CN102031298B - DNA (deoxyribose nucleic acid) probe and gene chip for detecting rhizomucor pusillus and applications thereof - Google Patents
DNA (deoxyribose nucleic acid) probe and gene chip for detecting rhizomucor pusillus and applications thereof Download PDFInfo
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Abstract
The invention relates to the technical field of biology and discloses a DNA (deoxyribose nucleic acid) probe for detecting rhizomucor pusillus. The DNA probe for detecting the rhizomucor pusillus has nucleotide sequence shown in the SEQ ID No.1 in the sequence table and has strong specificity and sensitivity as high as 0.04ng in detecting rhizomucor pusillus. The invention also provides a gene chip containing the DNA probe. By utilizing the gene chip, high-throughput, automatic and rapid detectionon samples possibly containing rhizomucor pusillus can be realized; and the gene chip has strong specificity, can ensure the accuracy of the detection result and has wide application prospect.
Description
Technical field
The present invention relates to biological technical field, be specifically related to a kind of dna probe, gene chip and application thereof that detects Rhizomucor pusillus.
Background technology
Invasive infections with fungi is fungi invasion human body and the severe infections that causes blood infection, each organs infection or whole body to be sent out.The morbidity of invasive infections with fungi is the trend of rising appreciably over nearly 20 years, and this operates relevant with extensive application Broad spectrum antibiotics, anticancer radiotherapy, chemotherapy and other immunosuppressor, steroid medicine, organ transplantation, venous cannula and various aggressive.
The main pathomycete of invasive infections with fungi has the condition pathomycetes such as Candida, Aspergillus, genera cryptococcus, also has the endemicity pathomycetes such as histoplasma capsulatum, Blastomyces dermatitidis, sporothrix.Under the varying environment, the fungal species of infection is different.Invasive infections with fungi does not have specific findings in early days, so be easy to be covered, case fatality rate is very high.
At present the technology of clinical detection fungi commonly used mainly contains:
(1) microscopy and cultivation, evaluation: be clinical the most frequently used identification of means such as silver hexamine staining, PAS dyeing, silver-colored ammonia G-S staining, 10%KOH method and method and fungi culture, but directly microscopy poor specificity, susceptibility deficiency can not identify fungal species.Though fungus culture can be identified fungal species, culture condition is harsh, and incubation time is long, and false negative is high, and susceptibility is not enough, can't assist clinical treatment.
(2) immunological method: the ultimate principle of immunological method is to utilize the specific combination of antigen and antibody, reaches mutual special seeking and visiting.This method is mainly for detection of antigen, antibody, meta-bolites and the cellular constituent of fungi.Can be according to the difference of fungal antigen with classification of fungi.But the specificity of antibody has limited the development of this technology.
Immunohistochemical methods, enzyme linked immunosorbent assay, immunoblotting and agglutination reaction all are based on the means that immunological method detects fungi, but being widely used at present the clinical G that only has tests, what the G test detected is the cell wall constituent of fungi: (1,3)-callose, after the phagocytic cell of human body is engulfed fungi, this material of energy sustained release makes increased content in blood and the body fluid (mycotic infection of superficial part is without similar phenomenon).Be applicable to the early diagnosis of all deep fungal infections except cryptococcus and zygomycetes, especially candidiasis and aspergillus tubigensis, but can not determine bacterial classification.Find to have many false positive possibilities in the clinical application, for example:
(1) use cellulose membrane to carry out the hemodialysis sample or the patient is exposed to gauze or other contain the material of dextran;
(2) Intravenous immunoglobulin, albumin, thrombin or blood products;
(3) strepticemia;
Exist when (4) operator processes sample and pollute.
In addition, the candidiasis that the mucosa injury that uses polyose cancer therapy drug, chemicotherapy to cause causes dextran in the food or field planting enters blood etc. through gi tract also may cause false positive.
(3) Protocols in Molecular Biology:
Comprise G+C mol% assay among in situ hybridization, polymerase chain reaction, the DNA, randomly amplified polymorphic DNA (RAPD) analysis, restriction enzyme polymorphism analysis (RFLP) etc., but above-mentioned technology all takes time and effort, do not have high-throughout characteristics, can not detect simultaneously fast the multiple fungi that may exist with a small amount of sample, therefore be confined to laboratory study more, still can not really be applied to clinical.
