CN101899512A - Non-enzymatic amplification detection gene chip based on rRNA genospecies specific sequence, detection system and detection method - Google Patents
Non-enzymatic amplification detection gene chip based on rRNA genospecies specific sequence, detection system and detection method Download PDFInfo
- Publication number
- CN101899512A CN101899512A CN2010102287559A CN201010228755A CN101899512A CN 101899512 A CN101899512 A CN 101899512A CN 2010102287559 A CN2010102287559 A CN 2010102287559A CN 201010228755 A CN201010228755 A CN 201010228755A CN 101899512 A CN101899512 A CN 101899512A
- Authority
- CN
- China
- Prior art keywords
- rrna
- detection
- gene
- gene chip
- probe
- 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
Abstract
The invention belongs to the technical field of gene chip applications, in particular to a non-enzymatic amplification detection gene chip based on the rRNA genospecies specific sequence, a detection system and a detection method. The non-enzymatic amplification detection gene chip based on the rRNA genospecies specific sequence comprises: (1) a matrix; (2) capture probe which is based on the 16 SrRNA specific sequence of prokaryote rRNA genospecies and directly fixed on the surface of the matrix; and (3) report probe which is based on the height conserved sequence of the rRNA genespecies and has the marked tail end. The non-enzymatic amplification detection gene chip can directly detect nucleic acid in rough samples in various forms without needing the links of extracting and amplifying the nucleic acid; and the capture probe is directly connected on the surface of the chip matrix, thereby simplifying the detection steps, reducing the retrieval cost and simultaneously having very high detection sensitivity.
Description
Technical field
The invention belongs to the gene chip applied technical field, particularly, the present invention relates to a kind of non-enzymatic amplification and detect gene chip, detection system and detection method based on the single-minded sequence of rRNA gene kind.
Background technology
Gene chip is to utilize the parallel analysis principle with the probe of a large amount of not homotactic nucleic acid molecule as target gene in the analytic sample, be solidificated in silicon chip in the microarray mode, glass is on the matrix such as nylon membrane, thereby make the gene chip on the ordinary meaning, be also referred to as dna microarray.
The infectious pathogen diagnosing chip is exactly that characterizing gene fragment (target gene) with pathogenic agent to be measured is fixed in and makes chip on the slide, will be from patient blood, extracting goes out the DNA or the RNA of pathogenic agent in tissue or the cell, in preparation target gene synthetic, nucleoside monomers dNTP and a part and the marker of vitamin H one class or the nucleoside monomers dNTP of fluorogene covalent coupling have been mixed, thereby the synthetic cDNA target gene that has various signal marks and be amplified, behind amplification label fluorescence, hybridize with chip, to have marker then hybridizes on the gene chip on the fixed probe fragment from the cDNA target of different cell tissue samples, hybridization signal is by scanner scanning, yin and yang attribute is judged in machine analysis as calculated again.
The preparation of target gene and mark are important steps of biochip technology flow process, present current techique be target gene must separate before with chip probe in conjunction with hybridization, amplification and mark.Marking method is source, chip type and research purpose different and difference to some extent per sample.Normally in pcr amplification, reverse transcription or the in-vitro transcription process of testing sample, realize mark to target gene.
Bacterial ribosome RNA (rRNA) is procaryotic sign, its 16S rRNA gene is to be present in all bacterial chromosome genes with the multiple copied form, this sequence conservative property in prokaryotic organism is extremely strong, some sequence fragment is total for all bacteriums, for close kind or relatively poor with the discriminating resolving power between the different strains in a kind of, but the difference on the sequence is enough to carry out the species evaluation between its kind.And contain the rRNA copy number in each cell and be approximately 10,000, make and walk around the amplification of front, directly carry out low concentration sample and open up the Molecular Detection application becoming possibility target sequence.
