CN102031292A - DNA probe for detecting candida krusei, gene chip and application thereof - Google Patents
DNA probe for detecting candida krusei, gene chip and application thereof Download PDFInfo
- Publication number
- CN102031292A CN102031292A CN201010258255XA CN201010258255A CN102031292A CN 102031292 A CN102031292 A CN 102031292A CN 201010258255X A CN201010258255X A CN 201010258255XA CN 201010258255 A CN201010258255 A CN 201010258255A CN 102031292 A CN102031292 A CN 102031292A
- Authority
- CN
- China
- Prior art keywords
- probe
- gene chip
- candida krusei
- seq
- sample
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Images
Landscapes
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention discloses a DNA probe for detecting candida krusei, which relates to the technical field of biology. The DNA probe for detecting candida krusei has a nucleotide sequence shown as a sequence table SEQ ID No.1, high candida krusei detectability specificity and sensitivity as high as 0.04ng. The invention also provides a gene chip containing the DNA probe. The gene chip of the invention can realize high-flux, automated and rapid detection on a sample which probably contains the candida krusei, has high specificity and wide application prospect and can ensure the precision of detecting results.
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 candida krusei.
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 condition pathomycetes such as Candida, Aspergillus, genera cryptococcus, also has endemicity pathomycetes such as histoplasma capsulatum, Blastomyces dermatitidis, sporothrix.Under the varying environment, the fungal species difference of infection.Invasive infections with fungi does not have specific findings in early days, so be easy to be covered, case fatality rate is very high.
The technology of at present clinical detection fungi commonly used mainly contains:
(1) microscopy and cultivation, evaluation: as silver hexamine staining, PAS dyeing, silver-colored ammonia G-S staining, 10%KOH method and method and fungi culture is clinical the most frequently used identification of means, but directly microscopy poor specificity, susceptibility deficiency can not identify fungal species.Though fungus culture can be identified fungal species, the culture condition harshness, incubation time is long, the false negative height, the susceptibility deficiency can't be assisted 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 used in antigen, antibody, meta-bolites and the cellular constituent that detects 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 in the clinical G that only has at present 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, can continue discharge this material, make that content increases (mycotic infection of superficial part does not have similar phenomenon) in blood and the body fluid.Be applicable to the early diagnosis of all deep fungal infections except that 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) venoclysis immunoglobulin (Ig), albumin, thrombin or blood products;
(3) strepticemia;
Exist when (4) operator handles sample and pollute.
In addition, the mucosa injury that uses polyose cancer therapy drug, chemicotherapy the to cause candidiasis that causes dextran in the food or field planting enters blood etc. through gi tract and 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 the multiple fungi that may exist fast simultaneously with a spot 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 candida krusei, have sequence shown in the SEQID No.1, it combines with candida krusei DNA specificity, and it is highly sensitive to detect candida krusei.
Dna probe of the present invention can be widely used in biology, medical field, as combining with nucleic acid hybridization technique based on round pcr, carries 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 candida krusei fast and automatically.A kind of gene chip that fungi detects that is used for 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.
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 adopted is selected from any material that can be used for preparing gene chip, includes but not limited to nitrocellulose filter, nylon membrane, poly-third ethene, sheet glass, silicon chip, particulate and plastic sheet.Described solid phase carrier after different chemically modifieds, have on its surface can immobilized nucleic acid molecule active group, these groups include but not limited to amino (NH
2), aldehyde radical (CHO), carboxyl (COOH) and sulfydryl (SH).
Gene chip of the present invention connects the specific dna probe at candida krusei on described solid phase carrier, described probe can be strand or double chain DNA molecule, and it can be hybridized with the candida krusei gene specific under suitable condition.Described probe can be 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, with probe stationary on carrier, thereby make gene chip.Simultaneously, on gene chip of the present invention, according to the order of sequence with the hybridization of different samples after, by whether the existence of candida krusei 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, it combines with the positive control sample specificity, and 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 that is used to detect candida krusei, comprises: with candida krusei specificity bonded probe, 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 the primer that is used for 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 candida krusei 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 detecting 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 a fungi 18SrRNA universal primer, has sequence shown in the sequence table SEQ ID No.2, and 3 ' primer is a 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, 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 detecting method that gene chip is carried out scanning analysis,, judge whether contain candida krusei in the described sample according to the positive signal addressing of being caught according to selected beacon molecule.
The present invention adopts the candida krusei specific oligonucleotide as probe, in the fungi testing process, utilize the fungi universal primer to sample in fungal gene amplification carry out mark.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 amplification PCR products can be used as probe equally 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 evaluation and the categorizing system based on genetic analysis gradually.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 candida krusei.
