CN104313114A - Detection kit and detection method for escherichia coli - Google Patents
Detection kit and detection method for escherichia coli Download PDFInfo
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- CN104313114A CN104313114A CN201410586920.6A CN201410586920A CN104313114A CN 104313114 A CN104313114 A CN 104313114A CN 201410586920 A CN201410586920 A CN 201410586920A CN 104313114 A CN104313114 A CN 104313114A
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Abstract
The invention discloses a detection kit and a detection method for escherichia coli, and belongs to the technical field of biological detection. The detection kit comprises lauryl trypsin culture solution, enzyme substrate solution, an enzyme inducing agent and cell lysis buffer solution, wherein the lauryl trypsin culture solution comprises the following components: trypsin, lactose, sodium chloride, dipotassium phosphate, monopotassium phosphate and sodium lauryl sulfate. The detection method is used for carrying out real-time detection through match of the detection kit and a fluorescence meter. The detection kit and the detection method have the advantage that the escherichia coli can be accurately and rapidly detected.
Description
Technical field
The invention belongs to technical field of biological, be specifically related to a kind of colibacillary detection kit and detection method thereof.
Background technology
Intestinal bacteria (E. Coli) are one way of lifes on the bacterium of the mankind or animal digestive tract, and it has broad variety, and wherein great majority are harmless.But some may cause bloody diarrhea.The most well-known intestinal bacteria type is O157:H7 type, and this bacterial classification can produce strong toxin, may cause severe anemia or renal failure, even dead.Therefore detect intestinal bacteria tool to be accurately and rapidly of great significance.
Traditional culture method detects intestinal bacteria and there is the cycle long (more than at least 48 hours), operate the shortcomings such as loaded down with trivial details, sensitivity is low, easy pollution, program are complicated and required reagent (biochemical test reagent and serum) is various, the colibacillary requirement of rapid detection can not be met far away.
Summary of the invention
the technical problem solved:the object of the invention is to overcome traditional culture method detect the deficiency of intestinal bacteria technology and provide one to detect colibacillary detection kit and detection method thereof accurately and rapidly.
in order to realize foregoing invention object, technical scheme of the present invention is as follows:
The invention discloses a kind of colibacillary detection kit, described test kit comprises bay Trypsin nutrient solution, enzyme substrate solution, enzyme inducer and cell lysis buffer solution, the composition of described bay Trypsin nutrient solution comprises: Trypsin, lactose, sodium-chlor, dipotassium hydrogen phosphate, potassium primary phosphate, sodium lauryl sulphate, described enzyme inducer is isopropyl ss-D-1-thiogalactoside (IPTG), described enzyme substrate solution is 4-methyl umbelliferone-B-D-glucuronide, and described cell lysis buffer solution is Hen egg-white lysozyme (HEWL).
Each constituent concentration of described bay Trypsin nutrient solution is respectively:
Trypsin: 20.0g/L
Lactose: 5.0g/L
Sodium-chlor: 5.0g/L
Dipotassium hydrogen phosphate: 2.75g/L
Potassium primary phosphate: 2.55g/L
Sodium lauryl sulphate: 0.1g/L.
The pH value of described bay Trypsin nutrient solution is 6.6 ~ 7.0.
Described test kit also comprises sterilized water.
The volume content of described bay Trypsin nutrient solution, enzyme substrate solution, enzyme inducer, cell lysis buffer solution, sterilized water is as follows:
Bay Trypsin nutrient solution 1 milliliter/pipe
Enzyme substrate solution 1 milliliter/pipe
Enzyme inducer 1 milliliter/pipe
Cell lysis buffer solution 1 milliliter/pipe
Sterilized water 1 milliliter/pipe.