In the Molecular Detection field, employed test set is a semiconductor device, and capture probe is preparation, a mark in solution, and capture probe can not be directly connected in matrix like this.For example, CN 200810117952.6 discloses molecule detecting system, it discloses the non-augmentation detection example of microbial pathogen, its capture probe is not to be directly fixed on the matrix, but with the polymer coupling after again indirect securement to chip matrix, though this method realizes non-enzymatic amplification and detects that the polymer coupling step is loaded down with trivial details, increase detects cost.
Summary of the invention
The purpose of this invention is to provide a kind of non-enzymatic amplification and detect gene chip based on the single-minded sequence of rRNA gene kind.
A further object of the present invention provides a kind of non-enzymatic amplification detection system based on the single-minded sequence of rRNA gene kind.
A further object of the present invention provides a kind of non-enzymatic amplification detection method based on the single-minded sequence of rRNA gene kind.
Non-enzymatic amplification based on the single-minded sequence of rRNA gene kind according to the present invention detects gene chip, and it comprises:
1) matrix;
2) capture probe, described capture probe are based on the single-minded sequence of 16SrRNA of prokaryotic organism rRNA gene kind, are directly fixed on stromal surface, constitute gene chip micro lattice;
3) reporter probe, described reporter probe are based on the highly conserved sequence between rRNA gene kind, and its end is labeled.
According to chip of the present invention, described matrix can be for the conventional substrate in gene chip field, as silicon chip, and glass, nylon membrane etc.
The acquisition of capture probe and fixing: according to the single-minded sequence fragment of the kind 16SrRNA of various pathogenic bacteria, design and synthesize one section specific oligonucleotide probe, be fixed in the gene chip solid phase surface, constitute gene chip micro lattice.This probe can be, but is not limited to the specific oligonucleotide fragment of following pathogenic bacteria: comprise non-pathogenic bacteria rDNA gene fragment common in respiratory tract, digestive tube and urogenital tract common clinical pathogenic bacteria and the clinical sample.
Above-mentioned oligonucleotide capture probe is to obtain by the compare of analysis to the rDNA sequences of all pathogenic bacterias, every kind of pathogenic bacteria obtains distinctive 16-25 base candidate bar of n (n≤10) bar sequence fragment, after further hybridization is verified, final every kind of pathogenic bacteria is determined 1 peculiar sequence, is called " planting single-minded capture probe ".
Capture probe is combined in the chip matrix surface can be realized by number of ways, for example can introduce epoxide group on the surface, with hydroxyl or the amino reaction that joins probe together, the surface can also be handled with poly-lysine, with binding site capture probe from the teeth outwards, uv irradiating can be combined in probe on the matrix such as passivation layer of closing on as slide glass or electronics, and oligonucleotide can be bonded to the sensor surface of introducing aldehyde radical, amino or isothiocyanate group.The manufacturing mechanism of array can be directly synthetic on pin mark, specking or the chip, and described matrix is glass or cmos image sensor surface.
The acquisition of reporter probe: based on rDNA sequence conserved regions design and filter out 30 the highest base candidate bar sequence fragments of homology between every kind of pathogen species, and at sequence 5 ' end mark vitamin H, after further hybridization is verified, final definite 1 oligonucleotide universal sequence, be called " versatility reporter probe ", preferably its length is 17-45 base.
Hybridization: the target rRNA in single sample source is hybridized simultaneously with the reporter probe of mark and the capture probe on the gene chip, form " capture probe-target rRNA-reporter probe " hybridization complex, add specific enzymes then, react with the marker (as vitamin H) of reporter probe, under substrate catalysis, can send signal.
According to gene chip of the present invention, wherein, described rRNA derives from infective pathogen body or non-infective pathogen body, and described infective pathogen body comprises respiratory tract, digestive tube and urogenital tract disease-related infective pathogen body.
According to the non-enzymatic amplification detection system based on the single-minded sequence of rRNA gene kind of the present invention, it comprises:
1) matrix;
2) capture probe, described capture probe are based on the single-minded sequence of 16SrRNA of prokaryotic organism rRNA gene kind, are directly fixed on stromal surface, constitute gene chip micro lattice;
3) reporter probe;
4) image sensor.
