CN102477456A - Fabrication procedure of pathogenic microorganism infection diagnosis type cell chip - Google Patents
Fabrication procedure of pathogenic microorganism infection diagnosis type cell chip Download PDFInfo
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- CN102477456A CN102477456A CN2010105534613A CN201010553461A CN102477456A CN 102477456 A CN102477456 A CN 102477456A CN 2010105534613 A CN2010105534613 A CN 2010105534613A CN 201010553461 A CN201010553461 A CN 201010553461A CN 102477456 A CN102477456 A CN 102477456A
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Abstract
The invention discloses a fabrication procedure of a pathogenic microorganism infection diagnosis type cell chip, which comprises the following steps of: arranging a reaction tank on a substrate of the cell chip; and arranging a cell slice in the reaction tank. A virus infection cell slice is prepared through the following steps: 1, preparation of the substrate: preparing the substrate by adopting organic macromolecular materials, metals or silicon, and fabricating the reaction tank on the substrate surface by adopting a micro-processing technology; 2, preparation of the cell slice: preparing the virus infection cell slice or a pathogenic microorganism cell slice according to detected pathogenic microorganism; 3, preparation of the cell chip: adhering the cell slice in the reaction tank. The invention provides the pathogenic microorganism infection diagnosis type cell chip which has the characteristics of high flux, low cost, rapidness and accuracy, and a preparation method thereof.
Description
Technical field
The present invention relates to a kind of production process of cause pathogeny imcrobe infection diagnosis type cell chip.
Background technology
Infection is the common disease and the frequently-occurring disease of harm people ' s health and life.The ubiquity of its infection, morbific complicacy make diagnosis and the treatment to such disease clinically very difficult, press for a kind of diagnostic techniques effectively and rapidly.For the detection of virus disease pathogenic agent, because the cultivation of virus is relatively more difficult, and the time is longer, and positive rate is low, not easy to operate, therefore mainly relies on the infection of serological method and molecular biology method diagnosis virus clinically.But above-mentioned two class methods have shortcoming or deficiency to some extent.For example: draw materials and limitation such as sensing range is narrow but neutralization test method, complement fixation test (CFT) method, hemagglutination-inhibition test method exist; Enzyme-linked immunosorbent assay adopts indirect method usually, because the quality standard disunity of manufacturer, so false positive rate is high; Radio immunoassay is had relatively high expectations to technical level of operators, and environment is had certain pollution.Pathogenic micro-organism such as bacterium, fungi mainly relies on cultured method to diagnose, and required time is longer.
The defective of preceding method is that also its detection to pathogenic micro-organism all is man-to-man detections, and the relation of pathogenic micro-organism and disease is clinically: a kind of disease can be caused by the several diseases pathogenic microorganism; A kind of pathogenic micro-organism can cause multiple disease again.Therefore, it can't satisfy clinical in the high-throughput of several diseases pathogenic microorganism, the needs of rapid detection.
Existing gene chip, protein chip are used for the pathogenic micro-organism detection both at home and abroad at present, and be still useless in the cell chip of pathogenic micro-organism diagnosis.Gene chip is based on the detection of clinical disease pathogenic microorganism nucleic acid level, and the content of pathogenic micro-organism is very low in the clinical samples, and nucleic acid must just can be used for gene chip and detect after pcr amplification amplifies; Multiple pathogen detection needs multiple PCR technique; And being of limited application of present multiple PCR technique can not satisfy the needs of high-throughput chip, and false positive appears in PCR easily; Therefore it is to the having relatively high expectations of experiment condition and operator, and the hospital that condition is general is difficult to carry out.The domestic existing protein chip product that tumour is diagnosed; In general; The making of protein chip needs highly purified specific antigens or monoclonal antibody specific; And highly purified specific antigens or monoclonal antibody specific complicated process of preparation, cost is high, causes adopting the protein chip of its making to cost an arm and a leg.
Yilai Gene Medicine Co., Ltd., Nanjing is about the Chinese patent " cell microarray chip and preparation method thereof " of cell chip, number of patent application: 00122065.9.What encapsulate on this technological cell chip is the normal cell of cultivating, and only is used for the high flux screening and the functional genomics research of compound, biomolecules or medicine, detects and can't be used for pathogenic micro-organism.