Apply above-mentioned colibacillary detection kit to carry out colibacillary detection method one and comprise step:
Step one, get a Boiling tube, and instill 0.5 ml sterile water wherein, and instill 1 enzyme inducer;
Step 2, get a cotton swab, with the sampling of cotton swab end, and be placed in above-mentioned Boiling tube, be mixed to form liquid to be measured;
Step 3, get a small test tube, instill 3 cell lysis buffer solution within it;
Step 4, get 200 microlitre drop to be measured with micropipette rifle or disposable liquid transfer gun head from above-mentioned Boiling tube and enter small test tube, fully mix, sealing, and wait for 5 ~ 10 minutes at ambient temperature;
Step 5, in above-mentioned small test tube instill 2 enzyme substrate solutions, fully mix;
Step 6, get 200 microlitre drop to be measured enter fluoroscopic examination sample tube with micropipette rifle or disposable liquid transfer gun head from above-mentioned small test tube, sealing, waits for 3 ~ 5 minutes at ambient temperature;
Step 7, sample test fluoroscopic examination sample tube being placed in luminoscope are indoor, and seal;
Open the test pattern of luminoscope, record first time test number P
1;
Wait for 20 minutes, again test, record second time test number P
2;
If test value P
2-P
1> 60, then sample is positive;
If test value P
2-P
1< 60, then sample is negative;
If test value P
2-P
1close to 60, then wait for 20 ~ 60 minutes again, again test, obtain new P
2;
Described enzyme inducer is isopropyl ss-D-1-thiogalactoside (IPTG), and described cell lysis buffer solution is Hen egg-white lysozyme (HEWL), and described enzyme substrate solution is 4-methyl umbelliferone-B-D-glucuronide.
Apply above-mentioned colibacillary detection kit to carry out colibacillary detection method two and comprise step:
Step one, get a Boiling tube, and instill 0.5 milliliter of above-mentioned bay Trypsin nutrient solution wherein, and instill 1 enzyme inducer;
Step 2, get a cotton swab, with the sampling of cotton swab end, and be placed in above-mentioned Boiling tube, be mixed to form liquid to be measured;
Step 3, the thermostat container above-mentioned liquid to be measured being placed in 38.5 DEG C are cultivated 3 ~ 5 hours;
Step 4, get a small test tube, instill 3 cell lysis buffer solution within it;
Step 5, get 200 microlitre drop to be measured with micropipette rifle or disposable liquid transfer gun head from above-mentioned Boiling tube and enter small test tube, fully mix, sealing, and wait for 5 ~ 10 minutes at ambient temperature;
Step 6, in above-mentioned small test tube instill 2 enzyme substrate solutions, fully mix;
Step 7, get 200 microlitre drop to be measured enter fluoroscopic examination sample tube with micropipette rifle or disposable liquid transfer gun head from above-mentioned small test tube, sealing, waits for 3 ~ 5 minutes at ambient temperature;
Step 8, sample test fluoroscopic examination sample tube being placed in luminoscope are indoor, and seal;
Open the test pattern of luminoscope, record first time test number P
1;
Wait for 20 minutes, again test, record second time test number P
2;
If test value P
2-P
1> 80, then sample is positive;
If test value P
2-P
1< 80, then sample is negative;
If test value P
2-P
1close to 80, then wait for 20 ~ 60 minutes again, again test, obtain new P
2.
The pH value of described bay Trypsin nutrient solution is 6.6 ~ 7.0.
Described enzyme inducer is isopropyl ss-D-1-thiogalactoside (IPTG), and described cell lysis buffer solution is Hen egg-white lysozyme (HEWL), and described enzyme substrate solution is 4-methyl umbelliferone-B-D-glucuronide.
Colibacillary detection kit of the present invention utilizes the feature biological enzyme existed in intestinal bacteria to be detected, is hydrolyzed the substrate of this enzyme and produces fluorescence, and recycling hand-held fluorescence instrument reads fluorescent value and quick and precisely identifies this colibacillary existence.
Beneficial effect of the present invention is:
Escherichia coli detection kit of the present invention detects sample, only needs 1 hour (not needing culturing bacterium situation) or about 6 hours (needing culturing bacterium situation) to complete detection, and method is simple, and accuracy is high; Simultaneously, adopt fluoroscopic examination intestinal bacteria specificity comparatively strong, effectively overcome the cycle existed in intestinal bacteria culture-based method long, operate the shortcomings such as loaded down with trivial details, sensitivity is low, easy pollution, program are complicated and required reagent (biochemical test reagent and serum) is various.