Can by digital image in other words " machine vision " sensing technology read bonded thing signal to be checked on the array, the characteristics that it has low background and relative high s/n ratio are bio-sensor systems of a kind of detection thing to be checked of reinforcement.This specifically bio-sensor system has been used the sensing technology of integrative approach of digital image pickup, the array that includes on the daughter board, the low light shell that has the transmitter heat sink and thing signal to be checked etc.
The optical signalling that array produced can be detected by digital image pickup, and wherein digital image pickup comprises the matrix of photosensor, and the capture probe of some system forms array by the surface that covalently or non-covalently is incorporated into digital image pickup.In another way, capture probe can be put built in can be near on the slide glass of digital image pickup, and directly has fiber coupler at slide glass and transmitter.
The regional area of single probe points can be greater than the single photosensor of digital image pickup on the array.
Same probe points size can be consistent with single photosensor.
According to detection system of the present invention, can use digital image pickup to detect the signal that capture probe-target rRNA-reporter probe hybridization back produces, wherein digital image pickup comprises the matrix of photosensor.
Digital image pickup can be the CMOS active pixel sensors, and its each pixel correspondence is connected with the photodiode of quanxtizer, amplifier and register.The top layer of CMOS active pixel sensors is a passivation layer, as the abundant silicon-dioxide of printing opacity, with as the thing solution to be checked of isolated array detection and the barrier of semiconductor circuits.
The optical detection that is suitable for comprises methods such as detecting fluorescence, chemoluminescence, noclilucence and quantum dot, marker or signaling molecule comprise radio isotope, fluorescent agent, chemoluminescence agent, chemical luminophor, luminescent biological agent, enzyme, antibody, particle such as magnetic bead and quantum dot attached on the reporter probe.The signaling molecule that is used to detect thing to be checked comprises radio isotope, fluorescence dye such as Cy3, Cy5, Alexa Fluor488, fluorescein, rhodamine, Texas are red, rose-red, dansyl chloride, the pyridine of bromination second, naphthylamines, pyrenes, porphyrin, chemiluminescence system such as luminol,3-aminophthalic acid cyclic hydrazide, luminol, acridine phenyl ester, ruthenium salt, chromophore and colorimetric probe such as Radioactive colloidal gold, azoic dyestuff, quinoline dye, cyanine dyes.
Marker comprises antagonist and antagonist, toxin, epitope, hormone, antibody, polypeptide, enzyme, oligonucleotide, peptide nucleic acid(PNA), lectin, carbohydrate, albumen and medicine, the enzyme that for example is used for the ELISA detection can be used for carrying out fluoroscopic examination, in addition, fluorescently-labeled antibiotin or streptavidin.
Can form multiple detection method by multiple marker to a kind of specific thing to be checked, for example reporter probe can connect multiple fluorescence or chemiluminescent labels, in addition, reporter probe also can be in conjunction with multiple target spot, so reporter probe can connect multiple targeted integration molecule and multiple different marker.
For fluoroscopic examination, the exciting light of fluorescence can be provided by the LED panel near array or close cmos sensor shell, near array between array point and the photodiode spike filter can be removed excitation signal from reading of array the signal, and emission light is detected.
Another kind method is that thing signal to be checked can read mensuration information by the optical detection chemoluminescence.Chemoluminescence comes from the light that chemical reaction produces, and can not had the wideband detector of spectral filter to detect, and as the CMOS active pixel sensors, the light that the array point sends is directly detected, and background signal mainly comes from " dark current ".Thing to be checked can mark on the chemoluminescence label be used for chemiluminescence detection, as alkaline phosphatase or horseradish peroxidase.A detection efficiency part depends on the selectivity of mark or specificity deposit efficiency, the mark recognizate can be this type of the material that can be discerned by the enzyme detection system of vitamin H or DigiTAb, chemiluminescence reaction takes place then release energy by chemical bond rupture and produce the photon of discrete wavelength.
