CN101906384A - Instrument device and method for bacterial flow count detection - Google Patents
Instrument device and method for bacterial flow count detection Download PDFInfo
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- CN101906384A CN101906384A CN2009100858825A CN200910085882A CN101906384A CN 101906384 A CN101906384 A CN 101906384A CN 2009100858825 A CN2009100858825 A CN 2009100858825A CN 200910085882 A CN200910085882 A CN 200910085882A CN 101906384 A CN101906384 A CN 101906384A
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- 238000001514 detection method Methods 0.000 title claims abstract description 45
- 230000001580 bacterial effect Effects 0.000 title claims abstract description 8
- 238000000034 method Methods 0.000 title abstract description 24
- 241000894006 Bacteria Species 0.000 claims abstract description 65
- 239000000523 sample Substances 0.000 claims abstract description 34
- 239000002096 quantum dot Substances 0.000 claims abstract description 26
- 239000011521 glass Substances 0.000 claims abstract description 14
- 230000005284 excitation Effects 0.000 claims abstract description 10
- 230000035945 sensitivity Effects 0.000 claims abstract description 3
- 230000014670 detection of bacterium Effects 0.000 claims abstract 2
- 238000006243 chemical reaction Methods 0.000 claims description 18
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- 238000003199 nucleic acid amplification method Methods 0.000 claims description 11
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- 230000008878 coupling Effects 0.000 claims description 6
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- 230000003595 spectral effect Effects 0.000 claims description 6
- 238000004891 communication Methods 0.000 claims description 5
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- 239000003153 chemical reaction reagent Substances 0.000 claims description 3
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- 238000004364 calculation method Methods 0.000 claims 5
- 230000005540 biological transmission Effects 0.000 claims 1
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- 239000012488 sample solution Substances 0.000 claims 1
- 238000005516 engineering process Methods 0.000 abstract description 5
- 230000008901 benefit Effects 0.000 abstract description 2
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- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 4
- 230000001795 light effect Effects 0.000 description 4
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- 230000008859 change Effects 0.000 description 3
- 230000036541 health Effects 0.000 description 3
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- 238000002347 injection Methods 0.000 description 3
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- 238000011160 research Methods 0.000 description 2
- 238000001228 spectrum Methods 0.000 description 2
- 206010000087 Abdominal pain upper Diseases 0.000 description 1
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- 101001038142 Escherichia coli O157:H7 Glutamine synthetase adenylyl transferase Proteins 0.000 description 1
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- C12M41/00—Means for regulation, monitoring, measurement or control, e.g. flow regulation
- C12M41/30—Means for regulation, monitoring, measurement or control, e.g. flow regulation of concentration
- C12M41/36—Means for regulation, monitoring, measurement or control, e.g. flow regulation of concentration of biomass, e.g. colony counters or by turbidity measurements
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Abstract
本发明公开了一种细菌流动计数检测的仪表装置和方法，涉及生物医学检测技术，该仪表装置内置玻璃微沟道及流动系统，可使量子点探针标记过的细菌单个通过，结合量子点荧光激发技术及高精度微弱光检测技术，实现对细菌的特异性高灵敏度快速检测。该方法是，将被测样品与特异性的量子点探针偶联后，放入仪器检测进样口，细菌随液体流动通过玻璃微沟道检测区，由激光二极管激发诱导量子点标记的细菌发出荧光，使用光电倍增管检测荧光信号，根据检测到的发光强度及脉冲个数，计算出被测细菌数。本发明的仪表装置整个检测时间不超过30分钟，相对常规的培养计数法，具有特异性强，灵敏度高，检测速度快等优点。
The invention discloses an instrument device and method for bacterial flow count detection, which relates to biomedical detection technology. The instrument device has a built-in glass microchannel and a flow system, which can allow bacteria marked with quantum dot probes to pass through individually, combined with quantum dots Fluorescence excitation technology and high-precision weak light detection technology realize specific, high-sensitivity and rapid detection of bacteria. The method is that after the sample to be tested is coupled with a specific quantum dot probe, it is put into the detection inlet of the instrument, and the bacteria flow through the glass microchannel detection area with the liquid, and the bacteria labeled with the quantum dot are excited by the laser diode. Fluorescence is emitted, the fluorescent signal is detected by a photomultiplier tube, and the number of bacteria to be tested is calculated according to the detected luminous intensity and the number of pulses. The entire detection time of the instrument device of the present invention does not exceed 30 minutes, and has the advantages of strong specificity, high sensitivity, fast detection speed and the like compared with the conventional culture counting method.
