CN101464409A - Apparatus and method for fast quantitative bacteria detection - Google Patents
Apparatus and method for fast quantitative bacteria detection Download PDFInfo
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- CN101464409A CN101464409A CNA2007101798644A CN200710179864A CN101464409A CN 101464409 A CN101464409 A CN 101464409A CN A2007101798644 A CNA2007101798644 A CN A2007101798644A CN 200710179864 A CN200710179864 A CN 200710179864A CN 101464409 A CN101464409 A CN 101464409A
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Abstract
The invention provides a device and a method for rapid quantitative detection of bacteria. The method comprises the following steps: the immune technique is adopted for marking bacteria in a sample with a fluorescent substance (such as FITC, fluorescence quantum dots, and the like); as a sample solution flows through a set of filtering device, the bacteria marked with the fluorescent substance settle on the surface of a filtering membrane; the fluorescent substance on the bacteria emits fluorescence by adopting laser to induce and arouse fluorescence; and the number of the bacteria in the sample can be obtained quickly and accurately by detecting the signal strength of all the fluorescence on the filtering membrane. The entire detecting process lasts for less than 30 minutes.
Description
Technical field
The present invention relates to field of biomedicine technology, be specifically related to a kind of device and method of fast quantitative bacteria detection.
Background technology
Bacterium, particularly pathogenic bacteria pollute the mankind's potable water, food etc., bring potential danger to health, for example enterohemorrhagic Escherichia coli can cause human hemorrhagic enteritis even death, large-scale outbreak also once took place in China in 1999 in the popular and outburst in country such as the U.S., Japan, Britain, Germany in succession in recent years.
Conventional sense method to total number of bacteria, mainly containing passing through in the National Standard Method cultivates sample and to carry out bacterial colony count with colony counting method after 24-48 hour in double dish, these detections need be carried out multinomial numerous and diverse analytical test and preparation nutrient culture media in the laboratory, owing to need to cultivate, required time is long, must need specialized equipment to count, and, can not distinguish the close pathogenic bacteria of form owing to there is not specificity.Said process often needs the professional and technical personnel to finish, and is difficult to realize field quick detection.At present both at home and abroad all at some quicker and sensitive method of detecting bacterium of research and development, there have been reagent set box or strip to sell on the market, mainly be to come number of bacteria in the sample estimates by the change color of contrast strip, though this method detection speed is fast, but sensitivity is low, is difficult to realize detection by quantitative.Also have on the market and adopt the ATP method to carry out the fast detecting of total number of bacteria in the sample, this is that a kind of bioluminescence method of utilizing is carried out the total number of bacteria detection, but this method does not have specificity, can't differentiate bacterium kind in the sample.In addition, the broad research detection method also has nucleic acid amplification technologies and micro-fluidic chip technology at present, but these technology still are in the laboratory study stage, do not reach the actual demand to the bacterium field quick detection.
Summary of the invention
The objective of the invention is to disclose a kind of device and method of fast quantitative bacteria detection, detection time is short, and is highly sensitive, has specificity, quantitatively accurate counting.
For achieving the above object, technical solution of the present invention is:
A kind of device of fast quantitative bacteria detection comprises the sample filtering part, and sample filtering partly comprises chamber, filtering membrane, back up pad up and down, wherein, be up and down filtering membrane and back up pad between the chamber, filtering membrane and back up pad are horizontally disposed with, and filtering membrane is affixed on support plate upper surface;
It also comprises light path part and circuit part;
Light path part comprises light source, light source optical filter, saturating anti-mirror, fluorescent optical filter, wherein, on the light emitted light path, is sequentially with light source optical filter, saturating anti-mirror, and fluorescent optical filter is positioned at anti-mirror side, on the light path of saturating anti-mirror reflection light;
Circuit part, comprise display unit, signal processing unit, the keyboard input block, host computer and photomultiplier, wherein, display unit, keyboard input block, host computer and photomultiplier are electrically connected with signal processing unit respectively, the photomultiplier receiving end is positioned on the light path of anti-mirror reflection light, is used to detect the light intensity of the fluorescence that is excited;
During use, filtering membrane to be measured is positioned on the light path of source emissioning light, and the fluorescent material on the filtering membrane can be sent fluorescence after the optical excitation of light emitted.
The device of described fast quantitative bacteria detection, its described three partly integrated be a portable apparatus.
The device of described fast quantitative bacteria detection, its described anti-mirror makes exciting light wave band light transmission, and makes the reflection of fluorescence band of light.