Summary of the invention
The purpose of this invention is to provide a kind of dna probe that detects Rhizomucor pusillus, have sequence shown in the SEQID No.1, itself and Rhizomucor pusillus DNA specific binding, it is highly sensitive to detect Rhizomucor pusillus.
Dna probe of the present invention can be widely used in biology, medical field, as with take round pcr as the basis nucleic acid hybridization technique be combined, carry out biological analysis or clinical diagnosis.
The present invention is in conjunction with biochip technology, can realize small sample, high-throughput, special, change the purpose of detection Rhizomucor pusillus fast and automatically.A kind of gene chip for the fungi detection provided by the invention forms by specific oligonucleotide probe being fixed on surface of solid phase carriers, and described oligonucleotide probe comprises the dna probe with sequence shown in the SEQ ID No.1.Relation between probe, primer and the fungi goal gene is as shown in table 1.
The employed primer of table 1. gene chip of the present invention and probe
As preferably, described solid phase carrier is selected from nitrocellulose filter, nylon membrane, polystyrene, sheet glass, silicon chip, particulate and plastic sheet.
The solid phase carrier that gene chip of the present invention adopts is selected from any material that can be used for preparing gene chip, includes but not limited to nitrocellulose filter, nylon membrane, poly-the third ethene, sheet glass, silicon chip, particulate and plastic sheet.Described solid phase carrier is after different chemically modifieds, and with active group that can immobilized nucleic acid molecule, these groups include but not limited to amino (NH on its surface
2), aldehyde radical (CHO), carboxyl (COOH) and sulfydryl (SH).
Connecting needle is to the specific dna probe of Rhizomucor pusillus on described solid phase carrier for gene chip of the present invention, and described probe can be strand or double chain DNA molecule, and it can be hybridized with the Rhizomucor pusillus gene specific under suitable condition.Described probe can by DNA automatically synthetic or pcr amplification obtain.Described probe can optionally add a purpose at its end that is connected with carrier and be to increase the connecting arm that the spatial activity of probe is beneficial to hybridize.
The preparation of gene chip of the present invention, be that point sample is to carrier in an orderly manner with described nucleic acid probe, active group and the carrier surface corresponding active group end modified by nucleic acid probe react, and form covalently bound, probe is fixed on the carrier, thereby makes gene chip.Simultaneously, on gene chip of the present invention, according to the order of sequence from the hybridization of different samples after, by whether the existence of Rhizomucor pusillus is arranged in can be clear and definite to the addressing of the catching positive signal corresponding sample.
As preferably, described surface of solid phase carriers also is fixed with positive control probe, negative control probe.
More preferably, described its sequence of positive control probe is shown in SEQ ID No.4, itself and positive control sample specific binding, the positive control sample fragment sequence is shown in SEQ ID No.6, and wherein the sequence with positive control probe joint position is CCCATGGGTCTTGTCTGGA.
More preferably, described negative control probe, its sequence is shown in SEQ ID No.5.
The present invention also provides a kind of test kit for detection of Rhizomucor pusillus, comprises: with the probe of Rhizomucor pusillus specific binding, described probe has in the nucleotide sequence shown in the sequence table SEQ ID No.1.Test kit of the present invention preferably also comprises for the primer to the fungal gene amplification, and it has the sequence shown in sequence table SEQ ID No.2 or the SEQ ID No.3.
The present invention also provides a kind of method that the sample that may contain Rhizomucor pusillus is detected, and comprises following steps:
Step 1: extract and the nucleotide sequence of amplification sample, described amplification with the beacon molecule marker to amplified production;
Step 2: get step 1 gained amplified production and hybridize with the probe that is fixed on the described gene chip;
Step 3: adopt corresponding detection method that gene chip is carried out scanning analysis according to described beacon molecule.
As preferably, 5 ' used primer of step 1 amplification step is fungi 18SrRNA universal primer, has sequence shown in the sequence table SEQ ID No.2, and 3 ' primer is fungi 28SrRNA universal primer, has the sequence shown in the SEQ ID No.3.
