CN109628313A - A kind of E. coli lysate and its preparation method and application - Google Patents
A kind of E. coli lysate and its preparation method and application Download PDFInfo
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- CN109628313A CN109628313A CN201910068025.8A CN201910068025A CN109628313A CN 109628313 A CN109628313 A CN 109628313A CN 201910068025 A CN201910068025 A CN 201910068025A CN 109628313 A CN109628313 A CN 109628313A
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Abstract
The present invention relates to Microorganism in Water detection technique field, a kind of E. coli lysate and its preparation method and application is disclosed.Lysate of the present invention includes dehydrating agent, bleeding agent and surfactant；Wherein the dehydrating agent is sodium chloride, EDTA-2Na or sodium hydroxide；The bleeding agent is glycerol, polyethylene glycol, tween or Qula logical -100；The surfactant is polyvinylpyrrolidone or ethyl cellulose.The present invention is grouped as E. coli lysate with suitable group, effectively quickly cracked for Escherichia coli in water quality and food, only need the i.e. releasable galactase of 10min time-consuming, detection of the aobvious blue completion to Escherichia coli is reacted with the test strips containing galactoside color developing agent (X-GAL), entire E. coli detection process can be foreshortened within 30min, compared to the longer time-consuming of existing product, the detection efficiency of Escherichia coli in water quality and food is significantly improved.
Description
Technical field
The present invention relates to Microorganism in Water detection technique fields, and in particular to a kind of E. coli lysate and its preparation side
Method and application.
Background technique
Escherichia coli is commonly known as Escherichia coli, is that Escherich had found in 1885, Escherichia coli belong to
Gramnegative bacterium, 0.5 × 1~3 microns of size, peritrichous can move, no gemma.The various saccharides that can ferment produce acid, produce
Gas, is the normal perch bacterium in humans and animals enteron aisle, enters enteron aisle with lactation after baby due, is accompanied all the life with people, almost
Account for the 1/3 of excrement dry weight.
National regulation, the total plate count in every milliliter of drinking water must not detect total large intestine in every 100 milliliters of water less than 100
Bacillus group.
Coliform group count is common Bacteriological Indexes in sewage quality analysis, with the coliform group count table in every liter of water
Show.Coliform includes several bacteriums being largely present in human body intestinal canal such as Escherichia coli, therefore is largely existed in excrement big
Intestinal flora.Under normal circumstances, coliform belongs to non-pathogenic bacteria.Coliform is such as detected in water sample, shows water by excrement
Toilet pollution.Since water causes the growing environment of disease carrying germ and virus and coliform essentially identical, and disease carrying germ is caused to water
Detection with virus is again relatively difficult, therefore usually uses coliform as indirect Testing index.If the coliform in water
Group's number is more than set quota, is considered as in these water that disease carrying germ and virus may be caused containing water, as direct body contact this
A little water may can be infected disease.
There are the water quality fecal coliform group test scraps of paper, Escherichia coli O 157: H7 Rapid detection test strip currently on the market.This two
It requires to sample in kind product use process, it is not only inconvenient for operation using constant incubator culture 24 hours, but also when detection
Between long (be greater than 24 hours), two kinds of products are prepared using microorganism detection (national standard) Method And Principle.And the said goods make
It is big with field limitation, it is only used for the test of food, tableware surface microorganism testing or enteropathogenic E. Coli, is not had
Standby general applicability.
In addition, there are also the lysates of Reagent Company's production in the market, such as Western and IP cell pyrolysis liquid, RIPA
Lysate (in by force, or weak), SDS lysate, NP-40 lysate, are mainly used as Western, IP or co-IP, ingredient mainly wraps
Include 1%Triton X-100,1%deoxycholate, 0.1%SDS and 1%NP-40 etc..However these lysates are not special
Door is used to detect Escherichia coli, and is used as other purposes, and the shortcomings that above-mentioned lysate is that pyrolysis time is very long,
Need 2-24 hour.
Summary of the invention
In view of this, the purpose of the present invention is to provide a kind of E. coli lysate and preparation method thereof, so that described
Lysate can Escherichia coli in rapid cleavage water quality or food, shorten time-consuming, and can be applied to water quality and food large intestine bar
In the detection of bacterium.
