CN105349664A - Gene chip and kit for detecting pathogenic bacteria in cerebrospinal fluid of central nervous system bacterial infester - Google Patents
Gene chip and kit for detecting pathogenic bacteria in cerebrospinal fluid of central nervous system bacterial infester Download PDFInfo
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Abstract
The invention discloses a gene chip and kit for detecting pathogenic bacteria in cerebrospinal fluid of a central nervous system bacterial infester. The gene chip comprises a solid-phase carrier and oligonucleotide probes fixed on the solid-phase carrier, the oligonucleotide probes comprise the arranged bacterium 16SrRNA universal probe, the gram-positive bacterium universal probe, the gram-negative bacterium universal probe and the probe for identifying the variety and genus specificity of bacteria. PCR amplification is performed on a genomic DNA of a sample to be detected through a designed primer, hybridization is performed with the gene chip, and then a hybridization result is analyzed and identified through interpretation software Baio Array Doctor V2.0. The gene chip and kit have the advantages that one or more pathogenic bacteria in the cerebrospinal fluid of the central nervous system bacterial infester can be quickly, accurately and reliably detected.
Description
Technical field
The present invention relates to biomedicine technical field, specifically multidigit point gene detection chip, especially the quick diagnosis gene chip integrated testing method of bacillary central nervous system infection pathogenic agent is applicable to, chip arranges the probe of various bacteria, infect some encountered pathogenic bacterias in cerebrospinal fluid comprising bacillary CNS, utilize pcr amplification product to carry out cross experiment detection, characterization test sample.
Background technology
Central nervous system (centralnervoussystem, CNS) infecting is one of neural system principal disease spectrum, showing as meninx (brain) inflammation shape clinical more, as heating, headache, vomiting, the disturbance of consciousness and meningeal irritation sign etc., case fatality rate and disability rate high, etiological diagnosis clear and definite is in early days the key of clinical correct anti-infective therapy.The pathogenic micro-organism kind causing CNS to infect is a lot, and bacterial pathogen is one of more common.For a long time, Clinical microorganism use for laboratory mainly comprises CSF smear microscopy and culture identification isophenous method, cerebrospinal fluid immunological detection method and biochemical markers detects and polymerase chain reaction (PCR) technology etc. in the method for diagnosing bacterial CNS pathogen infection, also constantly has both at home and abroad and utilizes bacteria 16 S rRNA genes constructional feature to build gene chip to identify the report of pathogenic agent.
CSF smear microscopy and cultivation isophenous method are still the most frequently used detection means of each clinical labororatory diagnosis central nervous system infection at present, therefrom find that pathogenic bacterium judge to infect " gold standard ", but these ordinary methods exist following not enough: (1) due to test in laboratory methodology reason cause pathogen infection to detect or sense cycle long, lag behind clinical demand make to make a definite diagnosis difficulty and miss treatment opportunity that the is early stage or critical period: 1. CSF smear microscopy, recall rate is low, and susceptibility is poor; 2. cerebrospinal fluid microbial culture required time long (2-5 days), cultivation positive rate low (about about 10-15%); 3. by antibiotic usage and the restriction of pathogenic bacteria self-condition, the examination that cannot realize broad spectrum of pathogens and monitoring.(2) due to pathogenic agent cannot or isolation identification fast and accurately, cause the blindness for the treatment of, resistant organism is increased and making patients's burden: the automatic bacterial assessing instrument 1. used at present may go wrong when identifying some bacterium, qualification accuracy is worth discussion; 2. the easy mistaken diagnosis of polyinfection or fail to pinpoint a disease in diagnosis; 3. the qualification of Mass Spectrometric Identification instrument still needs first to obtain positive bacterium colony at present, and direct-detection qualification pathogenic agent still needs great amount of samples to verify; 4. commercialization phenotypic evaluation system database is limited, is difficult to distinguish the close pathogenic bacteria of phenotype.