Array signal among the present invention detects can use digital image pickup, also can use charge coupled device (CCD), photomultiplier (PMT) or avalanche photodide to finish, the detection of thing to be checked also can be passed through, and for example electricity is led to detect and finished different array approaches.
According to the present invention, can be directly Array Construction on digital image pickup to strengthen the signal of its detection, this is to be formed on the chemoluminescence that the array on the passivation layer of digital image pickup top thin launches by detection to realize that the digital image pickup photosensitive part that signal can be approached array strengthens easily.
According to the non-enzymatic amplification detection method based on the single-minded sequence of rRNA gene kind of the present invention, it may further comprise the steps:
1) crude product of the rRNA in the preparation various ways sample;
2) directly fix one or more capture probes on chip matrix, described capture probe is based on the single-minded sequence of 16SrRNA of prokaryotic organism rRNA gene kind, is directly fixed on stromal surface;
3) reporter probe and capture probe are hybridized with the rRNA of same sample under the hybridization conditions of regulation, described reporter probe is based on highly conserved sequence between rRNA gene kind, the versatility oligonucleotide fragment of design and screening, length is 17-45 base, 5 ' end is through vitamin H (biotin) mark, constitute the versatility reporter probe, can be directly and target rRNA hybridize, add specific enzymes then, react with the marker (as vitamin H) of reporter probe, can send signal under substrate catalysis, detection signal directly obtains data from image sensor.
The invention provides the operative technique that a cover is easy and simple to handle, highly sensitive and the realization signal amplifies, is a kind of pair of probe (capture probe and reporter probe) gene chip signal amplification technique.According to technology of the present invention, can directly detect the nucleic acid in the rough sample of various ways, and not need the extracting and the amplification link of nucleic acid; Capture probe is directly connected to the chip matrix surface, simplify and detect step, minimizing retrieval cost, have high detection sensitivity simultaneously, this brand-new invention may change or substitute at present application present situation at gene type, transgenation and the gene expression atlas of clinical nucleic acid samples or other nucleic acid samples Application Areas.
Description of drawings
Fig. 1 detects Neisseria meningitidis in clinical sample 1.
Fig. 2 detects the listeria bacteria in clinical sample 2.
Fig. 3 detects simultaneously to causing meningitic 4 kinds of pathogenic bacterias.
Embodiment
The non-augmentation detection of microbial pathogen in embodiment 1 cerebrospinal fluid
Streptococcus pneumoniae (streptococcus pneumoniae), Neisseria meningitidis (neisseria meningitidis), listeria bacteria (listeria monocytogenes) and hemophilus influenzae bacteriums such as (hemophilus influenzae) are the The main pathogenic fungi that causes neonatal meningitis.Utilize the detection system among the present invention, under aseptic condition, extract cerebrospinal fluid clinically, need not pcr amplification, can carry out non-amplification amplification detection, realize making a definite diagnosis fast bacterial gene as clinical sample.
Have the feature of conservative property and variability concurrently according to bacterial 16 S rRNA gene, design can with the reporter probe of all bacterial 16 S rRNA hybridization, and the capture probe of 4 kinds of meningitis encountered pathogenic bacterias.Following probe is synthetic by Beijing AudioCodes biotechnology limited liability company.
The processing of clinical sample and the preparation of target gene: each 1mL of cerebrospinal fluid of 2 parts of different patients is joined the granulated glass sphere mixing that 1mL contains PBS damping fluid and the 1mL (volume) of 2%SDS respectively, on the whirlpool device, rotate mixing with top speed, use desk-top ultrasonication crusher machine one minute then, repeat more than 4 times, the ultrasonication product is centrifugal, get supernatant.
