo
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Abstract
The invention relates to a fungus colony PCR method and a pathogenic fungus identification method. The fungus colony PCR method aims to achieve the high positive rate of amplification, and the pathogenic fungus identification method aims to achieve short detection time and reliable results. The fungus colony PCR method comprises the following steps of: inoculating fungus cultures onto a culture medium for culture; taking fungus colonies which begin to appear on the culture medium as experimentally available colonies; and preparing a DNA template for colony PCR amplification. The pathogenic fungus identification method comprises the following steps of: inoculating clinical sample cultures or pathogenic fungus cultures onto the culture medium for culture; taking the colonies which begin to appear on the culture medium as the experimentally available colonies; preparing the DNA template for the PCR amplification; and detecting amplification products. The fungus colony PCR method and the pathogenic fungus identification method have the advantages of improving the positive rate of the fungus colony PCR amplification, solving the problems of complex steps, wall-breakage difficulty, long time and the like in fungus genomic DNA extraction, performing colony identification at the very beginning of the growth of pathogenic fungi, and achieving reliable results and identification time remarkably shorter than conventional fungus phenotype identification time.
Description
Technical field
The present invention relates to the method for colony polymerase chain reaction (PCR) method and the discriminating pathogenic epiphyte of a kind of fungi.
Background technology
At present, mycotic infection of superficial part is still keeping very high sickness rate and recurrence rate in the crowd, and wherein favus of the scalp, tinea unguium, dermatophytosis granuloma, recurrent monilial vaginitis etc. also exist the difficult problem of diagnosis and treatment aspect.And because immunocompromised host crowd's continuous expansion, the sickness rate of deep fungal infection increases especially year by year fast, and often threatening the life of institute's infection host, the success ratio of its treatment to depend on can early diagnosis and at the treatment of pathogenic bacteria.The pathogenic bacteria that causes various fungi infestations is kind surplus in the of 300 nearly, wherein common also kind surplus in the of 50 nearly.Different fungi kinds has than big-difference the susceptibility of different antifungal drugs, as Candida albicans, though Aspergillus fumigatus still is the The main pathogenic fungi of deep fungal infection, but the migration and variation with pathogen species of increasing along with preventative antimycotic treatment, much the insensitive even drug-fast pathogenic bacteria of routine clinical medication is obviously increased, present non-Candida albicans (as Candida parapsilosis), characteristics [the Arendrup MC.Invasive fungal infections:past achievements and challenges ahead.Clin Microbiol Infect that bent aspergillus of non-cigarette (as terreus etc.) and non-aspergillus filamentous fungus (as reaping hook mould etc.) infection proportion increases gradually, 2009,15 (7): 599-601.].Because deep fungal infection does not have a distinctive performance in early days clinical, there is very big difficulty in etiological diagnosis, so that the mortality ratio of deep fungal infection is up to 70%～90%[McCoy D, Depestel DD, Carver PL.Primary antifungal prophylaxis in adult hematopoietic stem cell transplant recipients:current therapeutic concepts.Pharmacotherapy, 2009,29 (11): 1306-1325.].At present clinically the diagnosis of fungi infestation is mainly relied on methods such as pathogenic bacteria morphology, serology and histopathology, comprise direct microscopy, fungus culture, little cultivation, polygalactomannan Detection of antigen (GM test) etc., length consuming time is arranged, susceptibility is low and is difficult to identify drawback such as bacterial classification, be difficult in the morbidity early detection and differentiate pathogenic bacteria.
Present stage Protocols in Molecular Biology develop rapidly differentiates that the molecular diagnostic techniques of bacterial classification emerges in an endless stream, and relies on quick, special, responsive advantage to be applied to the every field of Clinical Laboratory more and more.But these technology all are the successes with the bacterial classification template DNA is extracted as the basis.Fungal DNA preparation before the conventional pcr amplification needs through broken wall, extracting, precipitation, steps such as purifying, method commonly used has the CTAB method, liquid nitrogen method etc., the DNA extraction time was not waited by 6 hours by 2 hours, complex operation, sometimes need expensive reagent and some special processings, some reagent itself, as phenol, chloroforms etc. are unfavorable for that the operator's is healthy, and might have the residual of impurity such as thalline pigment, can influence subsequent P CR amplification equimolecular experiment [Fredricks DN, Smith C, Meier A.Comparison of Six DNA Extraction Methods for Recovery of Fungal DNA as Assessed by Quantitative PCR[J] .J Clin Microbiol, 2005,43 (10): 5122-5128.].