Cerebrospinal fluid immunological detection method (as suis latex agglutination detects) can only detect special pathogen, can not to detect in cerebrospinal fluid unknown bacterium and classify to pathogenic bacteria, there is the shortcomings such as false positive rate is high.PCR detection method mainly adopts fluorescent quantitative PCR technique and thymus nucleic acid sequencing technologies, and the former defect is to be limited to fluorescence detection channel number, and can only detect one or a few bacterial nucleic acid at present, flux is low simultaneously; The latter's complicated operation, is not easy to carry out in clinical laboratories routine, and the judgement of sequencing result also exists the risk of certain mistake, needs the two-way order-checking of multiple copied.And current numerous gene chip is prepared mainly with the different carriers such as nitrocellulose membrane and nylon membrane and the method such as digoxin, Radioactive colloidal gold colour developing or close the research of thuja acid chip detection Pathogens In Cerebrospinal Fluid based on the miniature widow of bacterial 16 S rRNA, but there are pathogenic bacterium to cover not comprehensively, lack the deficiencies such as large sample clinical verification data, thus limit it in clinical application.
There is many objective shortcomings in the conventional bacteria culture identification that current Clinical microorganism laboratory adopts, as relied on, phenotypic evaluation, susceptibility are low, sense cycle is long, be subject to the restriction of each side such as microbiotic application, qualification accuracy is also worth discussion, can not meet the requirement of clinical quick diagnosis.The method of carrying out strain typing at present mainly contains the methods such as PCR machine (PCR-RFLP), order-checking, sequence specific primers PCR, quantitative fluorescent PCR, not only complex operation, sense cycle is long, flux is little, and testing process influence factor is many, wayward, somatotype can not be carried out to multiple pathogens simultaneously, be difficult to the requirement meeting Clinical Laboratory, make clinically cannot carry out the wide spectrum of nervus centralis bacterial infection pathogenic agent, integrated detection so far.
Gene chip is one of great Progress & New Products of the most characteristics of the times occurred in high-tech area in recent years, its cardinal principle is fixed on carrier (as slide glass or silicon chip, nylon membrane, nitrocellulose filter, plastic sheet etc.) with a definite sequence and density by the gene probe (oligonucleotide probe, cDNA clone, PCR primer etc.) of lots of genes information in energy reflected sample to form array, carry out hybridization with actual sample (or nucleic acid amplification product), the information of all genes to be checked can be obtained by high-throughput by hybridization signal analysis.This technology with its high-throughput, fast, accuracy advantages of higher diagnoses for pathogenic infection and provide a new solution route, has vital role to the clinical treatment of patient infection's property disease, prognosis evaluation.
Bacterial ribosome RNA (rRNA) is the mark of bacterium life, and its 16SrRNA gene is present in all bacterial chromosomal genes with multiple copied form.Namely utilize the external nineties in 20th century bacteria 16 S rRNA genes constructional feature to build gene chip, and be applied to the report of Bacteria Identification.In recent years, the domestic people of having is devoted to the research for clinical detection cerebrospinal fluid bacterium of the oligonucleotide microarray prepared with the different carriers such as nitrocellulose membrane and nylon membrane and the method such as digoxin, Radioactive colloidal gold colour developing, but there are clinical common cerebrospinal fluid pathogenic bacterium and cover the deficiencies such as incomplete, hybridization time is long, particularly lack large sample clinical verification data and limit its clinical value.In the market topmost gene chip product be with the methods such as point sample prepare for genetic expression detect in, low density gene chip.An important development trend of gene chip is the stdn of chip preparation, sample preparation, hybridization, detection and data analysis, improves accuracy and the reliability of gene chip.Therefore, how namely to ensure to detect high-throughput, ensure that again chip performance superiority is one of its bottleneck problem.And utilize the multidigit point chip detection multiple nervus centralis bacterial infection pathogenic agent based on bacteria 16 S rRNA genes, play the features such as it is simple to operate, flux is large, result is accurate, clinical analysis nervus centralis bacterial infection pathogenic agent and kind are had important practical significance.