The preparation of gene chip: capture probe is dissolved in 20mM NaBO
3Among (pH 9.0)/5mM NaCl/20%DMSO, adopt the gene chip sample applying instrument to carry out point sample, 6 battle arrays of each chip point (4 kinds capture probe and positive and negative contrast) prepare chip according to ordinary method.
Hybridization:
A: at first have the chip of 6 battle arrays to add confining liquid I (6X SSPE/90% methane amide/1mg/ml BSA), leave standstill 30min in room temperature at point;
B: sucking-off confining liquid I, add hybridization solution (adding 10 μ L at 10 μ L supernatant liquors contains the solution of 90% methane amide and 6XSSPE and add 1 μ L vitamin H reporter probe), room temperature left standstill 1 hour;
C: forward to then and directly combine the experiencing on the chip of capture probe, in 30 ℃ of hybridization 1 hour.
D: the sucking-off hybridization solution, with washing lotion (0.1X SSPE/1mg/ml BSA/1% glycerine) washing 3 times, each 5min;
E: (1X TBS/5%BSA/1% glycerine/0.05%Tween-20) leaves standstill 30min to add the confining liquid II;
F: sucking-off confining liquid II, adding concentration are 1: 500 Straptavidine-HRP, reaction 30min;
G:, wash 3 times each 5min with washing lotion (1X TBS/1% glycerine/1mg/ml BSA) the unconjugated Straptavidine-HRP of flush away.
Detect: then chip sensor is inserted detector, add substrate catalysis, send signal, detection signal directly obtains data from image sensor.
According to the strong and weak existence of determining pathogenic bacteria of signal: the result shows from the battle array at the place of clinical sample 1 Neisseria meningitidis tangible bright spot, illustrates in the cerebrospinal fluid of sample 1 to have Neisseria meningitidis (the results are shown in Figure 1); From clinical sample 2, detect the listeria bacteria, and this gust brightness is higher, this pathogenic bacteria content in cerebrospinal fluid higher (the results are shown in Figure 2) is described.
In addition, native system not only can detect single pathogenic bacteria, also can detect simultaneously 4 kinds of pathogenic bacterias, with 4 kinds of pathogenic bacterias, streptococcus pneumoniae, Neisseria meningitidis, listeria bacteria and hemophilus influenzae are diluted to 10,000cfu/mL, after equal-volume mixes, get that 0.3mL contains the PBS damping fluid of 2%SDS with 0.3mL and the granulated glass sphere of 0.3mL (volume) mixes, on the whirlpool device, rotate mixing with top speed, centrifugal, get supernatant as the preparation target gene, can hybridize, detect by said procedure then.Clearly demonstrate the result that 4 kinds of pathogenic bacterias are detected simultaneously among Fig. 3.
The detection of embodiment 2 chlamydia trachomatises (CT) and gonococcus (NG)
When carrying out clinical detection, generally be to insert the vagina sampling with cotton ball for modal sexually transmitted disease CT/NT.Cotton ball after the sampling can wash down most of cell with the PBS damping fluid, be resuspended in then among the PBS of 1mL, get that 0.3mL contains the PBS damping fluid of 2%SDS with 0.3mL and the granulated glass sphere of 0.3mL (volume) mixes, on the whirlpool device, rotate mixing with top speed then, centrifugal, get the target gene solution that supernatant is preparation.
The design of capture probe and preparation: based on 16S rRNA specific gene group sequence between CT and NG bacterial classification, each designs a capture probe, 3 ' end mark-NH
2, give Beijing AudioCodes biotechnology limited liability company synthetic.Probe connects amination probe and the covalently bound method of epoxy activatory chip of adopting.Sequence is as follows:
CT-Pb：5′-GTTACTCGGATGCCCAAATATCGCC-NH
2-3′
NG-Pb：5′-GGATAGCGCAAGGCCCGAA-NH
2-3′
The design of reporter probe and preparation: check in multiple common bacteria 16S rRNA gene order at GenBank, have conserved sequence in conserved regions with seeking one section most of common bacteria, and 5 ' end coupling on vitamin H, then sequence is submitted to Beijing AudioCodes biotechnology limited liability company synthetic, as reporter probe.Its sequence is:
5’-Biotin-CGCTCGTTGCGGGACTTAACCCAACA-3
Hybridization and detection: hybridize and signal detection by embodiment 1 schedule of operation, hybridization temperature is 30 ℃, and the time is 1 hour.
