CN102041307B - DNA probe and gene chip for detecting aspergillus flavus, and application thereof - Google Patents
DNA probe and gene chip for detecting aspergillus flavus, and application thereof Download PDFInfo
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Abstract
The invention relates to the field of biotechnology and discloses a DNA probe for detecting aspergillus flavus. The DNA probe for detecting the aspergillus flavus has a nucleotide sequence shown as SEQ ID No.1. The DNA probe has high specificity for detecting the aspergillus flavus and high sensitivity of 0.04 ng. The invention also provides a gene chip comprising the DNA probe. The gene chip can realize high throughput, automatic and quick detection of a sample which may contain aspergillus flavus, has high specificity, ensures accuracy of a 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 flavus.
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 of infection is different.Invasive infections with fungi does not have specific findings in early days, so be easy to covered, case fatality rate is very high.
The technology of at present clinical detection fungi commonly used mainly contains:
(1) microscopy and cultivation, evaluation: like 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 the microscopy poor specificity, susceptibility is not enough, 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 used in antigen, antibody, meta-bolites and the cellular constituent that detects fungi.Can be according to the difference of fungal antigen property with classification of fungi.But the specificity of antibody has limited the development of this technology.
Immunohistochemical methods, EUSA, 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 be not sure of 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 VISOSE;
(2) venoclysis Tegeline, BSA, thrombin or blood products;
(3) strepticemia;
Exist when (4) operator handles sample and pollute.
In addition, the candidiasis that the mucosa injury that uses polyose cancer therapy drug, chemicotherapy to cause causes VISOSE or field planting in the food gets into blood etc. through gi tract also possibly 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 use a spot of sample to detect the multiple fungi that possibly exist fast simultaneously, 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 flavus, have sequence shown in the SEQ IDNo.1, it combines with the flavus dna specificity, and it is highly sensitive to detect flavus.
Dna probe according to the invention can be widely used in biology, medical field, as combining with the nucleic acid hybridization technique that with the round pcr is the basis, carries out biological analysis or clinical diagnosis.
The present invention combines biochip technology, can realize small sample, high-throughput, special, change the purpose of detection flavus fast and automatically.A kind of gene chip that fungi detects that is used for provided by the invention forms through specific oligonucleotide probe being fixed on surface of solid phase carriers, and said 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 according to the invention and probe
As preferably, said solid phase carrier is selected from nitrocellulose filter, nylon membrane, PS, 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, gathers 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 reactive group, these groups include but not limited to amino (NH
2), aldehyde radical (CHO), carboxyl (COOH) and sulfydryl (SH).
Gene chip according to the invention connects the specific dna probe to flavus on said solid phase carrier, said probe can be strand or double chain DNA molecule, and it can be hybridized with the flavus gene specific under suitable condition.Said probe can be through DNA automatically synthetic or pcr amplification obtain.Said 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 according to the invention; Be that point sample is to carrier in an orderly manner with said nucleic probe, reactive group and the carrier surface corresponding reactive group end modified through nucleic probe react, and form covalently bound; With probe stationary on carrier, thereby process gene chip.Simultaneously, on gene chip according to the invention, according to the order of sequence with the hybridization of different samples after, through whether the existence of flavus is arranged in can be clear and definite to the addressing of the catching positive signal corresponding sample.
As preferably, said surface of solid phase carriers also is fixed with positive control probe, negative control probe.
More preferably; Said 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, said 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 flavus, comprises: with flavus specificity bonded probe, said probe has in the nucleotide sequence shown in the sequence table SEQ ID No.1.Test kit according to the 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 possibly contain flavus is detected, and comprises following steps:
Step 1: extract and the nucleotide sequence of amplification sample, said 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 said gene chip;
Step 3: adopt corresponding detecting method that gene chip is carried out scanning analysis according to said 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 the goal gene marker beacon molecule in the sample; Said method includes but not limited to, methods such as PCR, RT-PCR, in-vitro transcription, random primer labelling, nick translation and terminal metastatic marker.Said beacon molecule can be resorcinolphthalein or isotropic substance; Said resorcinolphthalein includes but not limited to, Cy3, Cy5, fluorescein isothiocyanate, texas Red; Said isotropic substance includes but not limited to 32P.
Under suitable temperature and ionic strength conditions, said marked product and gene chip according to the 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 flavus in the said sample according to the positive signal addressing of being caught according to the beacon molecule of being selected for use.
The present invention adopts the flavus 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 according to the invention connects is listed in the table 1 with the used fungi universal primer of amplification.Need to prove; Oligonucleotide probe that the present invention adopted 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 said method, therefore said 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 it is the evaluation and the categorizing system on basis with the genetic analysis that medical mycology research has formed gradually.We utilize the high-throughput platform of chip based on Protocols in Molecular Biology, design perfect based on the technology of gene chip specific diagnosis infection by Aspergillus flavus.
Dna probe high specificity according to the invention, highly sensitive, contain the above flavus of 2-3CFU in the sample through detecting promptly visible positive signal, flavus PCR product reaches more than the 0.04ng and can be detected.
Gene chip according to the invention and to utilize said gene chip to carry out the required sample size of method of sample detection few, reaction volume is little, and reagent consumption is few; Can realize high-throughput, robotization and the rapid detection of sample; Analytic process is objective, high specificity, and the concordance rate through the detected result of present method and separation and Culture or order-checking comparison result reaches 100%; Guaranteed the particularity of detected result, be suitable for being applied to clinical.