In described sample labeling process, can utilize various nucleic acid labeling methods known in the art to make goal gene marker beacon molecule in the sample, described method includes but not limited to, the methods such as PCR, RT-PCR, in-vitro transcription, random primer labelling, nick translation and terminal metastatic marker.Described beacon molecule can be fluorescein or isotropic substance; Described fluorescein includes but not limited to, Cy3, Cy5, fluorescein isothiocyanate, texas Red; Described isotropic substance includes but not limited to 32P.
Under suitable temperature and ionic strength conditions, described marked product and gene chip of the present invention are hybridized.Hybridization conditions and method are known in the art.After the hybridization, adopt corresponding detection method that gene chip is carried out scanning analysis according to selected beacon molecule, according to the positive signal addressing of catching, judge whether contain Rhizomucor pusillus in the described sample.
The present invention adopts the Rhizomucor pusillus specific oligonucleotide as probe, in the fungi testing process, utilize the fungi universal primer to sample in fungal gene amplification carry out mark.The probe that gene chip of the present invention connects and the used fungi universal primer of amplification are listed in the table 1.Need to prove; oligonucleotide probe of the present invention and fungi universal primer also can be used for the regular-PCR amplification to goal gene; and the PCR product of amplification can be used as equally probe and prepares epiphyte detection gene chip according to described method, therefore described PCR product as the prepared gene chip of probe also within protection scope of the present invention.
Protocols in Molecular Biology is applied to fungal studies and has demonstrated many-sided advantage, and medical mycology research has formed gradually take evaluation and the categorizing system of genetic analysis as the basis.We utilize the high-throughput platform of chip based on Protocols in Molecular Biology, the technology that designs perfect to infect based on gene chip specific diagnosis Rhizomucor pusillus.
Dna probe high specificity of the present invention, highly sensitive, containing the above Rhizomucor pusillus of 2-3CFU in the sample is visible positive signal after testing, Rhizomucor pusillus PCR product reaches more than the 0.04ng and can be detected.
Gene chip of the present invention and to utilize described gene chip to carry out the required sample size of method of sample detection few, reaction volume is little, reagent consumption is few, can realize high-throughput, automatization and the rapid detection of sample, analytic process is objective, high specificity, and the concordance rate by the detected result of present method and separation and Culture or order-checking comparison result reaches 100%, guaranteed the accuracy of detected result, be suitable for being applied to clinical.
Description of drawings:
Fig. 1 shows the detected result of gene chip of the present invention and Rhizomucor pusillus strain hybrid;
Fig. 2 shows the DNA of different quantities Rhizomucor pusillus extraction of spores and the detected result of gene chip hybridization of the present invention;
Fig. 3 shows the agarose gel electrophoresis result of various dose Rhizomucor pusillus PCR product.
Embodiment:
The invention discloses a kind of dna probe, gene chip and application thereof that detects Rhizomucor pusillus, those skilled in the art can use for reference this paper content, suitably improve processing parameter and realize.Special needs to be pointed out is that all similarly replace and change apparent to those skilled in the art, they all are deemed to be included in the present invention.Product of the present invention and application are described by preferred embodiment, the related personnel obviously can change or suitably change and combination methods and applications as herein described within not breaking away from content of the present invention, spirit and scope, realizes and use the technology of the present invention.
The extraction of embodiment 1:DNA
Can extract according to this area ordinary method, sample can be flesh tissue, blood, urine, cerebrospinal fluid, secretory product (sputum, hydrothorax, ascites, bronchoalveolar lavage fluid etc.) etc.Use in the present embodiment the Qiagen minim DNA to extract test kit, take flesh tissue as example:
(1) flesh tissue is put into the mortar of high-temperature sterilization, ground after pouring liquid nitrogen into, the tissue that grinds is moved in the EP pipe of 1.5ml;
(2) add the 1ml normal saline flushing, centrifugal 3000rpm 5min abandons supernatant;
(3) add 200 μ l NaOH (50mM), hatch 10min for 95 ℃, centrifugal 5000rpm10min abandons supernatant;
(4) add 500 μ l Lyticase lywallzyme solution, hatch 30min for 37 ℃;
(5) centrifugal 14000rpm 10min abandons supernatant;
(6) add rapidly 180 μ l ATL and 55 ℃ of 15min of 20 μ l Proteinase Ks;
(7) add 100 μ l AL, the abundant mixing of concussion 15s..70 ℃ of 10min (adding 1 μ g vector rna in the 100 μ l AL damping fluids);
(8) 50 μ l ethanol (96-100%) concussion 15s. room temperature 5min (15-25 ℃);
(9) of short duration centrifugal;
(10) carefully all lysates are moved to post, avoid bedewing tube wall, pipettor does not touch film, and centrifugal 6000g (8000rpm) 1min. puts pillar into the 2ml collection tube, abandons collection tube.If lysate fully by film, does not use higher rotating speed centrifugal until pillar empty again;
(11) in pillar, add 500 μ l AW1 (shaking up), avoid bedewing tube wall.Centrifugal 6000g (8000rpm) 1min. puts pillar into the 2ml collection tube, abandons collection tube;
(12) in pillar, add 500 μ l AW2, avoid bedewing tube wall.Centrifugal 6000g (8000rpm) 1min. puts pillar into the 2ml collection tube, abandons collection tube;
(13) 14,000rpm, 3min make the film complete drying, and guaranteeing does not have ethanol;
(14) pillar is placed the 1.5ml centrifuge tube, abandon collection tube.Add 20-100 μ l AE distilled water (PH is greater than 7) in film central authorities.