Dna probe high specificity of the present invention, highly sensitive, containing the above candida krusei of 2-3CFU in the sample is visible positive signal after testing, candida krusei 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 candida krusei strain hybrid;
Fig. 2 shows the DNA of different quantities candida krusei spore extraction and the detected result of gene chip hybridization of the present invention;
Fig. 3 shows the agarose gel electrophoresis result of various dose candida krusei PCR product.
Embodiment:
The invention discloses a kind of dna probe, gene chip and application thereof that detects candida krusei, 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 regarded as being 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 in 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.Using the Qiagen minim DNA to extract test kit in the present embodiment, is example with the flesh tissue:
(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 180 μ l ATL and 55 ℃ of 15min of 20 μ l Proteinase Ks rapidly;
(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 the pillar sky once more;
(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, 20, (14,000rpm) centrifugal 1min is standby with fluid preservation in the collection tube for 000g.
Embodiment 2: the pcr amplification of sample
PCR reaction system: PCR reaction cumulative volume is 25 μ l, contains template DNA (candida krusei, flavus, Aspergillus fumigatus, aspergillus niger, terreus, Aspergillus nidulans, Candida kefyr, candida rugosa, the trichosporon asahii bacterium, rhizopus arrhizus, Rhizopus microsporus, Rhizomucor pusillus, absidia corymbifera, Candida glabrata, Candida albicans, 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 gone 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 under the room temperature with 200ml 20mM EDTA liquid.
(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 standby.
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
4And 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 150 μ l, 2 * SSPE/0.1%SDS mixing simultaneously 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 the X-ray sheet 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, it is standby to seal 4 ℃ of preservations.
Embodiment 5: result's judgement
On the egative film position of probe correspondence, stain appears, and ecbatic is positive.No stain, ecbatic is negative.
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 candida krusei, 5 Aspergillus fumigatus, 6 flavus, 7 aspergillus nigers, 8 terreus, 9 Aspergillus nidulanses, 10 Candida kefyrs, 11 candida rugosas, 12 trichosporon asahii bacterium, 13 rhizopus arrhizus, 14 Rhizopus microsporus, 15 Rhizomucor pusillus, 16 absidia corymbiferas, 17 Candida glabratas, 18 Candida albicanss, 19 Oidium tropicalees, 20 Candida parapsilosises, 21 monilia guilliermondiis, 22 Dublin candidiasis, 23 cryptococcus neoformans.
From Fig. 1 as seen, specific probe of the present invention only with the hybridization of the PCR product of candida krusei, not with other fungi PCR product generation non-specific hybridization, confirm that itself and candida krusei binding specificity are strong.
Embodiment 6: sensitivity and repeatability detect
After different quantities candida krusei spore extracts DNA, detect through gene chip 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 candida krusei 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 candida krusei 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 the above PCR product of 5ng to carry out electrophoresis at least 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 candida kruseis 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 the detected result of gene chip of the present invention through separation and Culture or order-checking comparison checking, 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 a 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. a dna probe that is used to detect candida krusei has sequence shown in the SEQ ID No.1.
2. one kind is used for the gene chip that candida krusei 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. a test kit that is used to detect candida krusei comprises the described dna probe of claim 1.
8. method that the sample that may contain candida krusei is detected 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 detecting method that gene chip is carried out scanning analysis according to described beacon molecule.
9. method according to claim 8 is characterized in that, the primer of the described amplification step of step 1 has the sequence shown in sequence table SEQ ID No.2 or the SEQ ID No.3.
10. method according to claim 8 is characterized in that, described beacon molecule can be fluorescein or isotropic substance, and described fluorescein comprises Cy3, Cy5, fluorescein isothiocyanate, texas Red; Described isotropic substance comprises 32P.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201010258255XA CN102031292A (en) | 2010-08-18 | 2010-08-18 | DNA probe for detecting candida krusei, gene chip and application thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201010258255XA CN102031292A (en) | 2010-08-18 | 2010-08-18 | DNA probe for detecting candida krusei, gene chip and application thereof |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN102031292A true CN102031292A (en) | 2011-04-27 |
Family
ID=43884744