The using method of described device the steps include: testing sample is carried out filtering after fluorescence labeling handles; Take out filtering membrane; Irradiation leaves the filtering membrane through the fluorescence labeling bacterium, induces the fluorescent material that excites on the filtering membrane to send fluorescence; The fluorescence signal that sends is injected the photomultiplier receiving end, and photomultiplier is converted to electric signal with fluorescence signal and exports signal processing unit to; Send into display unit after the signal processing unit processes and show last bacterial number.
Described method, its concrete steps are:
A). testing sample is carried out fluorescence labeling handle, select the filtering membrane in suitable aperture simultaneously as required;
B). the sample after the processing joins in the last chamber of sample filtering part, and pressurization makes sample solution flow through filtering membrane, back up pad, again through chamber outflow down; Stay the surface of filtering membrane in the sample through fluorescently-labeled bacterium, take out filtering membrane;
C). with b) filtering membrane to be measured that takes out of step places under the saturating anti-mirror of light path part, is positioned on the light path of source emissioning light;
D). open the light source of light path part, the light that light source sends obtains the exciting light of required wave band after the light source optical filter filters, and exciting light is radiated on the filtering membrane through saturating anti-mirror, induces the fluorescent material that excites on the filtering membrane to send fluorescence;
E) .d) fluorescence that sends of step together with the exciting light that is reflected after saturating anti-mirror reflection, filter through fluorescent optical filter again, only allow fluorescence signal to enter the photomultiplier receiving end, photomultiplier is converted to the signal processing unit that electric signal exports circuit part to fluorescence signal;
F). send into display unit after the signal processing unit of circuit part is handled the electric signal of photomultiplier output according to the indication of keyboard input block and show last bacterial number, or and upper machine communication, measurement result is sent in the database or network in the host computer.
Described method, its described b) take out filtering membrane in the step, in last chamber, add the buffer solution elution filtering membrane, to reduce the background fluorescence on the filtering membrane, improve signal to noise ratio (S/N ratio).
Described method, it finished to detection from obtaining sample, less than 30 minutes.
Beneficial effect of the present invention:
Compare with traditional cultural method bacterial detection, detection time of the present invention is short, finishes to detection from obtaining sample, and less than 30 minutes, and traditional detection method needed 24～48 hours at least; Compare with present method for quick, detection sensitivity height of the present invention has specificity, quantitative accurate counting, and method for quick generally all is a qualitative detection at present.
Description of drawings
Fig. 1 is a sample filtering installation drawing of the present invention;
Fig. 2 is light path part of the present invention and circuit part synoptic diagram.
Embodiment
The present invention's " device of fast quantitative bacteria detection " comprises sample filtering part, light path part and circuit part.
As shown in Figure 1, sample filtering partly has two cells 1,4 up and down, is filtering membrane 2 and back up pad 3 between the chamber 1,4 up and down, and filtering membrane 2 and back up pad 3 are horizontally disposed with, and filtering membrane 2 is affixed on back up pad 3 upper surfaces.The sample of handling through fluorescence labeling joins in the chamber 1, makes sample solution flow through filtering membrane 2, back up pad 3 by exerting pressure, again through chamber 4 outflows down.Select the filtering membrane 2 in suitable aperture as required, stay the surface of filtering membrane 2 in the sample through fluorescently-labeled bacterium, and unnecessary fluorescent dye warp flows out chamber 4 down in the sample.The effect of back up pad 3 is to support filtering membrane 2, makes its maintenance smooth, is unlikely to owing to exerting pressure filtering membrane 2 to be burst or the deformation effect testing result when filtering.Can in last chamber 1, add buffer solution elution filtering membrane 2 again,, improve signal to noise ratio (S/N ratio) to reduce the background fluorescence on the filtering membrane 2.
As shown in Figure 2, light path part is the interior part of frame of broken lines A among the figure, comprises light source A1, optical filter A2, saturating anti-mirror A3 and optical filter A4.The light that light source A1 sends obtains the exciting light of required wave band after optical filter A2 filters, exciting light is radiated on the filtering membrane 2 through saturating anti-mirror A3, induce the fluorescent material that excites on the filtering membrane 2 to send fluorescence, this fluorescence together with the exciting light that is reflected after saturating anti-mirror A3 reflection, after optical filter A4 filters, only allow fluorescence signal to enter photomultiplier B5, photomultiplier B5 is converted to electric signal output with fluorescence signal.Saturating anti-mirror A3 has the exciting light of making wave band light transmission, and makes the characteristics of fluorescence band of light reflection.
Circuit part is the interior part of solid box B among the figure, comprises display unit B1, signal processing unit B2, keyboard input block B3, host computer B4 and photomultiplier B5.Send into display unit B1 after signal processing unit B2 handles the electric signal of photomultiplier B5 output according to the indication of keyboard input block B3 and show last bacterial number, signal processing unit B2 also can with host computer B4 communication, measurement result is sent in the database or network among the host computer B4.