Summary of the invention
The invention solves that small throughput in the background technology, expensive, detection speed are slow, the technical problem of poor accuracy.
Technical solution of the present invention is: a kind of cause pathogeny imcrobe infection diagnosis type cell chip, comprise substrate 1 and reactive tank 2, and it is characterized in that: described substrate 1 is provided with reactive tank 2, is provided with cell sheet 3 in the reactive tank 2.
Above-mentioned cell sheet 3 be with the bacterium smear that contains pathogenic micro-organism or be coated with cut into by the eukaryotic cell sheet of virus infection with the corresponding thin slice of reactive tank 2 shapes.Above-mentioned reactive tank 2 be provided with recessed in or protrudingly all can in substrate 1 surface.Above-mentioned reactive tank 2 can be one, two or more, and the said cell sheet 3 that is arranged in the reactive tank 2 is one, two or more accordingly.Above-mentioned a plurality of reactive tank 2 is good with array arrangement.Above-mentioned substrate 1 can be organic polymer material, metal or silicon etc.; The material of described cell sheet 3 can be glass, plastics or cellulose nitrate film etc.
A kind of preparation method of the diagnosis of cause pathogeny imcrobe infection as stated type cell chip is characterized in that: should
1). the preparation of substrate
Employing machine macromolecular material, metal or silicon are prepared substrate 1, on substrate 1 surface, adopt micro-processing technology to produce reactive tank 2;
2). the preparation of cell sheet
According to detecting pathogenic micro-organism, preparation virus infected cell thin slice or pathogenic micro-organism cell sheet;
3). the preparation of cell chip
Cell sheet is bonded in the reactive tank 2.
Above-mentioned virus infected cell preparation of sections can comprise
(1). cell recovery; Select special host cell according to detecting virus;
(2). passage is cultivated;
(3). the preparation cell suspension;
(4). make the monolayer sheet;
(5). virus infection;
(6). fixing;
(7). cut into cell sheet;
The preparation of above-mentioned pathogenic micro-organism cell sheet can comprise
(1). the pure culture of other pathogenic micro-organisms such as bacterium, fungi;
(2). the preparation bacteria suspension;
(3). make the bacterium smear;
(4). fixing;
(5). cut into cell sheet.
The present invention has the following advantages:
1. high-throughput.The relation of pathogenic micro-organism and disease is clinically: a kind of disease can be caused by the several diseases pathogenic microorganism; A kind of pathogenic micro-organism can cause multiple disease again.Therefore, the present invention can synchronously detect the several diseases pathogenic microorganism, satisfies the requirement of clinical high throughput testing to pathogenic micro-organism;
2. low-cost.The chip material cost is low, and material is easy to get, and preparation technology is simple, and is cheap;
3. simple to operate, be easy to grasp, detect and need not special conditions, be convenient to popularize;
4. detect fast, accurately, can make a definite diagnosis fast infection;
5. environmentally safe;
6. applied range.Not only can be the clinical diagnosis basis that provides, also can be used for the mikrobe quarantine of livestock and poultry.
Description of drawings
Fig. 1,2,3 is respectively structural representation of the present invention.
Embodiment
Referring to Fig. 1,2,3, cause pathogeny imcrobe infection diagnosis type cell chip of the present invention mainly by substrate l,
Reactive tank 2, cell sheet 3 constitute.Substrate 1 can adopt organic polymer material, metal or silicon etc., is processed with the reactive tank 2 of placing cell sheet 3 on it, and reactive tank 2 need can be one, two or more according to detection, is good to be set to a plurality of of array arrangement.Cell sheet 3 is with the bacterium smear of pathogenic micro-organisms such as bacterium, fungi, spirochete or is coated with by the eukaryotic cell sheet of virus infection; Cut into the corresponding cell sheet 3 of reactive tank 2 shapes, the material of cell sheet can be glass, plastics or cellulose nitrate film etc.Cell sheet 3 is embedded in the reactive tank 2.Be coated with specific antigens on the cell sheet, so can directly be used for the detection of pathogenic micro-organism to a certain pathogenic micro-organism.During use, can deckglass be covered on the reactive tank 2.