Description of drawings:
Fig. 1 shows the detected result of gene chip according to the invention and aspergillus flavus strain hybridization;
Fig. 2 shows the DNA of different quantities aspergillus spore extraction and the detected result of gene chip hybridization according to the invention;
Fig. 3 shows the agarose gel electrophoresis result of various dose flavus PCR product.
Embodiment:
The invention discloses a kind of dna probe, gene chip and application thereof that detects flavus, 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 and are included in the present invention.Product of the present invention and application are described through 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 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 not fully through film, uses higher rotating speed centrifugal empty until pillar 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 zero(ppm) 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 subsequent use with fluid preservation in the collection tube for 000g.
Embodiment 2: the pcr amplification of sample
PCR reaction system: PCR reaction TV is 25 μ l, contains template DNA (flavus, Aspergillus fumigatus, black mold, terreus; Aspergillus nidulans, Candida kefyr, candida rugosa, trichosporon asahii bacterium, rhizopus arrhizus; Rhizopus microsporus, Rhizomucor pusillus, 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, primer I TS1, each 0.25 μ l of 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 according to the 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 with 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 add the EDTA of 10ml, sealing is stored in 4 ℃ of refrigerators, and is subsequent use.
Embodiment 4: utilize probe according to the invention to carry out the detection of sample
Use streptavidin-px (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 following:
(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) in magazine, put into the X-ray sheet, 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 add the EDTA of 10ml, it is subsequent use to seal 4 ℃ of preservations.
Embodiment 5: result's judgement
On the corresponding egative film position of probe, 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 gene chip marked is: 1 positive control (shown in SEQ ID No.4), 2 negative controls (shown in SEQ ID No.5), 3 blanks, 4-23 probe according to the invention;
Bacterial strain PCR product hybridization sequence: 1-4 flavus, 5 Aspergillus fumigatus, 6 black molds, 7 terreus, 8 Aspergillus nidulanses; 9 Candida kefyrs, 10 candida rugosas, 11 trichosporon asahii bacterium, 12 rhizopus arrhizus, 13 Rhizopus microsporus; 14 Rhizomucor pusillus, 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.
Visible from Fig. 1, specific probe according to the invention only with the PCR product hybridization of flavus, not with other fungi PCR product generation non-specific hybridization, confirm that itself and flavus binding specificity are strong.
Embodiment 6: sensitivity and repeatability detect
After the different quantities aspergillus spore extracts DNA, detect through gene chip according to the invention, the result is 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 flavus PCR product row agarose gel electrophoresis, the result is 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 contains the above flavus of 2-3CFU through detecting promptly visible positive signal in the sample; 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 according to the invention is described detects apparently higher than simple PCR.
Repeatability detects: the present invention has good repeatability, gets 10 parts of positive flavus 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 according to the invention through separation and Culture or order-checking comparison checking, and its detected result concordance rate reaches 100%, and the result sees table 2.
Table 2. the method for the invention is carried out clinical case and is detected
| Types of organization | Disease name | Fungus culture | Smear | The order-checking comparison | The inventive method |
| Phlegm | Bone marrow transplantation | - | - | Flavus | Flavus |
| Cornea secretory product | Keratitis | Flavus | It is thus clear that fungi composition | Flavus | Flavus |
| Sinus tissue | Sinusitis paranasal sinusitis | - | It is thus clear that fungi composition | Flavus | Flavus |
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 improvement and retouching, these improvement and retouching also should be regarded as protection scope of the present invention.
Claims (10)
1. one kind is used to detect the flavus dna probe of (the Latin name is called Aspergillus flavus), has sequence shown in the SEQ ID No.1.
2. one kind is used for the gene chip that flavus (the Latin name is called Aspergillus flavus) detects, and forms through oligonucleotide probe being fixed on surface of solid phase carriers, it is characterized in that said oligonucleotide probe comprises the said dna probe of claim 1.
3. gene chip according to claim 2 is characterized in that said solid phase carrier is selected from nitrocellulose filter, nylon membrane, PS, sheet glass, silicon chip, particulate and plastic sheet.
4. according to claim 2 or 3 described gene chips, it is characterized in that said oligonucleotide probe also comprises positive control probe, negative control probe.
5. gene chip according to claim 4 is characterized in that, said positive control probe is shown in SEQ ID No.4.
6. gene chip according to claim 4 is characterized in that, said negative control probe is shown in SEQ ID No.5.
7. one kind is used to detect the flavus test kit of (the Latin name is called Aspergillus flavus), comprises the said dna probe of claim 1.
8. method that the sample that possibly contain flavus (the Latin name is called Aspergillus flavus) is detected comprises following steps:
Step 1: extract and the nucleotide sequence of amplification sample, said 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 said gene chip of claim 2-6;
Step 3: adopt corresponding detecting method that gene chip is carried out scanning analysis according to said beacon molecule.
9. method according to claim 8 is characterized in that, the primer of the said 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, said beacon molecule can be resorcinolphthalein or isotropic substance, and said resorcinolphthalein comprises Cy3, Cy5, fluorescein isothiocyanate, texas Red; Said isotropic substance comprises 32P.
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| CN1286989C (en) * | 2005-04-22 | 2006-11-29 | 中国人民解放军军事医学科学院卫生学环境医学研究所 | Biochip for detecting pathogenesis fungus |
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| CN1286989C (en) * | 2005-04-22 | 2006-11-29 | 中国人民解放军军事医学科学院卫生学环境医学研究所 | Biochip for detecting pathogenesis fungus |
Non-Patent Citations (1)
| Title |
|---|
| 金敏 等.常见致病真菌通用引物PCR检测技术研究.《环境与健康杂志》.2009,第26卷(第11期),969－971. * |
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Granted publication date: 20120808 Termination date: 20160818 |