CN102031293A - DNA probe and gene chip for detecting Candida rugosa and application thereof - Google Patents
DNA probe and gene chip for detecting Candida rugosa and application thereof Download PDFInfo
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- CN102031293A CN102031293A CN 201010258256 CN201010258256A CN102031293A CN 102031293 A CN102031293 A CN 102031293A CN 201010258256 CN201010258256 CN 201010258256 CN 201010258256 A CN201010258256 A CN 201010258256A CN 102031293 A CN102031293 A CN 102031293A
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Abstract
The invention relates to the technical field of biology, and discloses a DNA probe for detecting Candida rugosa. The DNA probe for detecting Candida rugosa has a nucleotide sequence shown by a sequence table SEQ ID No.1, and the DNA probe has strong specificity for detecting the Candida rugosa, with sensibility of 0.04ng. The invention also provides a gene chip containing the DNA probe. By using the gene chip, high throughtput, automatic and rapid detection for samples which may contain the Candida rugosa can be realized, the specificity of the gene chip is strong, the accuracy of detection results can be ensured, and the gene chip has wide application prospects.
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 rugosa.
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 rugosa, have sequence shown in the SEQID No.1, it combines with candida rugosa DNA specificity, and it is highly sensitive to detect candida rugosa.
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 rugosa 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 (NH2), aldehyde radical (CHO), carboxyl (COOH) and sulfydryl (SH).
Gene chip of the present invention connects the specific dna probe at candida rugosa on described solid phase carrier, described probe can be strand or double chain DNA molecule, and it can be hybridized with the candida rugosa 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 rugosa 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 rugosa, comprises: with candida rugosa 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 rugosa 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 rugosa in the described sample according to the positive signal addressing of being caught according to selected beacon molecule.
The present invention adopts the candida rugosa 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 rugosa.
Dna probe high specificity of the present invention, highly sensitive, containing the above candida rugosa of 2-3CFU in the sample is visible positive signal after testing, candida rugosa 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 rugosa strain hybrid;
Fig. 2 shows the DNA of different quantities candida rugosa 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 rugosa PCR product.
Embodiment:
The invention discloses a kind of dna probe, gene chip and application thereof that detects candida rugosa, 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 rugosa, Aspergillus fumigatus, flavus, aspergillus niger, terreus, Aspergillus nidulans, Candida kefyr, 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, each 0.25 μ l of primer I TS1, ITS450pmol, 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 rugosa, 5 Aspergillus fumigatus, 6 flavus, 7 aspergillus nigers, 8 terreus, 9 Aspergillus nidulanses, 10 Candida kefyrs, 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.
From Fig. 1 as seen, specific probe of the present invention only with the hybridization of the PCR product of candida rugosa, not with other fungi PCR product generation non-specific hybridization, confirm that itself and candida rugosa binding specificity are strong.
Embodiment 6: sensitivity and repeatability detect
After different quantities candida rugosa 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 rugosa 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 rugosa 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 rugosas 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 rugosa has sequence shown in the SEQ ID No.1.
2. one kind is used for the gene chip that candida rugosa 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 rugosa comprises the described dna probe of claim 1.
8. method that the sample that may contain candida rugosa 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.
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Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| US6242178B1 (en) * | 1997-07-30 | 2001-06-05 | Centers For Disease Control And Prevention | Nucleic acid probes for detecting Candida species |
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| US6242178B1 (en) * | 1997-07-30 | 2001-06-05 | Centers For Disease Control And Prevention | Nucleic acid probes for detecting Candida species |
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