Accompanying drawing explanation
Fig. 1 is the schema that the present invention detects the detection method of colibacillary embodiment;
Fig. 2 the present invention detects the schematic diagram of the internal module of colibacillary embodiment hand-held fluorescence instrument used;
Fig. 3 is the optical module structure schematic diagram of Fig. 2;
Wherein, light source 1
Optical module 2
Lens 1
Spectral filter 1
Diaphragm 1
Diaphragm 2 2.4
Lens 2 2.5
Spectral filter 2 2.6
Spectral filter 3 2.7
Lens 3 2.8
Optical detector 3
Analysis and processing module 4
Cpu circuit 4.1
Amplifier 1
Amplifier 2 4.3
D/A converting circuit 4.4
Touch screen circuitry 4.5
Display circuit 4.6
USB circuit 4.7
Sample tube 5.
Embodiment
Now describe the present invention more fully hereinafter with reference to accompanying drawing, exemplary embodiment of the present invention shown in the drawings, thus scope of the present invention is conveyed to those skilled in the art by the disclosure fully.But the present invention can realize in many different forms, and should not be interpreted as being limited to the embodiment set forth here.
The embodiment of the present invention is developed and is provided a kind of colibacillary detection kit, this test kit comprises bay Trypsin nutrient solution, enzyme substrate solution, enzyme inducer, cell lysis buffer solution and sterilized water, wherein, the composition of bay Trypsin nutrient solution comprises: Trypsin, lactose, sodium-chlor, dipotassium hydrogen phosphate, potassium primary phosphate, sodium lauryl sulphate, enzyme inducer adopts isopropyl ss-D-1-thiogalactoside (IPTG), enzyme substrate solution is 4-methyl umbelliferone-B-D-glucuronide, using Hen egg-white lysozyme (HEWL) as cell lysis buffer solution.
The volume content of above-mentioned bay Trypsin nutrient solution, enzyme substrate solution, enzyme inducer, cell lysis buffer solution, sterilized water is preferably as follows:
Bay Trypsin nutrient solution 25 milliliters/pipe
Enzyme substrate solution 1 milliliter/pipe
Enzyme inducer 1 milliliter/pipe
Cell lysis buffer solution 1 milliliter/pipe
Sterilized water 1 milliliter/pipe.
The colibacillary detection kit of the present invention has the advantages such as quick, sensitive, safe.
Preferably, as embodiments of the invention, each constituent concentration of above-mentioned bay Trypsin nutrient solution is respectively:
Trypsin: 20.0g/L
Lactose: 5.0g/L
Sodium-chlor: 5.0g/L
Dipotassium hydrogen phosphate: 2.75g/L
Potassium primary phosphate: 2.55g/L
Sodium lauryl sulphate: 0.1g/L.
The embodiment of the present invention further provides a kind of colibacillary detection method---fluorescence detection method.Fluoroscopic examination has been widely used in the industries such as biology, chemistry, medicine, food safety, it is biological DNA detection and sequence, chemical molecular and material component identification, medical science cancer cells diagnose in requisite detection means.For this detection method, the invention provides a hand-held luminoscope, use the shortwave of 360nm, the front of this luminoscope is provided with sample test room with cover and touching display screen, a fluoroscopic examination sample tube 5 vertically placed can be held, as shown in Figures 2 and 3, during detection in sample test indoor, hold the fluoroscopic examination sample tube 5 detecting liquid and insert sample test indoor, cover lid can be tested.
Particularly, hand-held fluorescence instrument includes light source 1, optical module 2, optical detector 3 and analysis and processing module 4, as shown in Figure 2.Light source 1 by LED as excitation light source, wherein light source 1 is placed in optical module 2 front, optical detector 3 is placed in optical module 2 rear, during detection, sample tube 5 is placed in optical module 2, the light that light source 1 sends is received by optical detector 3 after optical module 2, and optical detector 3 is connected with analysis and processing module 4, the fluorescent signal detected is transferred to analysis and processing module 4 and analyzes.
Optical module 2 is made up of excitation module and receiver module, as shown in Figure 3, the confocal setting of optical system of excitation module and receiver module, wherein excitation module is disposed with lens 1, spectral filter 1 and diaphragm 1, receiver module is disposed with diaphragm 2 2.4, lens 2 2.5, spectral filter 2 2.6, spectral filter 3 2.7 and lens 3 2.8, and lens 1 are placed in light source 1 front, lens 3 2.8 are placed in optical detector 3 front.