Summary of the invention
The object of the invention is to overcome above-mentioned deficiency, a kind of gene chip and the test kit that detect pathogenic bacteria in central nervous system bacterial infection person cerebrospinal fluid are provided, which solve the problem utilized based on the multidigit point chip detection multiple nervus centralis bacterial infection pathogenic agent of bacteria 16 S rRNA genes.
An object of the present invention is to provide a kind of gene chip detecting pathogenic bacteria in central nervous system bacterial infection person cerebrospinal fluid, comprise solid phase carrier and be fixed on the oligonucleotide probe on this solid phase carrier, it is characterized in that: described oligonucleotide probe is included in the bacterial 16 S rRNA general probe that matrix is arranged, gram-positive bacteria general probe, the kind of gram-negative bacteria general probe and qualification bacterium, belong to specific probe, the kind of described qualification bacterium, belong to the DNA fragmentation that specific probe is the base sequence such as shown in SEQIDNO:1-NO:14, the DNA fragmentation that described bacterial 16 S rRNA general probe is the base sequence such as shown in SEQIDNO:15, the DNA fragmentation that described gram-positive bacteria general probe is the base sequence such as shown in SEQIDNO:16, the DNA fragmentation that described gram-negative bacteria general probe is the base sequence such as shown in SEQIDNO:17.
Further, the kind of above-mentioned qualification bacterium, genus specific probe 5 ' are held containing amido modified poly-dT string, and described amido modified poly-dT string is the 16 poly-deoxythymidylic acids gathered.
Further, this gene chip also comprises the DNA sequence dna being marked with biological vegetarian refreshments as shown in SEQIDNO:18.
Further, described solid phase carrier is for modifying slide or silicon chip.
Another object of the present invention is also to provide a kind of test kit for detecting pathogenic bacteria in central nervous system bacterial infection person cerebrospinal fluid, it is characterized in that: comprise gene chip as claimed in claim 1.
Further, described test kit also comprises the primer sequence shown in SEQIDNO:19-NO:20.
Further, 5 ' end band of described primer has labelling groups, and described labelling groups comprises: digoxin molecule, biotin molecule, fluorescein and derivative molecular thereof, Cy3, Cy5, alkaline phosphatase, horseradish peroxidase.
Another object of the present invention is to provide a kind of method detecting pathogenic bacterial infection in central nervous system bacterial infection person cerebrospinal fluid.It is characterized in that: comprise the steps:
(1) template DNA (extracting the DNA sample comprising PI bacterium from Cerebrospinal Fluid in Patients) is prepared by ordinary method;
(2) increased by PCR reaction by the template DNA of preparation in step (1) with primer sequence described in claim 6, obtaining 5 ' end band has labelling groups DNA cloning product;
(3) DNA cloning product in step (2) is mixed with hybridization buffer;
(4) DNA probe of EDS maps on gained mixed solution in step (3) and gene chip according to claim 3, Bio are carried out hybridization;
(5), after completing steps (4) hybridization, detect the hybridization signal of gene chip, treat measurement information according to acquisition of informations such as the position of marking signal on gene chip, intensity.
Further, first gene chip and pre-hybridization buffer are carried out prehybridization described step (4) is front.
Further, the step of PCR reaction in described step (2), also comprises:
1) 94 DEG C of denaturations 5 minutes; 2) 94 DEG C of sex change 45s; 3) 58 DEG C of annealing 45s; 4) 72 DEG C extend 60s, repeat 2)-4) 30 times; 5) reactions steps of last 72 DEG C of extension 7min.