Description
Technical field
The present invention relates to biomedical detection technique field, is instrumentation and method that a kind of bacterium flow count detects, can realize the specificity highly sensitive rapid detection to bacterium, is particularly useful for the pathogenic bacterium rapid detection.
Background technology
The pathogenic bacterium detection is the important content in public safety emergency case processing, biochemical anti-terrorism and the safe scouting field always.Its serious harm people's life and health causes heavy economic losses.The information of announcing according to the World Health Organization shows that the annual food origin disease that takes place in the whole world reaches billions of examples, the dead 0-15 of the annual diarrhoea that causes because of the food-borne pathogenic fungi pollution in whole world year children about 1,700,000.Even have at least 1/3 people suffer from food origin disease in developed country.About 7,600 ten thousand people of the U.S.'s annual generation food origin disease, wherein 5000 people's death.Annual infect the medical fee that produced and with a toll of 35,000,000,000 dollars by food origin disease.Some strong pathogenic bacterium such as anthrax, plague etc., as be used to the attack of terrorism and will cause bigger personal injury and loss.
Intestinal bacteria are the most general pathogenic bacterium, and HUMAN HEALTH is had great threat, and as Escherichia coli O 157: H7 at first was found in nineteen eighty-two, and 1999 at China's large-scale outbreak.Intestinal bacteria are ubiquitous bacteriums in people and the warm-blooded animal enteron aisle, are the main bacteria seed in the ight soil, and the symptom of people behind ehec infection is stomachache, vomiting, diarrhoea and heating.Infection may be fatefulue, especially to child and old man.In the U.S., the pathogenicity bo strain of 73000 people's ehec infections there is every year according to statistics, generally chemically examines needs about 24 hours and detect intestinal bacteria.And as the bacterium of anthrax and so on as being used to the attack of terrorism, then can cause immeasurable personal injury and loss, the a research report of being finished in 1993 by (United States Government) science and technology evaluation office shows: 100 kilograms of anthrax seedlings that an airplane is delivered---a kind of genus bacillus that is in dormant state can breeding rapidly in the human body body---can smog form appear at the quiet night sky, Washington D.C., produce toxin and make the poisoner that profuse bleeding take place rapidly, can seize 1,000,000 to 3,000,000 human lives at last.This is equivalent to 300 times of loss of life and personal injury quantity that 1000 kilograms of Schain poison gases of airplane delivery can cause.
Traditional bacteriologic test method is highly sensitive, and expense is low, can access the qualitative and quantitative result of aspect such as bacterial number and characteristic in the sample.But traditional detection method time and effort consuming, obtaining the result needs several days time usually, and the bacterial multiplication that requires to detect is visible bacterium colony.No matter daily life is still tackled Emergent Public Events such as the attack of terrorism, all presses for a kind of pathogenic bacterium detection method of fast high-sensitive degree.
All there is associated mechanisms to carry out the correlative study of bacterium method for quick both at home and abroad.Research method mainly contains ATP biloluminescence method, PCR method etc.But these methods generally all will be used the multiple instrument that comprises the pcr analysis instrument, and operation steps is comparatively loaded down with trivial details, is unfavorable for field quick detection.Some bacterium rapid detection apparatus based on the ATP biloluminescence method are arranged on the market, but the shortcoming of this method is that Bacteria Detection is not had specificity, only is used for the bacterial detection sum usually.
Summary of the invention
The invention provides instrumentation and method that a kind of bacterium flow count detects, this instrumentation, adopt the fluorescence quantum point mark method, little raceway groove of built-in glass and flow system, integrated laser shooting techniques and faint light detection technique, can realize specificity highly sensitive rapid detection, be particularly useful for the rapid detection of pathogenic bacterium bacterium.This method is used the tested bacterium sample of fluorescence quantum probe mark, and bacterium enters flow system, and is single by little raceway groove, under the exciting light effect, detect emission light light intensity, it is finished, and one-time detection is the shortest to be no more than 15 minutes, compares with existing culture method to have very big advantage.