The light sent by excitation light source LED converges in sample tube 5 via lens 1, exciting light arrives to be needed before sample first by a short logical or bandpass filter 1 and diaphragm 1, the effect of spectral filter 1 is filtered the part overlapped with fluorescent spectral component in exciting light, only through the spectral component that effectively can excite sample; The effect of diaphragm 1 spatially retrains exciting light, avoids excitation light irradiation in the larger scope of test tube wall, thus produce scattered light and impact measuring result.
Under exciting light effect, the fluorescence sent by sample, via after diaphragm 2 2.4, lens 2 2.5, spectral filter 2 2.6, spectral filter 3 2.7 and lens 3 2.8, finally arrives detector 3; The effect of diaphragm 2 2.4 is got rid of the scattered light be derived from test tube wall and fluorescence, only by being derived from the fluorescence of sample, lens 2 2.5 and lens 1 form the confocal system that a folding turn 90 degrees, their focuses overlap in sample, thus reach the collection of high efficiency fluorescent component, fluorescence after lens 2 2.5 collimate is more successively by spectral filter 2 2.6 and spectral filter 3 2.7, its effect is removed by exciting light, and only through fluorescent component, long pass filter or bandpass filter can be selected; Fluorescence through spectral filter 2 2.6 and spectral filter 3 2.7 converges on detector 3 by lens 3 2.8, and the Signal transmissions detected processes to analysis and processing module 4 by detector 3.
Analysis and processing module 4 includes cpu circuit 4.1, amplifier 1, amplifier 2 4.3, D/A converting circuit 4.4, touch screen circuitry 4.5, display circuit 4.6 and USB circuit 4.7, as shown in Figure 2, optical detector 3 is connected with cpu circuit 4.1 through amplifier 1, described cpu circuit 4.1 is connected with amplifier 2 4.3 through D/A converting circuit 4.4, described amplifier 2 4.3 is connected with light source 1, light source 1 is regulated and controled, described touch screen circuitry 4.5, display circuit 4.6 are all connected with cpu circuit 4.1 with USB circuit 4.7, complete corresponding good in interactive function.
This hand-held fluorescence instrument, because lens 2 2.5 and lens 1 form a confocal system, thus the collection that the fluorescence that sample is sent can be maximized; Sample and detector 3 are respectively relative to lens 2 2.5 and lens 3 2.8 symmetrically structure simultaneously, and be all in the focus of lens, this structure can ensure that the fluorescence that sample sends is parallel through spectral filter 2 2.6 and spectral filter 3 2.7.Thus eliminating the need because input angle difference can bring interference filter to lead to the shortcoming of optical wavelength displacement, improve accuracy of detection.
In addition, when sample at lower concentration, when only having the bacterium of several CFU, fluorescent emission may be atomic weak, and the interference of external environmental light will be extremely serious, now, this hand-held fluorescence instrument is modulated light source row by analysis and processing module, open and close the intensity of fluorescent signal under two states by contrast LED, draw the true intensity of fluorescent signal and there is no the interference of surround lighting, improve accuracy of detection.
Use, the embodiment of the present invention further provides the colibacillary detection method of a kind of detection, and this detection method carries out the schema detected, and as shown in Figure 1, comprises the steps:
Step S01: Zengjing Granule and sample disposal: provide and detect colibacillary sample;
Step S02: real-time fluorescence detects: carrying out detecting in real time needs to use above-mentioned colibacillary detection kit and hand-held fluorescence instrument.
Particularly, step S01 aseptically operates.
1) get a Boiling tube, and instill 0.5 milliliter of above-mentioned bay Trypsin nutrient solution wherein, and instill 1 enzyme inducer;
2) from test kit, get a cotton swab, use the surface of cotton swab end wiping test zone to collect sample, be placed in above-mentioned Boiling tube by the cotton swab end being moistened with sample, sealing, rocks gently, the sample that cotton swab is collected and liquid mixing, forms liquid to be measured;
3) 3 ~ 5 hours (being no more than 12 hours, was good with 5 hours) cultivated by thermostat container liquid to be measured being placed in 38.5 DEG C, to ensure to produce the enzyme required for enough detection intestinal bacteria.