The present invention to be applicable to from the clinical samples such as cerebrospinal fluid extracting directly pathogenic bacteria DNA for pcr amplification, and target gene is used for hybridization check, can be used for the pathogenic agent examination of doubtful bacterial infection.The present invention, is convenient to produce and operation for matrix with solid material carrier; With bacterial universal primers PCR method amplification bacterial target genes, avoid time-consuming cultivation stage, substantially reduce detection time (about 4 hours); The authentication method utilizing the oligonucleotide probe in the target sequence of mark and matrix to carry out hybridizing is more accurate compared with the authentication method based on physiology and chemistry, adds accuracy in detection, and is not subject to the impact of culture condition and Bacterial Physiological state; No matter bacterium " anyway ", genechip detection result all provides important clinical value.This technological method can not only in the shorter time clear and definite bacterium existence whether, also can the clinical Common infectious diseases pathogenetic bacteria of interpretation, for the early diagnosis that there is bacteriological infection and the unconspicuous disease of early clinical manifestation provides foundation, be conducive to antibiotic use, avoid antibiotic abuse.Along with increasing of specific probe quantity, its ability detected the sample of more sample or polyinfection also will be more and more higher, detection time can be shorter and shorter, diagnosis for clinical bacteria is provided a kind of brand-new, quick, sensitive method by this, replaces conventional bacteria Testing and appraisal method to a certain extent.
For above and other objects of the present invention, feature and advantage can be become apparent, below especially exemplified by preferred embodiment, and coordinate Figure of description, be described in detail below.
Accompanying drawing explanation
Accompanying drawing described herein is used to provide a further understanding of the present invention, forms a part of the present invention, and schematic description and description of the present invention, for explaining the present invention, does not form inappropriate limitation of the present invention.In the accompanying drawings:
Fig. 1 is the arrangement schematic diagram of 18 probes of gene chip of the present invention described in the embodiment of the present invention two, wherein
representative is marked with the DNA sequence dna of biological vegetarian refreshments.
Fig. 2 represents the figure of enterococcus faecalis analytical results.
Fig. 3 represents dung field coccus analytical results figure.
Fig. 4 represents the figure of streptococcus aureus analytical results.
Fig. 5 represents Listeria monocytogenes analytical results figure.
Fig. 6 represents streptococcus pneumoniae analytical results figure.
Fig. 7 represents the figure of escherichia coli analytical results.
Fig. 8 represents the figure of Klebsiella Pneumoniae analytical results.
Fig. 9 represents the figure of Pseudomonas aeruginosa analytical results.
Figure 10 represents the figure of Acinetobacter bauamnnii analytical results.
Figure 11 represents the figure of stenotrophomonas maltophilia result.
Figure 12 represents the figure of hemophilus influenzae result.
Figure 13 represents the figure of Enterobacter bacteria result.
Figure 14 represents the figure that there is G-bacterium result.
Figure 15 represents the figure that there is G+ bacterium result.
The principle of work schematic diagram of Figure 16 gene chip.
Embodiment
In order to further illustrate gene chip and the detection method thereof of pathogenic bacteria in detection central nervous system bacterial infection person cerebrospinal fluid of the present invention, be illustrated especially exemplified by following preferred embodiment, these embodiments are to illustrate instead of limiting the present invention in any form.
embodiment onethe design of probe and preparation (for escherichia coli)
1. sequence obtains: log in Genbank to the retrieval of escherichia coli standard samples DNA sequence dna, obtain base sequence, the 16SrRNA gene of selecting bacteria is target sequence;
2. probe design: consider the factor such as probe length, GC% and Tm value, the present invention, by PrimerPriemer5.0 software design probe, designs the DNA fragmentation of the base sequence as shown in SEQIDNO:4,
Further, sterically hindered during in order to reduce hybridization, the present invention holds at above-mentioned base sequence 5 ' increases amido modified poly-dT string;
Concrete, above-mentioned amido modified poly-dT string 16 poly-poly-deoxythymidylic acids;
3. the synthesis of gene probe: the probe of synthetic (entrusting Sangon Biotech (Shanghai) Co., Ltd.) above-mentioned design.
embodiment twothe synthesis of gene chip
1. design bacterium chip spot sample mode: each probe repeats 3 points, and make the micro-battle array of 7*12DNA as shown in Figure 1, in diagram, each probe reference numeral is in table 1.