Device and method of the present invention finished to detection from obtaining sample, less than 30 minutes.
The present invention utilizes immunological technique that fluorescent material is marked on the bacterium in the sample, adopt a kind of simple filtering technique bacterium to be trapped in the surface of filtering membrane, realize the bacterium fast quantification is detected by the induced with laser fluorescence excitation, owing to adopted immunological technique to make testing process have specificity, adopted light path design and the circuit design optimized to improve detection sensitivity, whole device is easy to be integrated into a portable apparatus, and the on-the-spot fast quantification that satisfies bacterium detects demand.
Described " fluorescent material ", the fluorescent dye that just is meant can be DAPI, FITC, fluorescence quantum or the like, any fluorescent dye can, but for different fluorescent dyes, used excitation source, optical filter, the technical parameter of photomultiplier may be not quite similar
Innovation part of the present invention:
The present invention does not at home and abroad also find similarly in the patent.
Abroad have and adopt filter method to carry out the bacterium patent of counting fast, but it is to estimate total number of bacteria by the change color of filtering membrane, and the present invention adopts the fluorescence light intensity to carry out count of bacteria, sensitivity and accuracy are higher; In addition, foreign patent is only protected with regard to the filtration fraction of front end, and the present invention has also protected the light path and the circuit part of rear end, and in the present invention, three parts constitute an intact device.
Claims (7)
1, a kind of device of fast quantitative bacteria detection comprises the sample filtering part, and sample filtering partly comprises chamber, filtering membrane, back up pad up and down, wherein, be up and down filtering membrane and back up pad between the chamber, filtering membrane and back up pad be arranged in parallel, and filtering membrane is affixed on support plate upper surface;
It is characterized in that, also comprise light path part and circuit part;
Light path part comprises light source, light source optical filter, saturating anti-mirror, fluorescent optical filter, wherein, on the light emitted light path, is sequentially with light source optical filter, saturating anti-mirror, and fluorescent optical filter is positioned at anti-mirror side, on the light path of saturating anti-mirror reflection light;
Circuit part, comprise display unit, signal processing unit, the keyboard input block, host computer and photomultiplier, wherein, display unit, keyboard input block, host computer and photomultiplier are electrically connected with signal processing unit respectively, the photomultiplier receiving end is positioned on the light path of anti-mirror reflection light, is used to detect the light intensity of the fluorescence that is excited;
During use, filtering membrane to be measured is positioned on the light path of source emissioning light, and the fluorescent material on the filtering membrane can be sent fluorescence after the optical excitation of light emitted.
2, the device of fast quantitative bacteria detection as claimed in claim 1 is characterized in that, described three parts: sample filtering part, light path part and circuit part are integrated into a portable apparatus.
3, the device of fast quantitative bacteria detection as claimed in claim 1 is characterized in that, described anti-mirror makes exciting light wave band light transmission, and makes the reflection of fluorescence band of light.
4, the using method of installing according to claim 1 is characterized in that step is: after testing sample is carried out the fluorescence labeling processing, filter; Take out filtering membrane; Irradiation leaves the filtering membrane through the fluorescence labeling bacterium, induces the fluorescent material that excites on the filtering membrane to send fluorescence; The fluorescence signal that sends is injected the photomultiplier receiving end, and photomultiplier is converted to electric signal with fluorescence signal and exports signal processing unit to; Send into display unit after the signal processing unit processes and show last bacterial number.
5, method as claimed in claim 4 is characterized in that, concrete steps are:
A). testing sample is carried out fluorescence labeling handle, select the filtering membrane in suitable aperture simultaneously as required;
B). the sample after the processing joins in the last chamber of sample filtering part, and pressurization makes sample solution flow through filtering membrane, back up pad, again through chamber outflow down; Stay the surface of filtering membrane in the sample through fluorescently-labeled bacterium, take out filtering membrane;
C). with b) filtering membrane to be measured that takes out of step places under the saturating anti-mirror of light path part, is positioned on the light path of source emissioning light;
D). open the light source of light path part, the light that light source sends obtains the exciting light of required wave band after the first light source optical filter filters, and exciting light is radiated on the filtering membrane through saturating anti-mirror, induces the fluorescent material that excites on the filtering membrane to send fluorescence;
E) .d) fluorescence that sends of step together with the exciting light that is reflected after saturating anti-mirror reflection, filter through second fluorescent optical filter again, only allow fluorescence signal to enter the photomultiplier receiving end, photomultiplier is converted to the signal processing unit that electric signal exports circuit part to fluorescence signal;
F). send into display unit after the signal processing unit of circuit part is handled the electric signal of photomultiplier output according to the indication of keyboard input block and show last bacterial number, or and upper machine communication, measurement result is sent in the database or network in the host computer.