And then carry out the cracking process of following sample:
4) small test tube (stirring) got in test kit, and toward interior instillation 3 cell lysis buffer solution;
5) get 200 microlitre drop to be measured with (replaceable) micropipette rifle or disposable liquid transfer gun head from Boiling tube and enter small test tube, sealing, rocks gently or in small test tube, repeatedly aspirates 8 times to reach abundant mixing effect, waiting for 5 ~ 10 minutes at ambient temperature;
6) instill 2 enzyme substrate solutions toward small test tube, rock gently;
7) get 200 microlitre drop to be measured with micropipette rifle or disposable liquid transfer gun head from small test tube and enter fluoroscopic examination sample tube, sealing, waits for 3 ~ 5 minutes at ambient temperature.
Step S02: real-time fluorescence detects.
8) get the sample test room that the fluoroscopic examination sample tube obtained in step S01 is placed in luminoscope, build lid, before carrying out this step, the outer wall of application lint-free cloth or lens wipe paper wiped clean fluoroscopic examination sample tube, and guarantee to manage interior bubble-free;
Open the test pattern of luminoscope, record first time test number P
1;
Wait for 20 minutes, again test, record second time test number P
2;
If test value P
2-P
1> 80, then sample is positive; Otherwise be negative;
If test value P
2-P
1close to 80, then wait for 20 minutes, again test, obtain new P
2;
Described P
2numerical value in 20 ~ 60 minutes effectively.
In waiting time section between first time test and second time are tested, fluoroscopic examination sample tube can be taken out and be placed in environment or incubator.
In above-mentioned steps S01, collecting sample, may contain intestinal bacteria in this sample, also may not contain intestinal bacteria, the detection method of the embodiment of the present invention to carry out detection and determines.If clearly know, cotton swab is moistened with in the sample of sample contains intestinal bacteria, then cotton swab directly can being placed in the Boiling tube of instillation 0.5 ml sterile water and 1 enzyme inducer, cultivating without the need to carrying out the cracking process can carrying out sample.
Particularly, a kind of colibacillary detection method, described method comprises step:
1) get a Boiling tube, and instill 0.5 ml sterile water wherein, and instill 1 enzyme inducer;
2) get a cotton swab, with the sampling of cotton swab end, and be placed in above-mentioned Boiling tube, be mixed to form liquid to be measured;
3) get a small test tube, instill 3 cell lysis buffer solution within it;
4) get 200 microlitre drop to be measured with micropipette rifle or disposable liquid transfer gun head from above-mentioned Boiling tube and enter small test tube, fully mix, sealing, and wait for 5 ~ 10 minutes at ambient temperature;
5) in above-mentioned small test tube, instill 2 enzyme substrate solutions, fully mix;
6) get 200 microlitre drop to be measured with micropipette rifle or disposable liquid transfer gun head from above-mentioned small test tube and enter fluoroscopic examination sample tube, sealing, waits for 3 ~ 5 minutes at ambient temperature;
7) sample test fluoroscopic examination sample tube being placed in luminoscope is indoor, and seals;
Open the test pattern of luminoscope, record first time test number P
1;
Wait for 20 minutes, again test, record second time test number P
2;
If test value P
2-P
1> 60, then sample is positive; Otherwise be negative;
If test value P
2-P
1close to 60, then wait for 20 minutes, again test, obtain new P
2;
Described P
2numerical value in 20 ~ 60 minutes effectively.
In waiting time section between first time test and second time are tested, described fluoroscopic examination sample tube can take out and be placed in environment or incubator.
Detect colibacillary detection method from above-mentioned hand-held fluorescence instrument, if carry out confirmatory detection, namely do not need to carry out cultivation and detect, the colibacillary required time of this detection can complete in 1 hour; Even if needing to carry out cultivating detects (cultivating five hours in the thermostat container of 38.5 DEG C), make the colibacillary required time of whole detection extend to 6 hours, but this detection method is still very simple.
Above-mentioned hand-held fluorescence instrument detects the accuracy comparison that colibacillary detection method and traditional culture method detect colibacillary method:
As can be seen from the above table, the result that hand-held fluorescence instrument detects colibacillary detection method is the positive totally 12, feminine gender totally 10; The result that traditional culture method detects colibacillary method is the positive totally 10, feminine gender totally 12.It can thus be appreciated that the accuracy adopting hand-held fluorescence instrument of the present invention to detect colibacillary detection method is higher.