Table 1 bacterial probe reference numeral
The corresponding bacterial classification of concrete numeral number in table 1 Fig. 1
The point sample of gene chip:
1) on the iArrayer gene chip sample applying instrument of Shanghai Bioon Technology Co., Ltd.'s production, press chip mode figure, point sample program is set;
2) by the 100uM probe solution that diluted and Shanghai Bioon Technology Co., Ltd.
spotting buffer mixes with certain proportion, joins the specified location of 384 orifice plates according to point sample program;
3) aldehyde radical substrate is placed on the sample application platform of point sample instrument successively;
4) point template is placed in point sample instrument, starts point sample program, complete chip point sample;
5) 12 hours are left standstill by under gene chip fixed temperature and humidity condition.
6) quality inspection: gene chip is placed in basis of microscopic observation successively, array should be neat, complete, without leak source.
If oligonucleotide probe is not containing amido modified, then its preparation method also can refer to: " the gene diagnosis technology-on-radiation operational manual " of Wang Shenwu chief editor; Derisi, JL equals " inquiring into metabolism and the Genetic Control of genetic expression in genome " (Dersi delivered in " science " 278 (5338): 680-686 in 1997, JL, IyerVR, BrownPO.Exploringthemetablicandgeneticcontrolofgeneexpre ssiononagenomicscale.Science.1997; 278 (5338): 680-686) and horse found the biochip of the chief editor such as people, Chemical Industry Press.
embodiment threeprimer synthesizes
1. sequence obtains: log in Genbank to Klebsiella Pneumoniae, Acinetobacter bauamnnii, Pseudomonas aeruginosa, escherichia coli, enterobacter, hemophilus influenzae, stenotrophomonas maltophilia, Neisseria meningitidis, enterococcus faecalis, faecium, Listeria monocytogenes, streptococcus aureus, streptococcus pneumoniae, the retrieval of coagulase negative staphylococcus DNA sequence dna, analysis.
2. design of primers: the primer being gone out base sequence as shown in SEQIDNO:19-NO:20 by PrimerPriemer5.0 software design;
Further, 5 ' end of described primer can also be modified with labelling groups,
Concrete, above-mentioned primer is marked with digoxin molecule (DIG), biotin molecule (Bio), fluorescein and derivative molecular (FITC etc.) thereof, other fluorescence molecules (as Cy3, Cy5 etc.), alkaline phosphatase (AP), horseradish peroxidase (HRP);
3. the synthesis of primer sequence: the primer of synthetic (entrusting Sangon Biotech (Shanghai) Co., Ltd.) above-mentioned design.
embodiment fourgene chip principle of work
3 ' of the amplified fragments produced after the primer amplification of design is held with the specific probe be fixed on chip complementary, and 5 ' end band has labelling groups.If probe is hold complementary probe with this amplified production 3 ', the specific probe hybridization on one end of this amplified production and chip, and the other end is with labelling groups, labelling groups is carried out color development treatment, signal can be detected, otherwise can't detect signal.Figure 16 is the principle of work schematic diagram of gene chip.