In order to achieve the above-mentioned object of the invention, the present invention provides the following technical scheme that
A kind of E. coli lysate, including dehydrating agent, bleeding agent and surfactant；Wherein the dehydrating agent is chlorination
Sodium, EDTA-2Na or sodium hydroxide；The bleeding agent is glycerol, polyethylene glycol, tween or Qula logical -100；The surface-active
Agent is polyvinylpyrrolidone or ethyl cellulose.
Preferably, the mass percent concentration of the dehydrating agent is 0.1-30%, more preferably 1-2%；The infiltration
The mass percent concentration of agent is 0.1-10%, more preferably 0.1-0.2%；The mass percent concentration of the surfactant
For 0.01-5%.In the specific embodiment of the invention, the mass percent concentration of the dehydrating agent is 1% or 2%, the infiltration
The mass percent concentration of saturating agent is 0.1% or 0.2%, and the mass percent concentration of the surfactant is 0.01%.
In the specific embodiment of the invention, the polyethylene glycol is polyethylene glycol 2000, the polyvinylpyrrolidone
For K30, the tween is Tween 80.
Meanwhile the present invention also provides the preparation methods of the lysate, comprising:
Dehydrating agent, bleeding agent and surfactant are weighed, water is added to be configured to lysate；
Escherichia coli bacteria liquid, sterile water and other bacterium colony bacterium solutions are detected using lysate of the present invention, are as a result shown
Show, 10min cracking is carried out using lysate, then (has galactoside color developing agent X- in test strips with matched test strip
GAL react aobvious blue with the releaser after Escherichia coli cracking) immerse sample to be tested 10min after, only Escherichia coli bacteria liquid is shown
Show blue, sterile water and other bacterium colony bacterium solutions are colourless, this illustrates that only time-consuming 10min can complete water quality to lysate of the present invention
The detection of Escherichia coli in sample, and accuracy is high, is less prone to false positive results.Based on excellent test result, this hair
It is bright propose the lysate detection water quality in Escherichia coli and/or preparation water quality Escherichia coli detection kit in answering
With.
From the above technical scheme, the present invention is grouped as E. coli lysate with suitable group, is effectively directed to water quality
Quickly cracked with Escherichia coli in food, it is only necessary to 10min time-consuming, that is, releasable galactase, with contain galactoside color developing agent
(X-GAL) the aobvious blue of test strips reaction completes the detection to Escherichia coli, can foreshorten to entire E. coli detection process
Within 30min, the longer time-consuming of existing product is compared, the detection efficiency of Escherichia coli in water quality and food is significantly improved.
Detailed description of the invention
Fig. 1 show colour developing result of the different samples after kit of the present invention detection；Wherein, A is Escherichia coli bacterium
Liquid colour developing result；B is sterile water colour developing result；C is mould bacterium solution colour developing result；D is staphylococcus bacterium solution colour developing result.
Specific embodiment
The invention discloses a kind of E. coli lysate and its preparation method and application, those skilled in the art can be borrowed
Reflect present disclosure, is suitably modified realization of process parameters.In particular, it should be pointed out that all similar substitutions and modifications are to this field
It is it will be apparent that they are considered as being included in the present invention for technical staff.Lysate of the present invention and its preparation side
Method and application be described by preferred embodiment, related personnel obviously can not depart from the content of present invention, spirit and
Lysate described herein and its preparation method and application is modified in range or appropriate changes and combinations, carrys out implementation and application
The technology of the present invention.
Water quality of the present invention includes Drinking Water, tap water, seawater, fishery water, swimming pool water, field irrigation
Water, underground water, surface water, trade effluent, test water etc.；Food includes food liquid and solid-state food；Food liquid is main
It needs first to filter out insoluble matter, uses active carbon before detecting including fruit juice, milk, beverage, food soup, liquid charging stock etc.
Decoloration, is then detected again；And solid-state food needs are first dissolved with water, filter out insoluble matter, using can be examined after active carbon decoloring
It surveys.
Just a kind of E. coli lysate provided by the present invention and its preparation method and application is described further below.
Embodiment 1: lysate of the present invention
Water is added to prepare lysate according to the concentration of a line any in table 1.