6, method as claimed in claim 5 is characterized in that, described b) take out filtering membrane in the step, in last chamber, add the buffer solution elution filtering membrane, to reduce the background fluorescence on the filtering membrane, improve signal to noise ratio (S/N ratio).
7, as claim 4 or 5 described methods, it is characterized in that, finish to detection from obtaining sample, less than 30 minutes.
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| CN 200710179864 CN101464409B (en) | 2007-12-19 | 2007-12-19 | Apparatus and method for fast quantitative bacteria detection |
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| CN 200710179864 CN101464409B (en) | 2007-12-19 | 2007-12-19 | Apparatus and method for fast quantitative bacteria detection |
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Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102253090A (en) * | 2010-04-15 | 2011-11-23 | 艾力股份公司-卡皮贾尼集团 | Device for detecting bacterial charge in liquid or semi-liquid food product |
| CN103305404A (en) * | 2013-05-29 | 2013-09-18 | 陕西延长石油(集团)有限责任公司研究院 | Microporous cell for evaluating bacteria chemotaxis and evaluation method thereof |
| CN105424667A (en) * | 2015-12-14 | 2016-03-23 | 中国人民解放军军事医学科学院卫生装备研究所 | Rapid detection method for trace microorganisms |
| CN105548178A (en) * | 2014-10-23 | 2016-05-04 | 免疫制药公司 | Fluorescence reader of medical diagnostic kits |
| CN106308887A (en) * | 2016-08-30 | 2017-01-11 | 苏州品诺维新医疗科技有限公司 | Debridement water jet cutter and timeliness monitoring method |
| CN106399075A (en) * | 2016-08-26 | 2017-02-15 | 浙江科技学院 | Reflector based fluorescent quantitative PCR detection system |
| CN108474792A (en) * | 2015-11-20 | 2018-08-31 | 辛纳米拉股份公司 | Method and apparatus for detection bacterium |
| CN114904397A (en) * | 2021-02-09 | 2022-08-16 | 上海工程技术大学 | Method for measuring aperture and aperture distribution of filter membrane |
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2007
- 2007-12-19 CN CN 200710179864 patent/CN101464409B/en active Active
Cited By (13)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102253090B (en) * | 2010-04-15 | 2016-01-20 | 艾力股份公司-卡皮贾尼集团 | For detecting the device of bacteria content in liquid state or semi-fluid food |
| CN102253090A (en) * | 2010-04-15 | 2011-11-23 | 艾力股份公司-卡皮贾尼集团 | Device for detecting bacterial charge in liquid or semi-liquid food product |
| CN103305404A (en) * | 2013-05-29 | 2013-09-18 | 陕西延长石油(集团)有限责任公司研究院 | Microporous cell for evaluating bacteria chemotaxis and evaluation method thereof |
| CN103305404B (en) * | 2013-05-29 | 2014-11-26 | 陕西延长石油(集团)有限责任公司研究院 | Microporous cell for evaluating bacteria chemotaxis and evaluation method thereof |
| CN105548178A (en) * | 2014-10-23 | 2016-05-04 | 免疫制药公司 | Fluorescence reader of medical diagnostic kits |
| CN108474792A (en) * | 2015-11-20 | 2018-08-31 | 辛纳米拉股份公司 | Method and apparatus for detection bacterium |
| CN108474792B (en) * | 2015-11-20 | 2021-02-02 | 辛纳米拉股份公司 | Method and apparatus for detecting bacteria |
| CN105424667B (en) * | 2015-12-14 | 2019-02-05 | 中国人民解放军军事医学科学院卫生装备研究所 | Trace method for rapid inspecting animalcule |
| CN105424667A (en) * | 2015-12-14 | 2016-03-23 | 中国人民解放军军事医学科学院卫生装备研究所 | Rapid detection method for trace microorganisms |
| CN106399075A (en) * | 2016-08-26 | 2017-02-15 | 浙江科技学院 | Reflector based fluorescent quantitative PCR detection system |
| CN106308887A (en) * | 2016-08-30 | 2017-01-11 | 苏州品诺维新医疗科技有限公司 | Debridement water jet cutter and timeliness monitoring method |
| CN114904397A (en) * | 2021-02-09 | 2022-08-16 | 上海工程技术大学 | Method for measuring aperture and aperture distribution of filter membrane |
| CN114904397B (en) * | 2021-02-09 | 2023-09-19 | 上海工程技术大学 | Method for measuring pore diameter and pore diameter distribution of filter membrane |
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| CN101464409B (en) | 2013-10-30 |
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