embodiment fivedetect test kit and the working method of pathogenic bacteria in central nervous system bacterial infection person cerebrospinal fluid
Detect in the method for pathogenic bacteria in central nervous system bacterial infection person cerebrospinal fluid utilizing the test kit containing the above-mentioned gene chip for preparing and primer, the present invention has all done a series of experiment to the factor such as detecting step, testing conditions and has groped, as component proportions each in bacteria PCR reaction mixture, hybridization temperature, hybridization time etc. and main agents formula, as hybridization solution, washing lotion etc. (formula refers to following examples).Each composition wherein in PCR reaction mixture, especially primer concentration, the selection of Buffer, Taq enzyme concentration, the kind of fluorescein and consumption, and template concentrations be repeatedly contrast experiment after the optimum combination that obtains.The annealing temperature that PCR adopts, time and extension time, the excellent condition of number of cycles also for selecting after gradient experiment.Pcr amplification is through 1) 94 DEG C of denaturations 5 minutes; 2) 94 DEG C of sex change 45s; 3) 58 DEG C of annealing 45s; 4) 72 DEG C extend 60s, repeat 2)-4) 30 times; 5) process of last 72 DEG C of extension 7min.Detect the experiment flow of the detection chip of pathogenic bacteria in central nervous system bacterial infection person cerebrospinal fluid also for optimizing rear result.Preferred embodiment below for utilizing mentioned reagent box to detect pathogenic bacteria in central nervous system bacterial infection person cerebrospinal fluid, is described as follows:
1. the preparation of positive controls sample
(1) apply TIANGEN test kit and extract positive control bacterial genomes DNA
1) streptococcus aureus (ATCC25923) and escherichia coli (ATCC25922) the bacterium liquid 5ml of stroke-physiological saline solution preparation is got, bacterium liquid is made 0.5 Maxwell unit, adopt 10 times of serial dilutions, make bacteria suspension concentration be respectively 10-10
8cFU/ml;
2) centrifugal 5 minutes of bacterium liquid 12000rpm/min, abandons supernatant; Add 20mg/ml N,O-Diacetylmuramidase 200 μ l, mixing, 37 DEG C of water-baths 30 minutes;
3) 20 μ l Proteinase K Solution are added, mixing; Add 220 μ l solution B, shake mixing in 15 seconds, 70 DEG C of metal baths 10 minutes;
4) add 220 μ l dehydrated alcohols, shake mixing in 15 seconds, transferred to by gained solution in clean gel column, centrifugal 30 seconds of 12000rpm/min, abandons waste liquid;
5) add 500 μ l solution D, centrifugal 30 seconds of 12000rpm/min, abandons waste liquid;
6) add 700 μ l solution PW, centrifugal 30 seconds of 12000rpm/min, abandons waste liquid; To repeat after above-mentioned steps 1 time 12000rpm/min centrifugal 2 minutes, abandon waste liquid, room temperature is placed and is dried residual liquid;
7) add 50 μ l elutriant TE, room temperature places 2-5 minute, 12000r/min centrifugal 2 minutes, and gained supernatant liquor is bacterial genomes DNA.
(2) .PCR amplification template DNA
The liquid that increases in test kit of the present invention contain upstream and downstream primer (biotin labeling) and PCR reaction needed for all ingredients (as dNTP, H
2o, Buffer etc.), the another pipe of Taq enzyme provides.Target dna fragment is expanded adding the good target DNA of Taq enzyme, extracting in amplification liquid rapidly, for hybridization and color reaction by PCR reaction.25 μ l amplification reaction systems consist of amplification liquid A as shown in table 2 (22 μ l)+2 μ l extract product (DNA)+1 μ lTaq enzyme.
Table 2 bacteria PCR amplification system amplification liquid A formula
| Reagent name | Consumption |
| 10×Buffer | 2.5μl |
| DNTP (each 2.5mM) | 2.5μl |
| Upstream primer (10uM) | 0.3μl |
| Downstream primer (10uM) | 1μl |
| DDW | 15.7μl |
| Total | 22μl |
Above mixed solution is put in PCR instrument, performs following program:
94℃5min
94℃45s
58℃45s
72 DEG C of 60s return second step and carry out 30 circulations
72℃7min
2. the preparation of negative control group sample
With the preparation of above-mentioned positive controls sample, bacterial template DNA is replaced to operate by 5ml stroke-physiological saline solution.