Clinical common sample has serum (slurry), whole blood, secretory product, fester, body fluid, flesh tissue etc., human body cell or Saliva Orthana are arranged in these samples, the mixing of impurity, need pre-treatment when extracting DNA, handle insufficient or incorrect, not only can react inhibition by residual PCR, also can cause the loss of fungi template DNA, make original fewer with regard to the very little DNA of content, conventional PCR may detect less than, very easily obtain false-negative result [Bretagne S, Costa JM.Towards a molecular diagnosis of invasive aspergillosis and disseminated candidosis[J] .FEMS Immunol Med Microbiol, 2005,45 (3): 361-318.].The few method of directly extracting DNA from clinical samples of using of China Clinical Laboratory section office just because of the difficult operation of this method, realizes that this leap may also can need for some time now.
Bacterium colony PCR (Colony PCR) technology sees article [the G ü ssow D of D.Gussow and T.Clackson the earliest, Clackson T.Direct clone characterization from plaques and colonies by the poIymerase chain reaction[J] .Nucleic Acids Res, 1989,17 (10): 4000.], be meant that with the bacterium colony of growing on the substratum be target, through simply boiling and the freezing physical method that waits, can extract genomic dna, be that template is carried out pcr amplification directly with the DNA that exposes after the thalline pyrolysis, saved loaded down with trivial details extraction DNA process, save the time, utilized this method to carry out the screening or the dna sequencing analysis of recombinant chou usually.But because present method is only destroyed thalline with the mode of thermo-cracking, be most commonly used to bacterium in the past, and fungal cell's skin has thicker cell walls, constitute by skeleton (microfibre that the insoluble polysaccharide crystal is formed) and matrix (polymer composite), structure is very fine and close, uses the heating power lysing cell difficult merely, so rare useful flora round pcr carries out the experimental study of mycology aspect, only see the application of odd relevant yeast aspect, do not see the research report that is used for filamentous fungus.
Summary of the invention
The invention provides the colony polymerase chain reaction (PCR) method of a kind of fungi, the positive rate height of amplification.
The present invention also provides the method for differentiating pathogenic epiphyte, adopts the colony polymerase chain reaction (PCR) method of above-mentioned fungi, detection time short, reliable results.
The colony polymerase chain reaction (PCR) method of described fungi is, fungal cultures is inoculated on the substratum, cultivates, and just grown macroscopic bacterium colony on the substratum, and as the available bacterium colony of experiment, the preparation dna profiling carries out colony PCR amplification.
The method of described discriminating pathogenic epiphyte is inoculated in clinical samples or pathogenic epiphyte culture on the substratum, cultivates, just grown macroscopic bacterium colony on the substratum, as the available bacterium colony of experiment, the preparation dna profiling, carry out colony PCR amplification, detect amplified production.
Above-mentioned substratum and culture temperature are common practise, and one adopts the PDA substratum, cultivate down at 28 ℃.
The reaction system of bacterium colony PCR is identical with the common PCR system, comprises: 4 μ l, 10 * damping fluid, 1.5 μ l MgCl
2Solution, 2 μ l dNTP solution, each 0.5 μ l of upstream and downstream primer, dna profiling (0.5～2) μ l, Taq archaeal dna polymerase 2U, wherein MgCl
2The concentration of solution, dNTP solution, primer is respectively 10mmol/L, 10 μ mol/L, 10 μ mol/L.
The preparation method of dna profiling is:
When fungi is filamentous fungus, the method for preparing dna profiling is: add autoclaved quartz sand in the aseptic 1 * TE damping fluid of 20 μ l, add the available bacterium colony mycelium of experiment, boil 5min, freezing immediately then 5～10min, 2 times repeatedly, vortex 3～5min then, centrifugal 5～the 10min of 12000～14000r/min gets supernatant liquor as dna profiling; When fungi is yeast, the method for preparing dna profiling is: add the available bacterium colony thalline of experiment at the aseptic 1 * TE damping fluid of 20 μ l, boil 3min, freezing immediately then 5～10min, centrifugal 5～the 10min of 12000～14000r/min gets supernatant liquor as dna profiling.