(15) room temperature (15-25 ℃) 1min.
(16) pillar is placed on the collection tube, and 20,000g (14,000rpm) centrifugal 1min, fluid preservation in the collection tube is for subsequent use.
Embodiment 2: the pcr amplification of sample
PCR reaction system: PCR reaction cumulative volume is 25 μ l, contains template DNA (Rhizomucor pusillus, flavus, Aspergillus fumigatus, aspergillus niger, terreus, Aspergillus nidulans, Candida kefyr, candida rugosa, Trichosporon asahii, rhizopus arrhizus, Rhizopus microsporus, absidia corymbifera, Candida albicans, candida krusei, Candida glabrata, Oidium tropicale, Candida parapsilosis, monilia guilliermondii, Dublin candidiasis or cryptococcus neoformans DNA) 5 μ l, 10 * Buffer2.5 μ l, 2.5mM dNTP 1.25 μ l, 25mM MgCl
23 μ l, each 0.25 μ l of primer I TS1, ITS4 50pmol, Hotstart Taq enzyme (5U/ μ l) 0.1 μ l mends ultrapure water to 25 μ l.
The PCR reaction conditions: 95 ℃ 15 minutes, the condition of amplification sex change, annealing, extension is respectively 94 ℃ of sex change 30 seconds, 60 ℃ of annealing 30 seconds, 72 ℃ were extended 1 minute, 35 circulations, last 72 ℃ were extended 10 minutes.Get 8 μ l PCR products electrophoresis 30 minutes in 1.5% agarose, electrophoresis poststaining 30 minutes is taken a picture in gel imaging system.Remaining PCR product is used for hybridization (5 μ l PCR products get final product).
Embodiment 3: the mark of probe and the preparation of gene chip
(1) uses 0.5M NaHCO
3(PH 8.4) dilution probe of the present invention is got the 4ul probe solution and is entered in the microwell plate to suitable concentration, and every hole adds the 4ul sampling liquid, abundant mixing, and blank is the above-mentioned NaHCO of equivalent
3Mixed solution with sampling liquid.
(2) the BiodyneC nylon membrane is cut to suitable size.
(3) under the room temperature, 16%w/v EDAC was hatched Biodyne C nylon membrane 10 minutes.
(4) rinsed with deionized water Biodyne C nylon membrane is 1 minute.
(5) blot nylon membrane with filter paper.
(6) get probe solution and blank solution point sample on the nylon membrane for preparing with point sample instrument.
(7) hatch 5 minutes under the room temperature.
(8) absorb the nylon membrane surface liquid, nylon membrane is put into 0.1M NaOH liquid deactivation 9 minutes.
(9) in shaking table, use 100ml 2 * SSPE rinsing Biodyne C nylon membrane 5 minutes.
(10) in shaking table with 60 ℃ of 2 * SSPE/0.5%SDS liquid rinsing Biodyne C nylon membranes 5 minutes.
(11) in shaking table, cleaning Biodyne C nylon membrane 20 minutes with 200ml 20mM EDTA liquid under the room temperature.
(12) Biodyne C nylon membrane is put into clean plastics bag, and added the EDTA of 10ml, sealing is stored in 4 ℃ of refrigerators, and is for subsequent use.