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201010258255XA Pending CN102031292A (en) | 2010-08-18 | 2010-08-18 | DNA probe for detecting candida krusei, gene chip and application thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN102031292A (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105950740A (en) * | 2016-05-30 | 2016-09-21 | 中国人民解放军第二军医大学 | LAMP (loop-mediated isothermal amplification) kit for early diagnosis of candida krusei and special primer |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1356395A (en) * | 2001-11-30 | 2002-07-03 | 中国科学院上海微系统与信息技术研究所 | Gene test chip of candida and its preparing process and application |
| CN1696931A (en) * | 2004-05-11 | 2005-11-16 | 希森美康株式会社 | Simulation system for simulating material concentration in a living body and storage medium |
| CN101348829A (en) * | 2007-12-29 | 2009-01-21 | 兰州百源基因技术有限公司 | Gene chip for detecting Mycotoruloides |
-
2010
- 2010-08-18 CN CN201010258255XA patent/CN102031292A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1356395A (en) * | 2001-11-30 | 2002-07-03 | 中国科学院上海微系统与信息技术研究所 | Gene test chip of candida and its preparing process and application |
| CN1696931A (en) * | 2004-05-11 | 2005-11-16 | 希森美康株式会社 | Simulation system for simulating material concentration in a living body and storage medium |
| CN101348829A (en) * | 2007-12-29 | 2009-01-21 | 兰州百源基因技术有限公司 | Gene chip for detecting Mycotoruloides |
Non-Patent Citations (5)
| Title |
|---|
| 《Communicating Current Research and Educational Topics and Trends in Applied Microbiolgy》 20071231 M.korabecna The Variability in the Fungal Ribosomal DNA(ITS1,ITS2, and 5.8S rRNA Gene): Its Biolgical Meaning and Application in Medical Mycology 784 1-7 第1卷, 第1期 * |
| 《Eur J Clin Microbiol Infect Dis》 20020228 Rodriguez-Villalobos H, etc Cross Reaction Between a Pan-Candida Genus Probe and Fusarium Spp.in a Fatal Case of Fusarium Oxysporum Pneumonia 全文 1-7 第21卷, 第2期 * |
| 《Journal of Medical Microbiolgy》 20090615 Soeta N,etc An Improved Rapid Quantitative Detection and Identification Method for a Wide Range of Fungi 全文 1-7 第58卷, 第8期 * |
| 《Journal of Microbiological Methods》 20070202 Xiang H，etc Rapid Simultaneous Detection and identification of Six Species Candida Using Polymerase Chain Reaction and Reverse Line Hybridization Assay 283 1-7 第69卷, 第2期 * |
| G.H.凯勒: "《DNA探针技术》", 30 September 1992 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105950740A (en) * | 2016-05-30 | 2016-09-21 | 中国人民解放军第二军医大学 | LAMP (loop-mediated isothermal amplification) kit for early diagnosis of candida krusei and special primer |
| CN105950740B (en) * | 2016-05-30 | 2019-12-10 | 中国人民解放军第二军医大学 | LAMP kit for early diagnosis of candida krusei and special primer thereof |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| JP5196863B2 (en) | Probe set, probe carrier and inspection method | |
| JP5196844B2 (en) | Probe set, probe carrier and inspection method | |
| JP2008278868A (en) | Probe, probe set, probe carrier and examination method | |
| JP2008278845A (en) | Probe, probe set, probe carrier and method for testing | |
| JP2008278857A (en) | Probe, probe set, probe carrier and examination method | |
| CN102041306B (en) | DNA probe and gene chip for detecting aspergillus fumigatus, and application thereof | |
| CN103119163A (en) | Method for acquisition of small RNA | |
| CN102041305B (en) | DNA probe and gene chip for detecting cryptococus neoformans and application thereof | |
| CN102031300B (en) | DNA probe and gene chip for detecting aspergillus terreus and application thereof | |
| US20120171661A1 (en) | Detection of Antigens | |
| CN104263833A (en) | Nucleic acid film strip and kit for identification of candida species and detection of gene mutation | |
| CN102031289B (en) | DNA (deoxyribose nucleic acid) probe and gene chip for detecting candida parapsilosis and applications thereof | |
| CN102041304B (en) | DNA probe and gene chip for detecting absidia corymbifera and application thereof | |
| CN102041308B (en) | DNA probe and gene chip for detecting candida glabrata and application thereof | |
| CN102031299B (en) | DNA probe for detecting Aspergillus niger, gene chip and application thereof | |
| CN102031296B (en) | DNA probe for detecting aspergillus nidulans, gene chip and application thereof | |
| CN101638688A (en) | Special probe and gene chip for identifying pathogenic Candida | |
| CN102031292A (en) | DNA probe for detecting candida krusei, gene chip and application thereof | |
| CN102031291B (en) | Deoxyribonucleic acid (DNA) probe for detecting candida dubliniensis, gene chip and application thereof | |
| CN102031293B (en) | DNA probe and gene chip for detecting Candida rugosa and application thereof | |
| CN102031290B (en) | Deoxyribonucleic acid (DNA) probe for detecting candida guilliermondii, gene chip and application thereof | |
| CN102031298B (en) | DNA (deoxyribose nucleic acid) probe and gene chip for detecting rhizomucor pusillus and applications thereof | |
| CN102031295B (en) | DNA probe for detecting rhizopus oligosporus, gene chip and application thereof | |
| CN102031294B (en) | DNA probe and gene chip for detecting trichosporon asahii and application thereof | |
| CN102031297B (en) | DNA probe and gene chip for detecting candida kefyr and application of DNA probe and gene chip |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C12 | Rejection of a patent application after its publication | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20110427 |