Concrete making method of the present invention is following:
1. the preparation of substrate
Adopt printing opacity organic polymer material such as PS etc. to prepare substrate 1, adopt micro-processing technology to produce square or rectangular reactive tank 2 above that.
2. the preparation of cell sheet
Difference according to detecting pathogenic micro-organism can prepare the different cells thin slice.1]. the virus infected cell preparation of sections:
[1]. cell recovery.When detecting virus, select special host cell according to different virus.
[2]. passage is cultivated.
[3]. the preparation cell suspension.
[4]. make the monolayer sheet.
[5]. virus infection.
[6]. fixing.
[7]. cut into cell sheet.
2]. the preparation of bacterium, other pathogenic micro-organism cell sheets of Mycophyta:
[1]. the pure culture of other pathogenic micro-organisms such as bacterium, fungi
[2]. the preparation bacteria suspension
[3]. make the bacterium smear
[4]. fixing
[5]. cut into cell sheet
3. the preparation of cell chip:
Cell sheet 3 usefulness optics transparent adhesive tapes are bonded in the reactive tank 2.
Making method embodiment of the present invention:
1. the preparation of substrate:
Employing has strong organic polymer material of transparence such as PS to prepare substrate 1, and the marginarium of substrate 1 is omited high than the intermediate zone and is step-like, and small stair is used for fixing deckglass.Adopt micro-processing technology to prepare some reactive tanks 2 as required on the substrate 1, the wall of reactive tank 2 a little more than the thickness of cell sheet 3 and with small stair with high, can be in order to the supporting cover slide.Reactive tank 3 internal fixing cell sheets 3.Reactive tank 2 can also be depressed in the substrate 1.
2. the preparation of cell sheet:
1). the virus infected cell preparation of sections:
According to the difference of infective virus, can adopt different cells system, like Hep-2, Vero-E.2BS, NSl, SP-2/0 etc.
(1). cell recovery: after the frozen pipe of plastics that freeze-stored cell will be housed takes out, put into 37 ℃ of water baths immediately, 3 minutes in liquid nitrogen container; Centrifugal, 1000rpm, 5 minutes; Abandon supernatant, add the RPMll640 nutrient solution that nutrient solution contains the lO% foetal calf serum,, process suspension with blowing and beating cell get off at the bottom of the suction pipe bottle.
(2). passage is cultivated: at CO: in the incubator of preserving moisture 37 ℃, 5%C0.Cultivate.Passage cultivated for 2-3 generations, and is stable until proterties.
(3). the preparation cell suspension: the nutrient solution that sucking-off is old, in culturing bottle, add and contain O.25% tryptic Digestive system, the wave and culture bottle makes Digestive system flow through all cells surface gently, 37 ℃, acts on after 2 minutes the sucking-off Digestive system.Add nutrient solution then and stop digestion, draw nutrient solution with suction pipe again and blow and beat a bottle parietal cell repeatedly, process cell suspension.
(4). make the monolayer sheet: the deckglass that will pass through cleaning and sterilization is put into aseptic petridish, draws cell suspension with suction pipe and drips on deckglass, and cell suspension is covered on the deckglass equably.37 ℃, to cultivate and foretell 2 days, observation of cell grows up to individual layer, inhales and removes cell suspension.
(5). virus infection: the viral suspension of certain titre is dropped on the monolayer sheet, at C0: in the incubator of preserving moisture 37 ℃, behind the absorption 1.5h, inhale the virus removal suspension, add the liquid of keeping of nutrient solution, at C0.Preserve moisture in the incubator 37 ℃, cultivated 2-3 days, inhale the virus removal suspension, take out cell sheet through virus infection.
(6). fixing: fixing more than 2 hours with acetone soln.
(7). cut into cell sheet: the remaining stationary liquid with on the cell sheet of PBS damping fluid and distilled water flush away virus infection, dry.By hand or the robotization cutting device cell sheet of virus infection is cut into several millimeters square cell sheets.