The foregoing is only preferred embodiment of the present invention, not in order to limit the present invention, any amendment done within all the spirit and principles in the present invention, equivalent replacement or improvement etc., all should be included within protection scope of the present invention.
Claims (10)
1. a colibacillary detection kit, it is characterized in that: described test kit comprises bay Trypsin nutrient solution, enzyme substrate solution, enzyme inducer and cell lysis buffer solution, the composition of described bay Trypsin nutrient solution comprises: Trypsin, lactose, sodium-chlor, dipotassium hydrogen phosphate, potassium primary phosphate, sodium lauryl sulphate, described enzyme inducer is isopropyl ss-D-1-thiogalactoside (IPTG), described enzyme substrate solution is 4-methyl umbelliferone-B-D-glucuronide, and described cell lysis buffer solution is Hen egg-white lysozyme (HEWL).
2. detection kit according to claim 1, is characterized in that: each constituent concentration of described bay Trypsin nutrient solution is respectively:
Trypsin: 20.0g/L
Lactose: 5.0g/L
Sodium-chlor: 5.0g/L
Dipotassium hydrogen phosphate: 2.75g/L
Potassium primary phosphate: 2.55g/L
Sodium lauryl sulphate: 0.1g/L.
3. detection kit according to claim 1 and 2, is characterized in that: the pH value of described bay Trypsin nutrient solution is 6.6 ~ 7.0.
4. detection kit according to claim 3, is characterized in that: described test kit also comprises sterilized water.
5. detection kit according to claim 4, is characterized in that: the volume content of described bay Trypsin nutrient solution, enzyme substrate solution, enzyme inducer, cell lysis buffer solution, sterilized water is as follows:
Bay Trypsin nutrient solution 1 milliliter/pipe
Enzyme substrate solution 1 milliliter/pipe
Enzyme inducer 1 milliliter/pipe
Cell lysis buffer solution 1 milliliter/pipe
Sterilized water 1 milliliter/pipe.
6. a colibacillary detection method, described method comprises step:
Step one, get a Boiling tube, and instill 0.5 ml sterile water wherein, and instill 1 enzyme inducer;
Step 2, get a cotton swab, with the sampling of cotton swab end, and be placed in above-mentioned Boiling tube, be mixed to form liquid to be measured;
Step 3, get a small test tube, instill 3 cell lysis buffer solution within it;
Step 4, get 200 microlitre drop to be measured with micropipette rifle or disposable liquid transfer gun head from above-mentioned Boiling tube and enter small test tube, fully mix, sealing, and wait for 5 ~ 10 minutes at ambient temperature;
Step 5, in above-mentioned small test tube instill 2 enzyme substrate solutions, fully mix;
Step 6, get 200 microlitre drop to be measured enter fluoroscopic examination sample tube with micropipette rifle or disposable liquid transfer gun head from above-mentioned small test tube, sealing, waits for 3 ~ 5 minutes at ambient temperature;
Step 7, sample test fluoroscopic examination sample tube being placed in luminoscope are indoor, and seal;
Open the test pattern of luminoscope, record first time test number P
1;
Wait for 20 minutes, again test, record second time test number P
2;
If test value P
2-P
1> 60, then sample is positive;
If test value P
2-P
1< 60, then sample is negative;
If test value P
2-P
1close to 60, then wait for 20 ~ 60 minutes again, again test, obtain new P
2.
7. colibacillary detection method according to claim 6, it is characterized in that: described enzyme inducer is isopropyl ss-D-1-thiogalactoside (IPTG), described cell lysis buffer solution is Hen egg-white lysozyme (HEWL), and described enzyme substrate solution is 4-methyl umbelliferone-B-D-glucuronide.