We have also carried out the pcr amplification of CT/NG to sample when 10 parts of clinical samples being carried out the gene chip detection, the result shows: the bright spot position is accurate, clear.Have 4 increments originally to present the two positive hybridization signals of CT/NG, wherein 2 parts detect the CT strong positive, the NG signal a little less than; All the other 6 people are negative.This result and pcr amplification result are in full accord, and this detection method high specificity is described, and be highly sensitive.
The rapid detection of bacterium in embodiment 3 sewage
Contain various mineral contaminants and organic dirt in the food enterprise sewage, the pathogenic bacterium of wherein carrying secretly can cause the outburst of multiple disease and popular.Existing detection method mainly is to utilize traditional micro-biological process, serological method and PCR method, and there are some restrictions in its method more: incubation time is long, can not distinguish the close bacterium of sibship, sensitivity is low etc.And biochip technology provided by the invention can realize quick, accurate, specific detection.Choose intestinal bacteria common in the food sewage (escherichia coli), faecalis (enterococcus), Shigellae (shigella flexneri), Salmonellas (salmonella enterica) and amount to 4 kinds of bacterium as detected object.
Design 4 species specific capture probes respectively in 16s RNA variable region; At the reporter probe of the stable region of 16s RNA design versatility, its sequence such as following table:
The preparation of target gene: the sewage sample 10mL that gathers was placed desk-top ultrasonication crusher machine one minute, repeat more than 4 times the centrifuging and taking supernatant.
Hybridization and detection: hybridize and signal detection by embodiment 1 schedule of operation, hybridization temperature is 30 ℃, and the time is 1 hour.
5 parts of sewage samples that we gather different areas have simultaneously carried out the comparison that gene chip detects and microbial culture detects, the result shows gene chip detected result and bacteria cultivation results basically identical, and gene chip has higher sensitivity than microbial culture.Concrete outcome sees the following form.
| The water sample numbering | Gene chip detects the bacterium of gene | The bacterium of turning out |
| 1 | SF、EC | SF、EC |
| 2 | SE | SE |
| 3 | EF | Do not detect |
| 4 | SF、EC、SE | SF、EC |
| 5 | SF、EC | EC |
This detection technique is that the good technical basis has been established in later more massive detection research, can be applied to many fields such as environmental monitoring, Food Hygiene Surveillance, commodity inspection quarantine.
Claims (9)
1. the non-enzymatic amplification based on the single-minded sequence of rRNA gene kind detects gene chip, it is characterized in that described gene chip comprises:
1) matrix;
2) capture probe, described capture probe are the single-minded sequence of 16S rRNA based on rRNA gene kind, are directly fixed on stromal surface, constitute gene chip micro lattice;
3) reporter probe, described reporter probe are based on highly conserved sequence between rRNA gene kind, and its end is labeled.
2. gene chip according to claim 1 is characterized in that, the length of described capture probe is 16-25 base.
3. gene chip according to claim 1 is characterized in that, the length of described reporter probe is 17-45 base.
4. gene chip according to claim 1 is characterized in that, described rRNA derives from infective pathogen body or non-infective pathogen body.
5. gene chip according to claim 4 is characterized in that, described infective pathogen body comprises blood, respiratory tract, digestive tube and urogenital tract disease-related infective pathogen body.
6. gene chip according to claim 1 is characterized in that, described matrix is glass or cmos image sensor surface.