As general knowledge known in this field, freezing temp is-15～-20 ℃.
As preferred version, when fungi is filamentous fungus, the method for preparing dna profiling is: add autoclaved quartz sand in the aseptic 1 * TE damping fluid of 20 μ l, add a few experiments and can use the bacterium colony mycelium, boil 5min, place-20 ℃ of freezing 5min down then immediately, 2 times repeatedly, vortex 5min then, the centrifugal 5min of 14000r/min gets supernatant liquor as dna profiling; When fungi is yeast, the method for preparing dna profiling is: add a few experiments at the aseptic 1 * TE damping fluid of 20 μ l and can use the bacterium colony thalline, boil 3min, place-20 ℃ of freezing 5min down then immediately, the centrifugal 5min of 14000r/min gets supernatant liquor as dna profiling.
Concrete operation steps is as follows: filamentous fungus: prepare the aseptic PCR pipe of 0.2ml, add a small amount of autoclaved quartz sand, add the aseptic 1 * TE damping fluid of 20 μ l, and standby.Scrape the mycelium that takes a morsel with getting acicula, add in the ready PCR pipe, mark, lid is tight, boils, and it is freezing to place refrigerator to carry out immediately then, 2 times repeatedly, is put in the vortice vortex.Centrifugal then, get supernatant liquor 1 μ l and carry out the PCR reaction as template.Wherein, the consumption of quartz sand one be 40-60 μ g.Yeast-like fungus: prepare the aseptic PCR pipe of 0.2ml, add the aseptic 1 * TE damping fluid of 20 μ l, standby.Scrape the thalline that takes a morsel with getting collarium, add in the ready PCR pipe, mark, lid is tight.Boil, place refrigerator and cooled to freeze then immediately, centrifugal after, get supernatant liquor 1 μ l and carry out PCR reaction as template.
Fungal cell's skin has thicker cell walls, and the effect of protection thalline is arranged.The main component of cell walls is a polysaccharide, forms the skeleton (microfibre that the insoluble polysaccharide crystal is formed) and the matrix (polymer composite) of cell walls, and structure is very fine and close.And form in the skeleton of microfibre, filamentous fungus is based on chitin, and yeast is based on dextran.The cell-wall component of a kind of fungi is not a fixed, at its different growth phases, the composition of cell walls has obvious difference [Griffin DW, Kellogg CA, Peak KK, et al.A rapid and efficient assay for extracting DNA from fungi[J] .Lett Appl Microbiol, 2002,34 (3): 210-214.].Early stage in growth, fungal cell wall then is made of for four layers periostracum, protein layer, proteglycan layer and matter layer dextran layer in the growth ripening stage mainly by periostracum with protein layer is two-layer constitutes.The applicant finds after deliberation, carry out the dna profiling preparation in early days in the filamentous fungus growth, can successfully carry out pcr amplification, with the bacterium colony of growth and maturity prepare template then can not, be the key point of bacterium colony PCR Success in Experiment of the present invention so select the early stage bacterium colony of growth to experimentize.
Select early stage bacterium colony that following advantage is arranged: the DNA that the DNA of preparation and ripe bacterium colony extract is in full accord, and reduced the mixing of impurity such as melanochrome, improved the positive rate of pcr amplification, shortened Diagnostic Time when the most important thing is clinical application, for clinical treatment gains time.
In all bacterium colony PCR reactions, the applicant is provided with negative control and positive control, because the specificity of bacterium colony PCR is poor than regular-PCR, negative control and positive control are necessary very much, can reduce false positive or false-negative result, in order to avoid obscure the analysis of tester to the result.In addition, to carry out PCR reaction in time through boiling, freeze the DNA that comes out after separating, can not place too of a specified duration, otherwise the deleterious of amplification.
