CN101464409B - Apparatus and method for fast quantitative bacteria detection - Google Patents
Apparatus and method for fast quantitative bacteria detection Download PDFInfo
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- CN101464409B CN101464409B CN 200710179864 CN200710179864A CN101464409B CN 101464409 B CN101464409 B CN 101464409B CN 200710179864 CN200710179864 CN 200710179864 CN 200710179864 A CN200710179864 A CN 200710179864A CN 101464409 B CN101464409 B CN 101464409B
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- 230000014670 detection of bacterium Effects 0.000 claims abstract 2
- 238000001914 filtration Methods 0.000 claims description 46
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- 238000001215 fluorescent labelling Methods 0.000 claims description 5
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Abstract
本发明一种快速定量检测细菌的装置及方法。首先采用免疫技术将荧光物质(如FITC，荧光量子点等)标记在样品中的细菌上，让样品溶液流过一套过滤装置，使标记有荧光物质的细菌留在过滤膜的表面，采用激光诱导激发荧光使标记在细菌上的荧光物质发射出荧光，通过检测过滤膜上总荧光信号强度获得对样本中细菌的快速准确计数，整个检测时间小于30分钟。
The invention relates to a device and method for rapid quantitative detection of bacteria. First, fluorescent substances (such as FITC, fluorescent quantum dots, etc.) are marked on the bacteria in the sample by immunological technology, and the sample solution flows through a set of filter devices, so that the bacteria marked with fluorescent substances remain on the surface of the filter membrane. The induced excitation fluorescence causes the fluorescent substances marked on the bacteria to emit fluorescence, and the rapid and accurate count of bacteria in the sample is obtained by detecting the total fluorescence signal intensity on the filter membrane, and the entire detection time is 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 occured in China in 1999 in recent years in succession popular in the country such as the U.S., Japan, Britain, Germany in outburst.
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 to be carried out in the laboratory multinomial numerous and diverse analytical test and preparation nutrient culture media, owing to need to cultivate, required time is long, must need specialized equipment to count, and owing to there is not specificity, can not distinguish the close pathogenic bacteria of form.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 kit or strip to sell on the market, mainly be to come the quantity of bacterium in the sample estimates by the change color of contrast strip, although this method detection speed is fast, but sensitivity is low, is difficult to realize quantitative detection.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 Luminescence photography that utilizes carries out the total number of bacteria detection, but the 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 up and down chamber, filtering membrane, back up pad, 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 source emissioning light road, is sequentially with light source optical filter, saturating anti-mirror, and fluorescent optical filter is positioned at anti-mirror side, on the catoptrical light path of saturating anti-mirror;
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 catoptrical light path of anti-mirror, for detection of 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, sends fluorescence after the optical excitation that the fluorescent material on the filtering membrane can be launched by light source.
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 processes; 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 process, select as required the filtering membrane in suitable aperture simultaneously;
B). the sample after the processing joins in the upper chamber of sample filtering part, and pressurization makes sample solution flow through filtering membrane, back up pad, flows out through lower chamber again; 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 behind the Electric signal processing of indication with photomultiplier output of the signal processing unit of circuit part according to the 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 upper 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 was complete to detecting 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, and complete to detecting from obtaining sample, less than 30 minutes, and traditional detection method needed 24～48 hours at least; Compare with present method for quick, detection sensitivity of the present invention is high, has specificity, quantitative accurate counting, and present method for quick generally all is qualitative detection.
Description of drawings
Fig. 1 is sample filtration device figure 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 up and down two cells 1,4, is up and down filtering membrane 2 and back up pad 3 between the chamber 1,4, 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 processing through fluorescence labeling joins in the upper chamber 1, makes sample solution flow through filtering membrane 2, back up pad 3 by exerting pressure, and flows out through lower chamber 4 again.Select as required the filtering membrane 2 in suitable aperture, stay the surface of filtering membrane 2 in the sample through fluorescently-labeled bacterium, and unnecessary fluorescent dye flows out through lower chamber 4 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 upper chamber 1, add again buffer solution elution filtering membrane 2, to reduce the background fluorescence on the filtering membrane 2, improve signal to noise ratio (S/N ratio).
As shown in Figure 2, light path part is the interior part of dotted line frame 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 behind the Electric signal processing of indication with photomultiplier B5 output of signal processing unit B2 according to 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, complete to detecting 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 Quantitative detection by the induced with laser fluorescence excitation, owing to adopted immunological technique so that testing process has 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, satisfies the on-the-spot Quantitative detection demand to bacterium.
Described " fluorescent material ", the fluorescent dye that just refers to can be DAPI, FITC, fluorescence quantum etc., 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 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 patent of bacterium quick counter, but it is to estimate total number of bacteria by the change color of filtering membrane, and the present invention adopts fluorescent 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 light path and the circuit part of rear end, and in the present invention, three parts consist of an intact device.
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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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| Publication number | Priority date | Publication date | Assignee | Title |
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| IT1400195B1 (en) * | 2010-04-15 | 2013-05-24 | Carpigiani Group Ali Spa | BACTERIAL CHARGE CONTROL DEVICE FOR A LIQUID OR SEMILIQUID FOOD PRODUCT. |
| 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 |
| KR101621050B1 (en) * | 2014-10-23 | 2016-05-13 | 주식회사 이뮨메드 | Fluorescence reader of medical diagnostic kits |
| DK3171172T3 (en) * | 2015-11-20 | 2018-11-05 | Sinamira AG | 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 |
| CN106399075B (en) * | 2016-08-26 | 2019-05-03 | 浙江科技学院 | A kind of detection system of the quantitative fluorescent PCR based on reflecting mirror |
| 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 |
| CN114904397B (en) * | 2021-02-09 | 2023-09-19 | 上海工程技术大学 | A method for measuring filter membrane pore size and pore size distribution |
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