7. non-enzymatic amplification detection system based on the single-minded sequence of rRNA gene kind is characterized in that described detection system comprises:
1) matrix;
2) capture probe, described capture probe are the single-minded sequence of 16S rRNA based on rRNA gene kind, are directly fixed on stromal surface, constitute gene chip micro lattice;
3) reporter probe, described reporter probe are based on the highly conserved sequence between rRNA gene kind, and its end is labeled;
4) image sensor.
8. detection system according to claim 7 is characterized in that, described image sensor is a cmos image sensor.
9. non-enzymatic amplification detection method based on the single-minded sequence of rRNA gene kind is characterized in that said method comprising the steps of:
1) crude product of the rRNA in the preparation various ways sample;
2) directly fix one or more capture probes on chip matrix, described capture probe is the single-minded sequence of 16S rRNA based on rRNA gene kind, is directly fixed on stromal surface;
3) reporter probe and capture probe are hybridized with the rRNA of same sample under the hybridization conditions of regulation, and described reporter probe is based on the highly conserved sequence between rRNA gene kind, and its end is labeled;
4) detection signal, detection signal directly obtains data from image sensor.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2010102287559A CN101899512A (en) | 2010-07-09 | 2010-07-09 | Non-enzymatic amplification detection gene chip based on rRNA genospecies specific sequence, detection system and detection method |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2010102287559A CN101899512A (en) | 2010-07-09 | 2010-07-09 | Non-enzymatic amplification detection gene chip based on rRNA genospecies specific sequence, detection system and detection method |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN101899512A true CN101899512A (en) | 2010-12-01 |
Family
ID=43225429
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN2010102287559A Pending CN101899512A (en) | 2010-07-09 | 2010-07-09 | Non-enzymatic amplification detection gene chip based on rRNA genospecies specific sequence, detection system and detection method |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN101899512A (en) |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105861646A (en) * | 2016-03-02 | 2016-08-17 | 成都市妇女儿童中心医院 | Electrochemical biological chip for detecting urinary tract pathogen 16SrRNA and technical application thereof |
| CN106148502A (en) * | 2014-12-11 | 2016-11-23 | 中国科学院上海生命科学研究院 | A kind of high throughput method directly detecting food source pathogenic bacteria |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1730667A (en) * | 2004-12-17 | 2006-02-08 | 浙江大学 | Helicobacter pylori clarithromycin drug-resistant gene polymorphism detecting chip and its preparation method and uses |
| WO2007056680A2 (en) * | 2005-11-03 | 2007-05-18 | Forsyth Dental Infirmary For Children | Methods and arrays for identifying human microflora |
| CN101514956A (en) * | 2008-08-18 | 2009-08-26 | 北京九州泰康生物科技有限责任公司 | Molecule detecting system |
-
2010
- 2010-07-09 CN CN2010102287559A patent/CN101899512A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1730667A (en) * | 2004-12-17 | 2006-02-08 | 浙江大学 | Helicobacter pylori clarithromycin drug-resistant gene polymorphism detecting chip and its preparation method and uses |
| WO2007056680A2 (en) * | 2005-11-03 | 2007-05-18 | Forsyth Dental Infirmary For Children | Methods and arrays for identifying human microflora |
| CN101514956A (en) * | 2008-08-18 | 2009-08-26 | 北京九州泰康生物科技有限责任公司 | Molecule detecting system |
Non-Patent Citations (4)
| Title |
|---|
| 《APPLIED AND ENVIRONMENTAL MICROBIOLOGY》 20030331 J�rg Peplies et al. Optimization Strategies for DNA Microarray-Based Detection of Bacteria with 16S rRNA-Targeting Oligonucleotide Probes 1397-4107 1-8 第69卷, 第3期 2 * |