The present invention has improved the positive rate of fungal colony pcr amplification, solved fungal gene group DNA extraction complex steps, the broken wall difficulty, problems such as length consuming time, and can carry out strain identification at pathogenic bacteria growth incunabulum, the more conventional fungi phenotypic evaluation time obviously shortens, and reliable results, its clinical meaning and value are obviously to have shortened the affirmation bacterial classification, confirm the time of diagnosis, and studies show that deep fungal infection, treatment time differs significant difference [the Nolla-Salas J that will cause mortality ratio in 48 hours, Sitges-Serra A, Le ó n-Gil C, et al.Candidemia in non-neutropenic critically ill patients:analysis of prognostic factors and assessment of systemic antifungal therapy.Study Group of Fungal Infection in the ICU[J] .Intensive Care Med, 1997,23 (1): 23-30.].
Description of drawings
Fig. 1 is 16 strain bacterial classification bacterium colony PCR electrophoresis result M:100bp Ladder; 1: negative control; 2: positive control (Aspergillus fumigatus) 3: Aspergillus fumigatus; 4: flavus; 5: terreus; 6: aspergillus niger; 7: aspergillus versicolor; 8: the many spores of most advanced and sophisticated match; 9: fusarium moniliforme; 10: Fusarium oxysporum; 11: blue colter is mould; 12: changeable Mucor; 13: absidia corymbifera; 14: mucor hiemalis; 15: Mucor pusillus; 16: Penicillium digitatum; 17: Penicilllum expansum; 18: penicillium funiculosum.
Fig. 2 is bacterium colony PCR and conventional pcr amplification product electrophorogram M:100bp Ladder; 1: negative control; 2: positive control (Aspergillus fumigatus) 3,5,7,9,11,13,15 is conventional PCR result; 4,6,8,10,12,14,16 is bacterium colony PCR result; 3,4 is Aspergillus fumigatus; 5,6 is flavus; 7,8 is terreus; 9,10 is aspergillus niger; 11,12 is aspergillus versicolor; 13,14 is the many spores of most advanced and sophisticated match; 15,16 is fusarium moniliforme.
Fig. 3 is the colony PCR amplification M:50bp marker as a result of Application Example 1; 1: negative control; 2: positive control (Aspergillus fumigatus); 3: yeast colony; 4: filmanetous colony.
Fig. 4 is Application Example 2 reference culture multiplex PCR electrophorogram M:50bp markers; 1: negative control; 2: Aspergillus fumigatus; 3: trichophyton; 4: Candida parapsilosis.
Fig. 5 is the clinical strain multiplex PCR electrophorogram M:50bp marker of Application Example 2; 1: negative control; 2: positive control (Aspergillus fumigatus); 3～13 are respectively: clinical strain 1～11.
Embodiment
1. bacterium colony is cultivated:
The used filamentous fungus of experiment is being inoculated under the aseptic condition on the PDA substratum for preparing, cultivate 7～14d for 28 ℃, after treating the colony growth maturation, scrape and take a morsel mycelia or spore in 100 μ l stroke-physiological saline solution with the aseptic acicula of getting, the vibration vortex makes hypha body disperse as far as possible, the ready-made bacterium solution that takes a morsel is inoculated on the PDA plate culture medium, cultivate about 20～40h (specifically deciding) for 28 ℃, grow macroscopic bacterium colony on the substratum, be considered as testing available bacterium colony on the colony growth situation.
2. the dna profiling preparation method of bacterium colony PCR
Get the aseptic PCR pipe of 0.2ml, add a small amount of autoclaved quartz sand, add the aseptic 1 * TE damping fluid of 20 μ l, standby.Scrape the above-mentioned mycelium that takes a morsel with getting acicula, add in the ready PCR pipe, mark, lid is tight, and 100 ℃ are boiled 5min, place-20 ℃ of refrigerator 5min then immediately, 2 times repeatedly, are put in vortice vortex 5min.The centrifugal 5min of 14000r/min gets supernatant liquor 1 μ l and carries out the PCR reaction as template.