8. a colibacillary detection method, described method comprises step:
Step one, get a Boiling tube, and instill 0.5 milliliter of bay Trypsin nutrient solution according to claim 2 wherein, and instill 1 enzyme inducer;
Step 2, get a cotton swab, with the sampling of cotton swab end, and be placed in above-mentioned Boiling tube, be mixed to form liquid to be measured;
Step 3, the thermostat container above-mentioned liquid to be measured being placed in 38.5 DEG C are cultivated 3 ~ 5 hours;
Step 4, get a small test tube, instill 3 cell lysis buffer solution within it;
Step 5, get 200 microlitre drop to be measured with micropipette rifle or disposable liquid transfer gun head from above-mentioned Boiling tube and enter small test tube, fully mix, sealing, and wait for 5 ~ 10 minutes at ambient temperature;
Step 6, in above-mentioned small test tube instill 2 enzyme substrate solutions, fully mix;
Step 7, get 200 microlitre drop to be measured enter fluoroscopic examination sample tube with micropipette rifle or disposable liquid transfer gun head from above-mentioned small test tube, sealing, waits for 3 ~ 5 minutes at ambient temperature;
Step 8, sample test fluoroscopic examination sample tube being placed in luminoscope are indoor, and seal;
Open the test pattern of luminoscope, record first time test number P
1;
Wait for 20 minutes, again test, record second time test number P
2;
If test value P
2-P
1> 80, then sample is positive;
If test value P
2-P
1< 80, then sample is negative;
If test value P
2-P
1close to 80, then wait for 20 ~ 60 minutes again, again test, obtain new P
2.
9. colibacillary detection method according to claim 8, is characterized in that: the pH value of described bay Trypsin nutrient solution is 6.6 ~ 7.0.
10. colibacillary detection method according to claim 8, it is characterized in that: described enzyme inducer is isopropyl ss-D-1-thiogalactoside (IPTG), described cell lysis buffer solution is Hen egg-white lysozyme (HEWL), and described enzyme substrate solution is 4-methyl umbelliferone-B-D-glucuronide.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106086159A (en) * | 2016-06-20 | 2016-11-09 | 中国疾病预防控制中心环境与健康相关产品安全所 | A kind of zymolyte culture medium that can simultaneously detect two kinds of fecal pollution indicator bacterias and application thereof |
| CN108796031A (en) * | 2018-06-29 | 2018-11-13 | 中南民族大学 | A kind of Hymecromone enrichment material and application thereof |
| CN111073950A (en) * | 2019-12-31 | 2020-04-28 | 中国科学院长春应用化学研究所 | Fluorescence signal enhancement method for detecting escherichia coli by enzyme substrate method |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1122147A (en) * | 1993-03-12 | 1996-05-08 | 詹姆斯·D·堡格 | Rapid coliform detection system |
| CN101591695A (en) * | 2008-05-26 | 2009-12-02 | 刘侠 | The sodium lauryl sulphate neutralization increases bacterial context soup |
| CN101910409A (en) * | 2007-11-23 | 2010-12-08 | 荷兰应用自然科学研究组织Tno | Detect the system of microbial contamination |
-
2014
- 2014-10-28 CN CN201410586920.6A patent/CN104313114A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1122147A (en) * | 1993-03-12 | 1996-05-08 | 詹姆斯·D·堡格 | Rapid coliform detection system |
| CN101910409A (en) * | 2007-11-23 | 2010-12-08 | 荷兰应用自然科学研究组织Tno | Detect the system of microbial contamination |
| CN101591695A (en) * | 2008-05-26 | 2009-12-02 | 刘侠 | The sodium lauryl sulphate neutralization increases bacterial context soup |
Non-Patent Citations (1)
| Title |
|---|
| 苏德模 等: "MUG快速检定药品中大肠杆菌的方法研究", 《药物分析杂志》 * |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106086159A (en) * | 2016-06-20 | 2016-11-09 | 中国疾病预防控制中心环境与健康相关产品安全所 | A kind of zymolyte culture medium that can simultaneously detect two kinds of fecal pollution indicator bacterias and application thereof |
| CN106086159B (en) * | 2016-06-20 | 2019-10-29 | 中国疾病预防控制中心环境与健康相关产品安全所 | A kind of zymolyte culture medium that can detect two kinds of fecal pollution indicator bacterias simultaneously and its application |
| CN108796031A (en) * | 2018-06-29 | 2018-11-13 | 中南民族大学 | A kind of Hymecromone enrichment material and application thereof |
| CN111073950A (en) * | 2019-12-31 | 2020-04-28 | 中国科学院长春应用化学研究所 | Fluorescence signal enhancement method for detecting escherichia coli by enzyme substrate method |
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