For achieving the above object, technical solution of the present invention is:
The instrumentation that a kind of bacterium flow count detects, it comprises master control computing unit, analog to digital conversion signal treatment circuit, signal amplification circuit, Drive and Control Circuit, photomultiplier, laser diode, spectral filter and little raceway groove and flow system; Wherein, the master control computing unit respectively with analog to digital conversion signal treatment circuit, Drive and Control Circuit, demonstration, storage, communicate by letter, button control function module and power module directly be electrically connected; Drive and Control Circuit and laser diode, photomultiplier directly is electrically connected; The photomultiplier order is electrically connected with master control computing unit input terminus through signal amplification circuit, analog to digital conversion signal treatment circuit;
Laser diode places little raceway groove top, is used to produce exciting light, and photomultiplier is located at little raceway groove side, detects emission light every spectral filter; Little raceway groove is vertical with laser diode, photomultiplier respectively;
The external flow system of little raceway groove interlinks with flow system, and for the sample flow of solution provides power by little raceway groove, flow system is electrically connected with Drive and Control Circuit, controlled by the master control computing unit.
Described instrumentation, the peak wavelength of its described laser diode in the 300-450 nanometer range, wave spectrum width 10-20 nanometer; Or be peak wavelength in the 300-450 nanometer range, the photodiode of wave spectrum width 10-20 nanometer.
Described instrumentation, it is provided with condensing lens or optical fiber between laser diode and little raceway groove, to improve the launching efficiency of exciting light.
Described instrumentation, its described spectral filter between photomultiplier and little raceway groove has the 1-2 sheet, is used for the interference that the filtering exciting light detects emission light; Between little raceway groove and photomultiplier, condensing lens is set, improves photomultiplier radiative detection efficiency.
Described instrumentation, its described little raceway groove is the little raceway groove of glass, its diameter is the 10-20 micron, can guarantee that bacterium passes through smoothly, can make its single as far as possible passing through again, improves detection sensitivity.
A kind of detection method of described instrumentation, it uses the tested bacterium sample of fluorescence quantum probe mark, bacterium enters flow system, single by little raceway groove, luminous under the exciting light effect of laser diode, photomultiplier detects emission light light intensity, realizes the specificity highly sensitive rapid detection to bacterium.
Described detection method, it comprises step:
A) bacterium that detects as required, choose the antibody probe of specific quantum dot-labeled mistake, the antibody probe coupling of tested bacteria samples and quantum dot-labeled mistake, the reaction times is 5-30 minute, suitably adjust the reaction times according to temperature, reaction finishes the back and adds the apparatus injection port;
B) drive the good bacteria samples of quantum dot probe mark by little raceway groove by the master control computing unit through the drive control circuit flow system;
C) through 3-10 second, treat the sample flow velocity-stabilization after, start the laser diode launching excitation light by the master control computing unit through Drive and Control Circuit, start photomultiplier by the master control computing unit through Drive and Control Circuit simultaneously and detect the emission optical signal;
D) current signal of photomultiplier output through signal amplification circuit, modulus signal change over signal Acquisition Circuit, converts digital voltage signal to, feeds back to the master control computing unit;
E) the master control computing unit has judged whether that in conjunction with pre-defined algorithm bacterium passes through according to signal magnitude, and the number of bacteria that statistics is passed through calculates tested bacterium number, and storage, demonstration and the communications of control test result.
Described detection method, its described A) in the step, the antibody probe of quantum dot-labeled mistake is to utilize quantum dot and need the specific antibody coupling of tested bacterium to make, and cooperates instrument to use as reaction reagent.
Described detection method, its described C), D) in two steps, be 2-10 minute used detection time.
The present invention adopts fluorescence quantum dot marking method and flow count method, realizes bacterium highly sensitive specificity rapid detection, the shortlyest can in 15 minutes, finish one-shot measurement, obtain reliable results, compare with existing culture method, easy to operate, shorten detection time greatly.