3.PCR amplification
With fungi universal primer ITS 1 and ITS 4, PCR reaction system 25 μ l contain 4 μ l, 10 * damping fluid, 1.5 μ l MgCl
2(10mmol/L), 2 μ l dNTPmix (10umol/L), each 0.5 μ l of primer (10 μ mol/L), dna profiling 2 μ l, Taq archaeal dna polymerase 2U.Reaction conditions is .95 ℃ of 1min after 95 ℃ of pre-sex change of 5min, 52 ℃ of 1min, and 72 ℃ of 1min carry out 30 circulations altogether. and last 72 ℃ are extended 10min.Amplified production adopts 1.5% sepharose to carry out electrophoresis, each well point sample 4 μ l, and electrophoretic buffer is 1 * TAE, constant voltage 80V, the about 30min of electrophoresis.After electrophoresis finishes, get sepharose observations and take the photograph sheet under ultraviolet (uv) transmission on the gel imaging instrument.Negative control and positive control are established in reaction, and negative control replaces dna profiling with deionized water, the DNA genome of the Aspergillus fumigatus that positive control extracts and detected with ordinary method.
The result shows, in the used 16 kinds of bacterial classifications of 7 genus of all experiments, all successfully amplifies positive band, and PCR product electrophoresis concrete outcome is seen Fig. 1.
4.ITS district's gene sequencing
The PCR product is served sea living worker's biotechnology company limited and is carried out base sequence mensuration, and contrasts with online known bacterial classification ITS district fragment sequence.Sequencing result sees Table 1.
Table 1:16 strain bacterium sequencing result
5. the comparison of bacterium colony PCR and conventional PCR
In order to differentiate the reliability of bacterium colony PCR reaction result, under the same conditions, the conventional PCR reaction carried out of the genomic templates of 7 kinds of filamentous funguss carrying with previous experiments (Aspergillus fumigatus, flavus, terreus, aspergillus niger, aspergillus versicolor, fusarium moniliforme and the many spores of most advanced and sophisticated match) compares.Bacterium colony PCR result is consistent with conventional PCR electrophorogram result, but band is dark slightly, sees Fig. 2.
The research that bacterium colony PCR is applied to fungi does not appear in the newspapers as yet at home, it is many slightly to be applied to saccharomycetic report abroad, the research that is used for filamentous fungus is detected in two Japanese scholars, but is not simple direct bacterium colony PCR, but by special reagent or damping fluid.Is example with scholar Alshahni MM etc. research in 2009, belongs in 20 kind of 35 strain filamentous fungus used 3, and directly bacterium colony PCR experiment positive strain has only 11 examples, and positive rate is 31.4%.Aspergillus fumigatus, terreus, flavus, aspergillus niger, aspergillus versicolor, Penicilllum expansum and fusarium moniliforme used in their experiment all successfully do not increase, and the present invention has all successfully carried out bacterium colony PCR and obtained positive findings.Analyze the bigger reason of experimental result difference and be, used mycelium such as Alshahni MM is the ripe bacterium colony of growth 7～10d, and used bacterium colony is the bacterium colony of growth incunabulum among the present invention.
Application Example 1: the application in subcutaneous fungi infestation pathogen identification
1.1 clinical data
The patient man, July the baby, red papule takes place in place, canthus, right side in no obvious cause before 4 months, there is warts on the top, then ulceration, at skin lesions circumferece a plurality of similar skins take place and decrease, increase gradually to spread to skin, right side cheek, nose, forehead etc. near the eyes and locate, skin decreases and is wetting property erythema tubercle, contain more fester, repeatedly take out the purulence processing with syringe needle in local outpatient service and hospital, and give quiet of microbiotic such as penicillin, gentamicin, skin decreases and still continues expansion.Came Dermatosis Hospital, Chinese Academy of Medical Sciences's outpatient service to go to a doctor on March 29th, 2010.Dermatology Department checks: the visible large stretch of wetting property erythema tubercle of right side face, forehead, the bridge of the nose and left side inner eye corner, central skin decrease and merge in flakes, on cover thicker yellowish brown crust, right eyelid top skin decreases significant depressions, size is about 1cm * 3cm, right eye is opened eyes limited.It is the red tubercle of a plurality of protuberances that skin decreases the edge, meromixis, about 0.5cm * 1cm～1cm * 1.5cm size, nodule surface is smooth, the visible purulence head of part, matter is soft, and a 1cm * 3cm redness tubercle is arranged under the auris dextra, pressure fluctuation arranged.