| 《APPLIED AND ENVIRONMENTAL MICROBIOLOGY》 20030531 Darrell P. Chandler et al. Sequence versus Structure for the Direct Detection of 16S rRNA on Planar Oligonucleotide Microarrays 2950-2958 1-8 第69卷, 第5期 2 * |
| 《热带农业科学》 20091031 荣光 等 16S rRNA 在兽医病原菌分类鉴定中的应用 45-49 1-8 第29卷, 第10期 2 * |
| 《第二军医大学学报》 20070831 薛建亚等 细菌基因芯片的构建及其在细菌鉴定中的应用 第919页"1.3 基因芯片的制备"部分 1-8 第28卷, 第8期 2 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106148502A (en) * | 2014-12-11 | 2016-11-23 | 中国科学院上海生命科学研究院 | A kind of high throughput method directly detecting food source pathogenic bacteria |
| CN105861646A (en) * | 2016-03-02 | 2016-08-17 | 成都市妇女儿童中心医院 | Electrochemical biological chip for detecting urinary tract pathogen 16SrRNA and technical application thereof |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Dunbar et al. | Quantitative, multiplexed detection of bacterial pathogens: DNA and protein applications of the Luminex LabMAP™ system | |
| CN102165071A (en) | Methods of producing homogeneous plastic-adherent aptamer-magnetic bead-fluorophore and other sandwich assays | |
| CN103898208A (en) | Quick high-throughput intestines source pathogenic bacterium detection method | |
| Xing et al. | Microfluidic biosensor for one-step detection of multiplex foodborne bacteria ssDNA simultaneously by smartphone | |
| Li et al. | One-step synthesis of mannose-modified polyethyleneimine copolymer particles as fluorescent probes for the detection of Escherichia coli | |
| CN106399517A (en) | Multi-cross isothermal amplification and nanogold biosensing combined nucleic acid detection technology | |
| CN103958988A (en) | Dna chip for diagnosing urogenital infectious diseases | |
| CN104946634A (en) | Double-labelling fluorescence activation type probe and method for detecting DNA by adopting same | |
| CN105316391A (en) | Method of detecting salmonella, shigella and staphylococcus aureus | |
| Ikeda et al. | Rapid and simple detection of food poisoning bacteria by bead assay with a microfluidic chip-based system | |
| CN102978295B (en) | Pathogenic microorganism nucleic acid amplification-free detection and typing method | |
| CN106755358A (en) | It is intersect the method that amplification combines gold nano bio-sensing detection vibrio parahemolyticus more | |
| CN102140507A (en) | Detection genetic chip and detection kit for infectious diarrhea | |
| CN102424862B (en) | Genotyping chip for legionella pneumophila, and kit for detection of legionella pneumophila | |
| CN111088380A (en) | Brucella LF-RPA detection primer, probe and detection kit | |
| CN101768636B (en) | Composition and kit for detecting vibrio cholerae and detection method | |
| EP1651773A1 (en) | Diagnostics of diarrheagenic escherichia coli (dec) and shigella spp. | |
| CN102952850B (en) | Real-time fluorescent quantitative PCR method used for detecting Mycobacterium tuberculosis, and primer, probe and kit thereof | |
| CN103451310B (en) | Gene chip capable of simultaneously detecting various vibrios and method for detecting vibrios | |
| CN103160587B (en) | Genetic typing chip of 10 common pathogenic legionella and detection kit | |
| CN101899512A (en) | Non-enzymatic amplification detection gene chip based on rRNA genospecies specific sequence, detection system and detection method | |
| ES2299727T3 (en) | PROCEDURE FOR THE QUANTITATIVE DETERMINATION OF POLYNUCLEOTIDES IN A MIX. | |
| CN108624653A (en) | A kind of kit of quantum dot detection of nucleic acids for bloodstream infection pathogen | |
| CN106566889A (en) | Primers, probes, kit and method for detecting proteus mirabilis | |
| JP2013504319A (en) | Peptide nucleic acid probe, kit and method for detecting Helicobacter pylori and / or clarithromycin resistance, and use thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20101201 |
|
| RJ01 | Rejection of invention patent application after publication |