2]. the preparation of other pathogenic micro-organism cell sheets such as bacterium, fungi:
(1). the pure culture of other pathogenic micro-organisms such as bacterium, fungi: adopt the type strain or the clinical separation and Culture strain of other pathogenic micro-organisms such as bacterium, fungi, the solid medium separation and Culture.
(2). preparation bacteria suspension: collect single bacterium colony, process bacteria suspension.
(3). make the bacterium smear: get aseptic deckglass, drip an amount of bacteria suspension, evenly its seasoning is treated in coating.
(4). fixing: fixing more than 2 hours with acetone soln.
(5). cut into cell sheet: by hand or the robotization cutting device bacterium smear is cut into several millimeters square cell sheets.
3. the preparation of cell chip:
The cell sheet that cuts is sticked with glue agent to bond on the corresponding reactive tank of substrate.4 ℃ of preservations.
The present invention according to clinical needs can clinical common pathogenic micro-organism be combined into cns series, respiratory system series respectively, urogenital system is serial, prenatal and postnatal care is serial, cardiovascular systems is serial, eye nose ear larynx series detection chip.
Claims (4)
1. the production process of a cause pathogeny imcrobe infection diagnosis type cell chip, it is characterized in that: comprise substrate (1) and reactive tank (2), it is characterized in that: described substrate (1) is provided with reactive tank (2), is provided with cell sheet (3) in the reactive tank (2),
(1). described cell sheet (3) be with the bacterium smear that contains pathogenic micro-organism or be coated with cut into by the eukaryotic cell sheet of virus infection with the corresponding thin slice of reactive tank (2) shape;
(2). said reactive tank (2) be provided with recessed in or protruding in substrate (1) surface;
(3). described reactive tank (2) is one, two or more, and the said cell sheet (3) that is arranged in the reactive tank (2) is one, two or more accordingly;
(4). described a plurality of reactive tanks (2) are array arrangement;
(5). described substrate (1) is organic polymer material, metal or silicon; The material of described cell sheet (3) is glass, plastics or cellulose nitrate film.
2. the production process of an a kind of cause pathogeny imcrobe infection diagnosis type cell chip as claimed in claim 1, it is characterized in that: this method comprises
1) preparation of substrate adopts machine macromolecular material, metal or silicon to prepare substrate (1), on substrate (1) surface, adopts micro-processing technology to produce reactive tank (2);
2) preparation of cell sheet is according to detecting pathogenic micro-organism, preparation virus infected cell thin slice or pathogenic micro-organism cell sheet;
3) preparation of cell chip is bonded in cell sheet in the reactive tank (2).
3. like the production process of the said cause pathogeny imcrobe infection diagnosis of claim 2 type cell chip, it is characterized in that: said virus infected cell preparation of sections comprises
(1) cell recovery; Select special host cell according to detecting virus;
(2) passage is cultivated;
(3) preparation cell suspension;
(4) make the monolayer sheet;
(5) virus infection;
(6) fixing;
(7) cut into cell sheet.
4. like the preparation method of the said cause pathogeny imcrobe infection diagnosis of claim 3 type cell chip, it is characterized in that: the preparation of said pathogenic micro-organism cell sheet comprises
(1) pure culture of other pathogenic micro-organisms such as bacterium, fungi;
(2) preparation bacteria suspension;
(3) make the bacterium smear;
(4) fixing;
(5) cut into cell sheet.
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| CN2010105534613A CN102477456A (en) | 2010-11-22 | 2010-11-22 | Fabrication procedure of pathogenic microorganism infection diagnosis type cell chip |
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| CN2010105534613A CN102477456A (en) | 2010-11-22 | 2010-11-22 | Fabrication procedure of pathogenic microorganism infection diagnosis type cell chip |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111662959A (en) * | 2020-07-22 | 2020-09-15 | 中国医学科学院病原生物学研究所 | High-throughput rapid detection method for fungi |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111662959A (en) * | 2020-07-22 | 2020-09-15 | 中国医学科学院病原生物学研究所 | High-throughput rapid detection method for fungi |
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Application publication date: 20120530 |