1.2 method:
The mycology laboratory examination
The preparation of bacterium colony pcr gene group dna profiling: fungus culture goes out 2 kinds of bacterium colonies, be respectively filmanetous colony and yeast colony, filmanetous colony genomic dna method for preparing template is with embodiment 1, yeast colony genomic dna method for preparing template is: add a small amount of thalline at the aseptic 1 * TE damping fluid of 20 μ l, 100 ℃ are boiled 3min, place-20 ℃ of refrigerator and cooled to freeze 5min then immediately, the centrifugal 5min of 14000r/min gets supernatant liquor as dna profiling.
Pcr amplification
Each bacterium colony pcr gene group dna profiling to preparation carries out pcr amplification respectively, and concrete grammar is with embodiment 1.
ITS district gene sequencing
1.3 colony PCR amplification result
The results are shown in Figure 3.
1.4 product sequencing result
The order-checking fragment is the ITS region sequence between ITS1 and the ITS4 as shown above, and the yeast colony sequencing result is shown as 489bp, and the filmanetous colony sequencing result is shown as 557bp, through BLAST (network address
Http:// blast.ncbi.nlm.nih.gov/Blast.cgi) after the comparison, yeast colony is a Candida parapsilosis, filmanetous colony is an Alternaria tenuissima.The order-checking diagnosis is consistent with the morphology diagnosis.
1.5 discuss
Collect 1 routine subcutaneous fungi infestation patient in this experiment, this patient is big baby in July, and site of pathological change is at face, and pathologic sampling is restricted, and can't obtain histopathology and specific stain result, and in this case, it is particularly important that mycologic evidence seems.Repeatedly fungus microscope examination is all positive with cultivation under this patient's skin damage surface and the scab, after fungus culture the 3rd day, grow macroscopic two kinds of bacterium colonies, picking carries out the detection of bacterium colony PCR, finish to need the 3h time altogether to pcr amplification from handling sample, send order-checking then, can obtain sequencing result on the 2nd day, obtaining sequencing result from drawing materials to needs 5 day time altogether.Traditional form method identifies that yeast colony is Candida parapsilosis 7d consuming time approximately, identifies that filamentous fungus is chain lattice spore 10d consuming time approximately.Bacterium colony PCR sequencing result is consistent with afterwards little cultivation of filamentous fungus and the good colour developing of oidiomycetic kerma culture identification result, has verified the exactness of bacterium colony PCR.
1. bacterium colony is cultivated:
Fungi is adopted standard bacteria strain Aspergillus fumigatus, and cultural method is with embodiment 1.
2. the dna profiling preparation method of bacterium colony PCR
With embodiment 1.
3.PCR amplification
Multiplex PCR system primer fungi universal primer NS, dermatophytosis Auele Specific Primer and yeast Auele Specific Primer (seeing Table 2).PCR reaction system 25 μ l contain 4 μ l, 10 * damping fluid, 1.5 μ l MgCl
2(10mmol/L), 2 μ l dNTPmix (10umol/L), each 0.5 μ l of primer (10 μ mol/L), dna profiling 2 μ l, Taq archaeal dna polymerase 2U.Reaction conditions is .95 ℃ of 1min after 95 ℃ of pre-sex change of 5min, 52 ℃ of 1min, and 72 ℃ of 1min carry out 30 circulations altogether. and last 72 ℃ are extended 10min.Amplified production adopts 1.5% sepharose to carry out electrophoresis, each well point sample 4 μ l, and electrophoretic buffer is 1 * TAE, constant voltage 80V, the about 30min of electrophoresis.After electrophoresis finishes, get sepharose observations and take the photograph sheet under ultraviolet (uv) transmission on the gel imaging instrument.Negative control is established in reaction, and negative control replaces dna profiling with deionized water.
Remove fungi and adopt standard bacteria strain trichophyton, all the other are with embodiment 2.
1. bacterium colony is cultivated:
Fungi is adopted standard bacteria strain Candida parapsilosis, and cultural method is with embodiment 1.
2. the dna profiling preparation method of bacterium colony PCR
Add a small amount of thalline at the aseptic 1 * TE damping fluid of 20 μ l, 100 ℃ are boiled 3min, place-20 ℃ of refrigerator and cooled to freeze 5min then immediately, and the centrifugal 5min of 14000r/min gets supernatant liquor as dna profiling.
3.PCR amplification
With embodiment 2.
The result as shown in Figure 4, embodiment 2-4 can amplify the fragment of 310bp, 450bp and 271bp size respectively.