Embodiment 4: utilize probe of the present invention to carry out the detection of sample
Use streptavidin-peroxidase (Roche Diagnostics Co.) with 2 * SSPE (SSPE is 0.18M NaCl, 10mM NaH
2PO
4, and 1mM EDTA[pH 7.7]-0.5%SDS) 1: 4,000 dilution; Hybridization temperature is 60 ℃; Film (Hyperfilm; Amersham) time shutter is 2 minutes.Concrete steps are as follows:
(1) get 5 μ l pcr amplification products, add simultaneously 150 μ l, 2 * SSPE/0.1%SDS mixing with 1 μ l positive control fragment (sequence is shown in SEQ IDNo.6) sample, add in the EP pipe.
(2) 99 ℃ were heated 10 minutes, then the EP pipe were positioned over rapidly at least 5 minutes on ice.
(3) in the hybridization case with 2 * SSPE/0.1%SDS liquid of 60 ℃ of 250ml shake rinsing mark the Biodyne C nylon membrane 5 minutes of probe.
(4) absorb the nylon membrane surface liquid.
(5) use from the pcr amplification product 150 μ ls of molding jig adding behind 2 * SSPE/0.1%SDS solution dilution in correspondence position.
(6) in the hybridization case, hybridized 60 minutes for 60 ℃.
(7) in shaking table, wash Biodyne C nylon membrane twice with 60 ℃ of 2 * SSPE/0.5%SDS solution of 250ml, each 10 minutes.
(8) add 15ml 2 * SSPE/0.5%SDS and 1/5000 streptavidin-superoxide enzyme conjugates mixed solution, hatched 45 minutes for 42 ℃.
(9) in shaking table, wash Biodyne C nylon membrane twice with 42 ℃ of 2 * SSPE/0.5%SDS liquid of 250ml, each 10 minutes.
(10) in shaking table, wash Biodyne C nylon membrane twice with 250ml 2 * SSPE liquid under the room temperature, each 5 minutes.
(11) add 20ml ECL photographic developer on Biodyne C nylon membrane, incubated at room 1 minute.
(12) Biodyne C nylon membrane is put into magazine.
(13) put into X-ray in magazine, observations is developed a film in exposure.
(14) Biodyne C nylon membrane is shaken rinsing twice with 80 ℃ of 250ml 1%SDS liquid, each 30 minutes.
(15) in shaking table, cleaned Biodyne C nylon membrane 15 minutes with 200ml 20mM EDTA liquid again.
(16) Biodyne C nylon membrane is put into clean plastics bag, and added the EDTA of 10ml, seal 4 ℃ and save backup.
Embodiment 5: result's judgement
On egative film position corresponding to probe, stain appears, and the result is positive in expression.Without stain, the result is negative in expression.
Fig. 1 has shown according to the mark of embodiment 1-4 preparation the gene chip hybridization result of probe of the present invention.The order of the probe of mark is on the gene chip: 1 positive control (shown in SEQ ID No.4), 2 negative controls (shown in SEQ ID No.5), 3 blanks, 4-23 probe of the present invention;
Bacterial strain PCR product hybridization sequence: 1-4 Rhizomucor pusillus, 5 Aspergillus fumigatus, 6 flavus, 7 aspergillus nigers, 8 terreus, 9 Aspergillus nidulanses, 10 Candida kefyrs, 11 candida rugosas, 12 Trichosporon asahiis, 13 rhizopus arrhizus, 14 Rhizopus microsporus, 15 absidia corymbiferas, 16 Candida albicanss, 17 candida kruseis, 18 Candida glabratas, 19 Oidium tropicalees, 20 Candida parapsilosises, 21 monilia guilliermondiis, 22 Dublin candidiasis, 23 cryptococcus neoformans.
As seen from Figure 1, specific probe of the present invention only with the hybridization of the PCR product of Rhizomucor pusillus, not with other fungi PCR product generation non-specific hybridization, confirm that itself and Rhizomucor pusillus binding specificity are strong.