Table 1
Embodiment 2: lysate detection test of the present invention
Choosing the Escherichia coli bacteria liquid that OD value is 0.3, the mould bacterium solution that sterile water, OD value are 0.3 and OD value is 0.3
Staphylococcus bacterium solution is detected as subjects；
The lysate 4 in embodiment 1 is added into water sample to be measured, stands 10-20min；Then in the kit
Test strip immerses water sample to be measured, after keeping 10min, if test strip shows the blue of arbitrary extent, and as positive, inspection
Test paper slip is colourless or other colors are feminine gender.
Testing result is shown in Table 2 and Fig. 1；
Table 2
| Sample name | Drying chemical reagent paper testing result (stands cracking 10min) |
| Escherichia coli bacteria liquid (OD value 0.3) | The positive (blue) 20min |
| Sterile water | Feminine gender (colourless) 20min |
| Mould bacterium solution (OD value 0.3) | Feminine gender (colourless) 20min |
| Staphylococcus bacterium solution (OD value 0.3) | Feminine gender (colourless) 20min |
Only Escherichia coli bacteria liquid occurs discoloration and is displayed in blue it can be seen from the above results, other two groups without colour developing,
Whole process only expends 20min (10min cracking+10min reaction) simultaneously, this illustrates that kit of the present invention is not only time-consuming short, and
And accuracy is high, is less prone to false positive results.In addition, being detected using lysate 1-3, three only has Escherichia coli bacterium
Liquid occurs discoloration and is displayed in blue, other three groups without colour developing.
Meanwhile according to above-mentioned test method, the present invention is detected using the Escherichia coli bacteria liquid of different OD values, is as a result seen
Table 3.
Table 3
| Sample name | Drying chemical reagent paper testing result (20min cracking+10min reaction) |
| Escherichia coli bacteria liquid (OD value 0.3) | Positive (blue+) 30min |
| Escherichia coli bacteria liquid (OD value 0.6) | Positive (blue ++) 30min |
| Escherichia coli bacteria liquid (OD value 0.9) | Positive (blue +++) 30min |
| Escherichia coli bacteria liquid (OD value 1.2) | Positive (blue ++++) 30min |
| Escherichia coli bacteria liquid (OD value 1.5) | Positive (blue +++ ++) 30min |
The Escherichia coli bacteria liquid of different OD values can be described by the present invention lysate cracking it can be seen from 3 result of table
After detect, and with the increase of OD value, colored intensity is more and more obvious.In addition, being detected using lysate 1-3, San Zhebiao
Reveal and the identical colour developing trend of table 3.
The above is only a preferred embodiment of the present invention, it is noted that for the ordinary skill people of the art
For member, various improvements and modifications may be made without departing from the principle of the present invention, these improvements and modifications are also answered
It is considered as protection scope of the present invention.
Claims (9)
1. a kind of E. coli lysate, which is characterized in that including dehydrating agent, bleeding agent and surfactant；It is wherein described de-
Aqua is sodium chloride, EDTA-2Na or sodium hydroxide；The bleeding agent is glycerol, polyethylene glycol, tween or Qula logical -100；Institute
Stating surfactant is polyvinylpyrrolidone or ethyl cellulose.
2. lysate according to claim 1, which is characterized in that the concentration of the dehydrating agent is 0.1-30%.
3. lysate according to claim 1, which is characterized in that the concentration of the bleeding agent is 0.1-10%.
4. lysate according to claim 1, which is characterized in that the concentration of the surfactant is 0.01-5%.
5. lysate according to claim 1, which is characterized in that the polyethylene glycol is polyethylene glycol 2000.
6. lysate according to claim 1, which is characterized in that the tween is Tween 80.
7. lysate according to claim 1, which is characterized in that the polyvinylpyrrolidone is K30.
8. lysate described in claim 1-7 any one detection water quality or food in Escherichia coli and/or preparation water quality or
Application in food Escherichia coli detection kit.
9. the preparation method of lysate described in claim 1, which is characterized in that dehydrating agent, bleeding agent and surfactant are weighed,
Water is added to be configured to lysate.
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| CN1677106A (en) * | 2005-02-02 | 2005-10-05 | 东北农业大学 | Technology for treating excrement and urine in pig's infectious gastroenteritis quick testing method |
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| CN102242189A (en) * | 2010-03-11 | 2011-11-16 | 三星泰科威株式会社 | Nucleic Acid Template preparation for real-time PCR |
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Application publication date: 20190416 |