Application Example 2
2.1 clinical data: the clinical samples object of collection is the superficial mycosis patient that medical courses in general institute skin grinds institute's fungi testing laboratory, is the fungus microscope examination positive sample, jock itch 6 examples wherein, the tinea manuum 2 examples, tinea unguium 3 examples.
2.2 method:
The mycology laboratory examination
Bacterium colony pcr gene group dna profiling preparation: with clinical implementation example 1.
Pcr amplification: each the bacterium colony pcr gene group dna profiling to preparation, carry out pcr amplification respectively, concrete grammar is with embodiment 2.
Table 2: the used Auele Specific Primer situation of multiplex PCR
2.3 multi-PRC reaction result
11 strain clinical strains identify that with above-mentioned multi-PRC reaction system electrophorogram is seen Fig. 5 as a result, can differentiate relatively that according to 50bp Ladder contrast and with the type strain collection of illustrative plates fungi arrives kind.Clinical strain 1～7 and 11 is dermatophytosis, and clinical strain 8 and 10 at first is thought of as filamentous fungus and belongs to.
Superficial mycosis is modal fungal infectious disease, and crowd's morbidity is 20%～25% in the world wide, and the The main pathogenic fungi of superficial mycosis is a dermatophytosis, but the shared ratio of yeast has significantly than before and increases.Medication is of great importance to clinical guidance to the kind level with the pathogenic bacteria Rapid identification.Existing fungus culture identifies that bacterial classification needs 14 days, and obtaining drug sensitivity tests needs 21 days, and indivedual growth are slower, need the time longer.
The used multiplex PCR system of this experiment can be differentiated the bacterial classification of 3 kinds, i.e. dermatophytosis, yeast and filamentous fungus by setting up in investigator's previous work of the present invention in same system.If 3 pairs of primer amplifications are all positive, then prompting may be dermatophytosis, yeast and mould, or dermatophytosis and zymic polyinfection; If preceding 2 pairs of primer amplification positives, then prompting may be the polyinfection of dermatophytosis and mould or the simple infection of dermatophytosis; If the 1st pair and the 3rd pair of primer amplification positive, then prompting may be polyinfection or the zymic simple infection of yeast and mould; If only the 1st pair of primer amplification positive arranged, then may be the simple infection of mould.Like this, within a short period of time, for the clinician provides the reference information of pathogenic bacteria, the doctor can select the suitableeest medicine at a certain patient in conjunction with clinical manifestation.
Experimental result shows that also this reaction system is more stable, can amplify purpose fragment clearly, does not see the interference of obvious non-specific band, has carried out fungus culture with the PDA substratum simultaneously, and culture identification result is with bacterium colony multiplex PCR unanimity as a result.Illustrate that the bacterium colony round pcr can be used in the clinical detection of mycotic infection of superficial part, preliminary evaluation needs 3-5 days time to kind.Owing to time is pressing, routine sample surplus investigator of the present invention has only collected ten, be intended to verify the feasibility of bacterium colony PCR, do not carry out the experimental study of large-scale clinical data and bacterial classification data.The success of this experiment is that certain experiment basis has been established in later research, and this method is simple to operate, and agents useful for same is less, and is time saving and energy saving, is particularly suitable for large-scale experimental study, obviously shortens the experimental study cycle.
Claims (8)
1. the colony polymerase chain reaction (PCR) method of a fungi is characterized in that, fungal cultures is inoculated on the substratum, cultivates, and has just grown macroscopic bacterium colony on the substratum, and as the available bacterium colony of experiment, the preparation dna profiling carries out colony PCR amplification.
2. the colony polymerase chain reaction (PCR) method of fungi as claimed in claim 1, it is characterized in that, when fungi was filamentous fungus, the method for preparing dna profiling was: add autoclaved quartz sand in the aseptic 1 * TE damping fluid of 20 μ l, add the available bacterium colony mycelium of experiment, boil 5min, freezing immediately then 5～10min, 2 times repeatedly, vortex 3～5min then, centrifugal 5～the 10min of 12000～14000r/min gets supernatant liquor as dna profiling.