Embodiment 6: sensitivity and repeatability detect
Behind the different quantities Rhizomucor pusillus extraction of spores DNA, through genechip detection of the present invention, the result as shown in Figure 2, (1-2.5ng being equivalent to 100CFU), (2-1.25ng being equivalent to 50CFU), (3-0.63ng being equivalent to 25CFU), (4-0.31ng being equivalent to 12.5CFU), (5-0.15ng being equivalent to 6CFU), (6-0.08ng being equivalent to 3CFU), (7-0.04ng being equivalent to 1.5CFU), 8-0.02ng (being equivalent to 0.75CFU), 9-0.01ng (being equivalent to 0.4CFU).
Get various dose Rhizomucor pusillus PCR product row agarose gel electrophoresis, the result as shown in Figure 3.Marker is 750bp, 500bp, the negative contrast of C, 1-1 * 10
3Ng, 2-500ng, 3-250ng, 4-50ng, 5-5ng, 6-0.5ng, 7-0.05ng, 8-5 * 10-
3Ng.
Above presentation of results, containing the above Rhizomucor pusillus of 2-3CFU in the sample is visible positive signal after testing; Fungi PCR product reaches more than the 0.04ng and can be detected.And when utilizing agarose gel electrophoresis to detect the PCR product, want at least the above PCR product of 5ng to carry out electrophoresis and can see the finding of naked eye band.The susceptibility that detection scheme of the present invention is described detects apparently higher than simple PCR.
Repeatability detects: the present invention has good repeatability, gets 10 parts of positive Rhizomucor pusillus and carries out duplicate detection, and positive rate reaches 100%.
Embodiment 8: clinical isolates strain detected result
The bacterial strain of detection separation and Culture in clinical samples utilizes result that genechip detection of the present invention goes out through separation and Culture or order-checking comparison, and its detected result concordance rate reaches 100%, the results are shown in Table 2.
Table 2. the method for the invention is carried out clinical case and is detected
The above only is preferred implementation of the present invention; should be pointed out that for those skilled in the art, under the prerequisite that does not break away from the principle of the invention; can also make some improvements and modifications, these improvements and modifications also should be considered as protection scope of the present invention.
Claims (10)
1. dna probe for detection of Rhizomucor pusillus, its sequence is shown in SEQ ID No.1.
2. one kind is used for the gene chip that Rhizomucor pusillus detects, and forms by oligonucleotide probe being fixed on surface of solid phase carriers, it is characterized in that described oligonucleotide probe comprises the described dna probe of claim 1.
3. gene chip according to claim 2 is characterized in that, described solid phase carrier is selected from nitrocellulose filter, nylon membrane, polystyrene, sheet glass, silicon chip, particulate and plastic sheet.
4. according to claim 2 or 3 described gene chips, it is characterized in that described oligonucleotide probe also comprises positive control probe, negative control probe.
5. gene chip according to claim 4 is characterized in that, described positive control probe is shown in SEQ ID No.4.
6. gene chip according to claim 4 is characterized in that, described negative control probe is shown in SEQ ID No.5.
7. the test kit for detection of Rhizomucor pusillus comprises the described dna probe of claim 1.
8. the method that detects of the sample to containing Rhizomucor pusillus of a non-medical diagnosis on disease or therapeutic purpose comprises following steps:
Step 1: extract and the nucleotide sequence of amplification sample, described amplification with the beacon molecule marker to amplified production;
Step 2: get step 1 gained amplified production and hybridize with the probe that is fixed on each described gene chip of claim 2-6;
Step 3: adopt corresponding method of detection that gene chip is carried out scanning analysis according to described beacon molecule.
9. method according to claim 8 is characterized in that, the sequence of the primer of the described amplification step of step 1 is shown in sequence table SEQ ID No.2 and SEQ ID No.3.
10. method according to claim 8 is characterized in that, described beacon molecule is fluorescein or isotropic substance, and described fluorescein comprises Cy3, Cy5, fluorescein isothiocyanate, texas Red; Described isotropic substance comprises 32P.
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| JPH099971A (en) * | 1995-06-30 | 1997-01-14 | Meito Sangyo Kk | New selected marker gene and transformation system of rhizomucor pusillus |
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| Title |
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| JP特开平9-9971A 1997.01.14 |
| 汪昭贤等.畜禽毛霉病主要病原真菌形态及生化特性.《中国兽医学报》.2000,第20卷(第4期),375-378. * |
| 韩黎等.微小根毛霉致病株的原生质体形成条件.《微生物学通报》.1998,第25卷(第1期),27-29. * |
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