3. the preparation of clinical sample
(1). cerebrospinal fluid cultivates the preparation of extraction with above-mentioned positive controls sample of positive separation of bacterial genomic dna;
(2). the extraction and application QIAampUCPPathogenMiniKit test kit of clinical suspected infection CSF sample DNA.1.5ml cerebrospinal fluid is added in PathogenLysisTube, the centrifugal 5min of 13400r/min, abandon supernatant, add the supporting buffer A TL of test kit (containing reagent D X) suspended particle, turbula shaker vibrates 10 minutes, of short duration centrifugal, draw 400 μ l supernatant liquors in aseptic EP pipe, subsequent experimental operation is undertaken by QIAampUCPPathogenMiniKit specification sheets.By positive controls sample P CR amplification condition, amplification obtains with biotin labeled clinical sample nucleic acid DNA.
4. hybridize
Adopt the hybridization colouring reagents box (BST03021) that Shanghai Bioon Technology Co., Ltd. produces,
e-Hyb Full-automatic hybridization appliance: in BSE03011, carries out by the following method:
(1) hybridization reaction solution preparation: draw 190 μ l hybridization buffers, each 10 μ l of the amplified production prepared in above-mentioned steps 1-3, mixing.
(2) arrange response procedures and each reagent of packing by table 3, working procedure, hybridization color reaction is carried out automatically.
Table 3. hybridizes system and response procedures
(3) take out gene chip, carry out mark
5. the detection of chip hybridization signal:
To hybridize and wash complete gene chip and put into
bE-2.0 biochip identification reading instrument detects, and uses
gene-chip Image analysis software BaioArrayDoctorV2.0 carries out image scanning and data analysis, Output rusults.As shown in Fig. 2-15, for utilizing test kit detected result scintigram provided by the invention.
The present invention to be applicable to from the clinical samples such as cerebrospinal fluid extracting directly pathogenic bacteria DNA for pcr amplification, and target gene is used for hybridization check, can be used for the pathogenic agent examination of doubtful bacterial infection.The present invention, is convenient to produce and operation for matrix with solid material carrier; With bacterial universal primers PCR method amplification bacterial target genes, avoid time-consuming cultivation stage, substantially reduce detection time (about 4 hours); The authentication method utilizing the oligonucleotide probe in the target sequence of mark and matrix to carry out hybridizing is more accurate compared with the authentication method based on physiology and chemistry, adds accuracy in detection, and is not subject to the impact of culture condition and Bacterial Physiological state; No matter bacterium " anyway ", genechip detection result all provides important clinical value.This technological method can not only in the shorter time clear and definite bacterium existence whether, also can the clinical Common infectious diseases pathogenetic bacteria of interpretation, for the early diagnosis that there is bacteriological infection and the unconspicuous disease of early clinical manifestation provides foundation, be conducive to antibiotic use, avoid antibiotic abuse.Along with increasing of specific probe quantity, its ability detected the sample of more sample or polyinfection also will be more and more higher, detection time can be shorter and shorter, diagnosis for clinical bacteria is provided a kind of brand-new, quick, sensitive method by this, replaces conventional bacteria Testing and appraisal method to a certain extent.
Above-mentioned explanation illustrate and describes some preferred embodiments of the application, but as previously mentioned, be to be understood that the application is not limited to the form disclosed by this paper, should not regard the eliminating to other embodiments as, and can be used for other combinations various, amendment and environment, and can in application contemplated scope described herein, changed by the technology of above-mentioned instruction or association area or knowledge.And the change that those skilled in the art carry out and change do not depart from the spirit and scope of the application, then all should in the protection domain of the application's claims.