Description of drawings
Fig. 1 is bacterium rapid detection instrumentation structure of the present invention and FB(flow block);
Fig. 2 is the bacterium rapid detection process synoptic diagram of the inventive method;
Fig. 3 is the bacterium rapid detection schematic diagram of the inventive method.
Embodiment
Describe the present invention with reference to the accompanying drawings in detail.
As shown in Figure 1, little raceway groove 8 of the built-in glass of this highly sensitive bacterium specificity device for fast detecting and flow system 14 also comprise master control computing unit 1, analog to digital conversion signal treatment circuit 11, signal amplification circuit 10, Drive and Control Circuit 12, photomultiplier 9, laser diode 7 and spectral filter 13; Wherein, master control computing unit 1 respectively with analog to digital conversion signal treatment circuit 11, Drive and Control Circuit 12, show 3, storage 4, communicate by letter 6, button control function module 5 and power module 2 directly be electrically connected; Drive and Control Circuit 12 and laser diode 7, photomultiplier 9 directly is electrically connected; Photomultiplier transit 9 pipes order is electrically connected with master control computing unit 1 input terminus through signal amplification circuit 10, analog to digital conversion signal treatment circuit 11.
Laser diode 7 places the little raceway groove of glass 8 tops, is used to produce exciting light, and photomultiplier 9 is located at the little raceway groove of glass 8 sides, is used for detecting emission light, and the little raceway groove 8 of glass is vertical with photomultiplier 9 planes with laser diode 7.
The little raceway groove 8 external flow systems 14 of glass are for the sample flow of solution provides power by the little raceway groove of glass.Flow system 14 is electrically connected with Drive and Control Circuit 12, is subjected to 1 control of master control computing unit.
When the bacterium of quantum dot-labeled mistake flows through the little raceway groove 8 of glass, under the exciting light effect that laser diode 7 sends, produce stronger emission optical signal, photomultiplier 9 detects the emission optical signal, converts electrical signal output to, through signal amplification circuit 10, analog to digital conversion signal treatment circuit 11 converts numerary signal to, is input to master control computing unit 1, and master control computing unit 1 has judged whether that according to preset algorithm tested bacterium passes through, statistical counting draws detected result.
As shown in Figure 2, the process that this device carries out bacterium specificity rapid detection has been described.The bacterium of Jian Ceing at first as required, choose the antibody probe of specific quantum dot-labeled mistake, the antibody probe coupling of tested bacteria samples and quantum dot-labeled mistake, the reaction times is 5-30 minute, suitably adjust the reaction times according to temperature, reaction finishes the back and adds the apparatus injection port.Drive the good bacteria samples of quantum dot probe mark by the little raceway groove of glass by the master control computing unit through the drive control circuit flow system.Through 3-10 second, treat the sample flow velocity-stabilization after, start the laser diode launching excitation light by the master control computing unit through Drive and Control Circuit, start photomultiplier by the master control computing unit through Drive and Control Circuit simultaneously and detect the emission optical signal.The current signal of output through signal amplification circuit, modulus signal change over signal Acquisition Circuit, converts digital voltage signal to, feeds back to the master control computing unit.The master control computing unit has judged whether that in conjunction with pre-defined algorithm bacterium passes through according to signal magnitude, and the number of bacteria that statistics is passed through calculates tested bacterium number, and storage, demonstration and the communications of control test result.
The concrete steps of the inventive method are:
A) bacterium that detects as required, choose the antibody probe of specific quantum dot-labeled mistake, the antibody probe coupling of tested bacteria samples and quantum dot-labeled mistake, the reaction times is 5-30 minute, suitably adjust the reaction times according to temperature, reaction finishes the back and adds the apparatus injection port;
B) drive the good bacteria samples of quantum dot probe mark by little raceway groove by the master control computing unit through the drive control circuit flow system;
C) through 3-10 second, treat the sample flow velocity-stabilization after, start the laser diode launching excitation light by the master control computing unit through Drive and Control Circuit, start photomultiplier by the master control computing unit through Drive and Control Circuit simultaneously and detect the emission optical signal;
D) current signal of photomultiplier output through signal amplification circuit, modulus signal change over signal Acquisition Circuit, converts digital voltage signal to, feeds back to the master control computing unit;
E) the master control computing unit has judged whether that in conjunction with pre-defined algorithm bacterium passes through according to signal magnitude, and the number of bacteria that statistics is passed through calculates tested bacterium number, and storage, demonstration and the communications of control test result.