3. the colony polymerase chain reaction (PCR) method of fungi as claimed in claim 1, it is characterized in that, when fungi is yeast, the method for preparing dna profiling is: add the available bacterium colony thalline of experiment at the aseptic 1 * TE damping fluid of 20 μ l, boil 3min, freezing immediately then 5～10min, the centrifugal 5～10min of 12000～14000r/min gets supernatant liquor as dna profiling.
4. as the colony polymerase chain reaction (PCR) method of claim 2 or 3 described fungies, it is characterized in that freezing temp is-15～-20 ℃.
5. a method of differentiating pathogenic epiphyte is characterized in that, clinical samples or pathogenic epiphyte culture are inoculated on the substratum, cultivate, just grown macroscopic bacterium colony on the substratum, be considered as testing available bacterium colony, the preparation dna profiling carries out colony PCR amplification, detects amplified production.
6. the method for discriminating pathogenic epiphyte as claimed in claim 5, it is characterized in that, when pathogenic epiphyte was filamentous fungus, the method for preparing dna profiling was: add autoclaved quartz sand in the aseptic 1 * TE damping fluid of 20 μ l, add a small amount of mycelium, boil 5min, freezing immediately then 5～10min, 2 times repeatedly, vortex 3～5min then, centrifugal 5～the 10min of 12000～14000r/min gets supernatant liquor as dna profiling.
7. the method for discriminating pathogenic epiphyte as claimed in claim 5, it is characterized in that, when pathogenic epiphyte is yeast, the method for preparing dna profiling is: add a small amount of thalline at the aseptic 1 * TE damping fluid of 20 μ l, boil 3min, freezing immediately then 5～10min, the centrifugal 5～10min of 12000～14000r/min gets supernatant liquor as dna profiling.
8. as the method for claim 6 or 7 described discriminating pathogenic epiphytes, it is characterized in that freezing temp is-15～-20 ℃.
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN103374604A (en) * | 2012-04-12 | 2013-10-30 | 中国科学院大连化学物理研究所 | Pre-treatment method of thallus of saccharomycetes colony for PCR (Polymerase Chain Reaction) |
| CN103627702A (en) * | 2013-12-04 | 2014-03-12 | 上海市农业科学院 | Method for extracting edible mushroom DNA |
| CN104560958A (en) * | 2015-01-22 | 2015-04-29 | 湖南农业大学 | Simple extracting method for DNA in colony polymerase chain reaction (PCR) amplification fungal rDNA-ITS |
| CN108642136A (en) * | 2018-05-21 | 2018-10-12 | 云南大学 | A kind of thermophilic preparation method for belonging to fungi pcr template |
| CN109852712A (en) * | 2019-01-27 | 2019-06-07 | 上海海洋大学 | A kind of simple and effective bacterial fungus bacterium colony PCR general program |
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| 《中国皮肤性病学杂志》 20040229 王英 等 聚合酶链反应快速检测丝状真菌 123-125页 1、5 第18卷, 第2期 2 * |
| 《中国皮肤性病学杂志》 20040229 王英 等 聚合酶链反应快速检测丝状真菌 123-125页 2-4、6-8 第18卷, 第2期 2 * |
| 《食品科技》 20090430 卢鑫 等 马克思克鲁维酵母DNA提取方法的比较 31-33、37页 2-4、6-8 第34卷, 第4期 2 * |
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103374604A (en) * | 2012-04-12 | 2013-10-30 | 中国科学院大连化学物理研究所 | Pre-treatment method of thallus of saccharomycetes colony for PCR (Polymerase Chain Reaction) |
| CN103627702A (en) * | 2013-12-04 | 2014-03-12 | 上海市农业科学院 | Method for extracting edible mushroom DNA |
| CN104560958A (en) * | 2015-01-22 | 2015-04-29 | 湖南农业大学 | Simple extracting method for DNA in colony polymerase chain reaction (PCR) amplification fungal rDNA-ITS |
| CN108642136A (en) * | 2018-05-21 | 2018-10-12 | 云南大学 | A kind of thermophilic preparation method for belonging to fungi pcr template |
| CN109852712A (en) * | 2019-01-27 | 2019-06-07 | 上海海洋大学 | A kind of simple and effective bacterial fungus bacterium colony PCR general program |
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| CN101851684B (en) | 2013-07-17 |
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