Claims (10)
1. one kind is detected the gene chip of pathogenic bacteria in central nervous system bacterial infection person cerebrospinal fluid, comprise solid phase carrier and be fixed on the oligonucleotide probe on this solid phase carrier, it is characterized in that: described oligonucleotide probe is included in the bacterial 16 S rRNA general probe that matrix is arranged, gram-positive bacteria general probe, the kind of gram-negative bacteria general probe and qualification bacterium, belong to specific probe, the kind of described qualification bacterium, belong to the DNA fragmentation that specific probe is the base sequence such as shown in SEQIDNO:1-NO:14, the DNA fragmentation that described bacterial 16 S rRNA general probe is the base sequence such as shown in SEQIDNO:15, the DNA fragmentation that described gram-positive bacteria general probe is the base sequence such as shown in SEQIDNO:16, the DNA fragmentation that described gram-negative bacteria general probe is the base sequence such as shown in SEQIDNO:17.
2. the gene chip of pathogenic bacteria in detection central nervous system bacterial infection person cerebrospinal fluid according to claim 1, it is characterized in that: the kind of above-mentioned qualification bacterium, genus specific probe 5 ' are held containing amido modified poly-dT string, described amido modified poly-dT string is the 16 poly-deoxythymidylic acids gathered.
3. the gene chip detecting pathogenic bacteria in central nervous system bacterial infection person cerebrospinal fluid according to claim 1, is characterized in that: this gene chip also comprises the DNA sequence dna being marked with biological vegetarian refreshments as shown in SEQIDNO:18.
4. the gene chip of pathogenic bacteria in detection central nervous system bacterial infection person cerebrospinal fluid according to claim 1, is characterized in that: described solid phase carrier is for modifying slide or silicon chip.
5., for detecting a test kit for pathogenic bacteria in central nervous system bacterial infection person cerebrospinal fluid, it is characterized in that: comprise gene chip as claimed in claim 1.
6. the test kit for detecting pathogenic bacteria in central nervous system bacterial infection person cerebrospinal fluid according to claim 5, is characterized in that: described test kit also comprises the primer sequence shown in SEQIDNO:19-NO:20.
7. the test kit for detecting pathogenic bacteria in central nervous system bacterial infection person cerebrospinal fluid according to claim 6, it is characterized in that: 5 ' end band of described primer has labelling groups, and described labelling groups comprises: digoxin molecule, biotin molecule, fluorescein and derivative molecular thereof, Cy3, Cy5, alkaline phosphatase, horseradish peroxidase.
8. detect a method for pathogenic bacteria in central nervous system bacterial infection person cerebrospinal fluid, be not used in the Diagnosis and Treat of disease, it is characterized in that: comprise the steps:
(1) template DNA (extracting the DNA sample comprising PI bacterium from Cerebrospinal Fluid in Patients) is prepared by ordinary method;
(2) increased by PCR reaction by the template DNA of preparation in step (1) with primer sequence described in claim 6, obtaining 5 ' end band has labelling groups DNA cloning product;
(3) DNA cloning product in step (2) is mixed with hybridization buffer;
(4) DNA probe of EDS maps on gained mixed solution in step (3) and gene chip according to claim 3, Bio are carried out hybridization;
(5), after completing steps (4) hybridization, detect the hybridization signal of gene chip, treat measurement information according to acquisition of informations such as the position of marking signal on gene chip, intensity.
9. the method for pathogenic bacteria in detection central nervous system bacterial infection person cerebrospinal fluid according to claim 8, is characterized in that: first gene chip and pre-hybridization buffer are carried out prehybridization described step (4) is front.
10. the method for pathogenic bacteria in detection central nervous system bacterial infection person cerebrospinal fluid according to claim 8, is characterized in that: the step of PCR reaction in described step (2), also comprises:
1) 94 DEG C of denaturations 5 minutes; 2) 94 DEG C of sex change 45s; 3) 58 DEG C of annealing 45s; 4) 72 DEG C extend 60s, repeat 2)-4) 30 times; 5) reactions steps of last 72 DEG C of extension 7min.
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| CN116144811A (en) * | 2022-12-21 | 2023-05-23 | 迪飞医学科技(南京)有限公司 | Multiplex primer set, method and kit for detecting cerebrospinal fluid pathogen |
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