At A) in the step, the antibody probe of quantum dot-labeled mistake is to utilize quantum dot and need the specific antibody coupling of tested bacterium to make, and cooperates instrument to use as reaction reagent.
At C), D) in two steps, be 2-10 minute used detection time.
As shown in Figure 3, the principle of work that this device carries out bacterium specificity rapid detection has been described.Bacterium that need be detected combines with quantum dot-labeled antibody probe specificity, when flowing through the little raceway groove of glass, under the exciting light effect that laser diode sends, can produce stronger emission optical signal, by detecting the emission optical signal, judged whether that tested bacterium passes through, statistical counting draws detected result.
Claims (8)
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Cited By (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2013016873A1 (en) * | 2011-08-04 | 2013-02-07 | Zhang Hongpeng | Ship domestic sewage detection device |
| CN103512841A (en) * | 2012-06-22 | 2014-01-15 | 纬创资通股份有限公司 | Biological sample detection device and biological sample detection method |
| CN103917859A (en) * | 2011-11-16 | 2014-07-09 | 索尼公司 | Biometric device, biometric method, program, and recording medium |
| CN104619828A (en) * | 2012-08-24 | 2015-05-13 | 株式会社佐竹 | Method for examining microorganism and device for same |
| CN105424662A (en) * | 2015-11-24 | 2016-03-23 | 江南大学 | Fluorescent biological 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 |
| CN109370895A (en) * | 2018-11-15 | 2019-02-22 | 湖北华龙西科生物科技有限公司 | A kind of liquid fermentation can system and microorganism Intelligent fermentation |
| CN109374584A (en) * | 2018-08-27 | 2019-02-22 | 九江精密测试技术研究所 | A kind of ballast water for ship biological effectiveness real time on-line monitoring device |
| CN109370894A (en) * | 2018-11-15 | 2019-02-22 | 湖北华龙西科生物科技有限公司 | A kind of Horizontal type solid fermentation can system and microorganism Intelligent fermentation |
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| CN1376800A (en) * | 2001-03-23 | 2002-10-30 | 广东省微生物研究所 | Counting card for bacteria, funguses and caliform floras and its preparing process |
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Cited By (11)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2013016873A1 (en) * | 2011-08-04 | 2013-02-07 | Zhang Hongpeng | Ship domestic sewage detection device |
| CN103917859A (en) * | 2011-11-16 | 2014-07-09 | 索尼公司 | Biometric device, biometric method, program, and recording medium |
| CN103512841A (en) * | 2012-06-22 | 2014-01-15 | 纬创资通股份有限公司 | Biological sample detection device and biological sample detection method |
| CN103512841B (en) * | 2012-06-22 | 2015-12-02 | 纬创资通股份有限公司 | Biological sample detection device and biological sample detection method |
| CN104619828A (en) * | 2012-08-24 | 2015-05-13 | 株式会社佐竹 | Method for examining microorganism and device for same |
| US9915601B2 (en) | 2012-08-24 | 2018-03-13 | Satake Corporation | Method for examining microorganisms and examination apparatus for microorganisms |
| CN105424662A (en) * | 2015-11-24 | 2016-03-23 | 江南大学 | Fluorescent biological 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 |
| CN109374584A (en) * | 2018-08-27 | 2019-02-22 | 九江精密测试技术研究所 | A kind of ballast water for ship biological effectiveness real time on-line monitoring device |
| CN109370895A (en) * | 2018-11-15 | 2019-02-22 | 湖北华龙西科生物科技有限公司 | A kind of liquid fermentation can system and microorganism Intelligent fermentation |
| CN109370894A (en) * | 2018-11-15 | 2019-02-22 | 湖北华龙西科生物科技有限公司 | A kind of Horizontal type solid fermentation can system and microorganism Intelligent fermentation |
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| CN101906384B (en) | 2013-03-27 |
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