CN102758004A - Detection kit for enteroadhesive E.Coli and detection method - Google Patents
Detection kit for enteroadhesive E.Coli and detection method Download PDFInfo
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- CN102758004A CN102758004A CN2012100680985A CN201210068098A CN102758004A CN 102758004 A CN102758004 A CN 102758004A CN 2012100680985 A CN2012100680985 A CN 2012100680985A CN 201210068098 A CN201210068098 A CN 201210068098A CN 102758004 A CN102758004 A CN 102758004A
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Abstract
The invention is applicable to the technical field of microorganisms, and provides a detection kit for enteroadhesive E.Coli and a detection method. The kit comprises a DNA extracting solution and a polymerase chain reaction (PCR) solution. Through the adoption of the detection kit for the enteroadhesive E.Coli and the detection method, the enteroadhesive E.Coli can be detected at the same time; and compared with the conventional culture method, the detection method has the advantages of speediness, sensitivity, safety and the like and can be applied to the detection of the enteroadhesive E.Coli.
Description
Technical field
The invention belongs to microbial technology field, relate in particular to a kind of adhesivity intestinal bacteria detection kit and detection method.
Background technology
(enteroadhesive E.Coli is just to know in recent years EAEC) to the adhesivity intestinal bacteria, and concrete morbific serotype is unclear fully as yet.EAEC does not invade intestinal epithelial cells, does not produce enterotoxin LT, ST or shiga-like toxin, does not therefore cause tissue injury.Unique ability that has with the Hep-2 cell adhesion that is characterized as.EAEC invades children's more, is main with children's in the groove, and the adult also can fall ill, and is prone to cause diarrhoea delay chronicity.Clinical manifestation does not have heating more, suffers from diarrhoea 3～5 times/day, and mostly stool is rare egg style or band milk lobe appearance, and amount is many, and enteroplegia and mucus blood sample stool can appear in severe patient.
General in the world adhesivity intestinal bacteria are adopted to cultivate and detect as follows:
(1) increases bacterium
Should check as early as possible behind the sample collecting.Except perishable foodstuff was answered the precooling Tibetan before check, generally do not refrigerate.Take by weighing check 25g with aseptic formality, be added in the 225mL nutrient broth, smash 1min or add the sterilization sand milling broken with mortar with homogenizer.Take out in right amount, the inoculation lactose bile salt medium, to measure coliform MPN, remaining moves in 500mL wide-necked bottle, cultivates 6h in 36 ± 1 ℃.Picking 1 ring is inoculated in the 1 pipe 30mL enterobacteria enrichment broth, cultivates 18h in 42 ℃.
(2) separate
With lactose fermentation male bile salt lactose fermentation tube and enrichment liquid difference streak inoculation Mai Kangkai or eosin methylene blue agar flat board; With serious pollution sample can be dull and stereotyped with the even direct streak inoculation Mai Kangkai of liquid of sample or Yihong methylene blue, cultivates 18～24h, the observation bacterium colony in 36 ± 1 ℃.Not only to note the bacterium colony of lactose fermentation, also will note the bacterium colony of lactose nonfermented and delayed reaction simultaneously.
(3) biochemical test
1. inoculate triple sugar iron agar (TSI) or kligler iron agar (KI) respectively from dull and stereotyped several bacterium colonies of direct picking of of discriminating.Simultaneously these cultures are inoculated peptone water, semisolid, pH7.2 urea agar, KCN meat soup and lysine decarboxylase test medium respectively.Above culture was all cultivated liquid at 36 ℃.
2. the TSI inclined-plane produces acid or does not produce acid, and bottom produces acid, and H2S culture negative and that urea is negative is intestinal bacteria.The TSI bottom does not produce acid, or H2S, KCN, urea have each positive culture, all non-intestinal bacteria.Do oxidase test and gramstaining in case of necessity.
(4) serological test
Do slide agglutination test with adhesivity intestinal bacteria multivalence O serum, as present strong agglutination reaction, be the test positive.
(5) result's report
Report is made in comprehensive above biochemical test, serological test.
According to above-mentioned universal method, simultaneously cycle of these three kinds of bacteriums of adhesivity intestinal bacteria long (at least 48 hours) in the test sample, operation is loaded down with trivial details, sensitivity is low, be prone to pollute, program complicacy and required reagent (biochemical test reagent and serum) are various etc., and shortcoming can not satisfy the colibacillary requirement of rapid detection adhesivity far away.
Summary of the invention
Technical problems such as in view of this, the present invention provides a kind of adhesivity intestinal bacteria detection kit, and it is low to detect adhesivity intestinal bacteria sensitive simultaneously in the solution prior art, and the cycle is long, and the colibacillary detection method of adhesivity.
The present invention is achieved in that
A kind of adhesivity intestinal bacteria detection kit; Comprise DNA extraction liquid, PCR reaction solution; Said PCR reaction solution comprises first reaction solution, second reaction solution and paraffin; Comprise adhesivity intestinal bacteria first primer, adhesivity intestinal bacteria second primer, adhesivity intestinal bacteria probe in said first reaction solution; Said adhesivity intestinal bacteria first primer sequence is shown in SEQ ID NO:1, and adhesivity intestinal bacteria second primer sequence is shown in SEQ ID NO:2, and adhesivity intestinal bacteria probe sequence is shown in SEQ ID NO:3.
The present invention further provides a kind of adhesivity intestinal bacteria detection method, comprises the steps:
Provide adhesivity colibacillary DNA sample;
Carry out fluorescence quantitative PCR detection, use aforesaid adhesivity intestinal bacteria detection kit in this fluorescence quantitative PCR detection step.
Adhesivity intestinal bacteria detection kit of the present invention and detection method can detect the adhesivity intestinal bacteria simultaneously; Need not increase bacterium for a long time to testing sample cultivates; Adding simultaneously has the primer of detection and probe; Compare with traditional culture method have fast, advantage such as sensitive, safety, can be applicable to the colibacillary detection of adhesivity.
Description of drawings
Fig. 1 is positive control pcr amplification figure in the embodiment of the invention adhesivity intestinal bacteria detection kit;
Fig. 2 is embodiment of the invention adhesivity intestinal bacteria detection kit negative control pcr amplification figure;
Fig. 3 is that the embodiment of the invention one adhesivity intestinal bacteria detect positive sample pcr amplification figure in the method for testing;
Fig. 4 is that the embodiment of the invention one adhesivity intestinal bacteria detect negatives pcr amplification figure in the method for testing.
Embodiment
In order to make the object of the invention, technical scheme and advantage clearer,, the present invention is further elaborated below in conjunction with accompanying drawing and embodiment.Should be appreciated that specific embodiment described herein only in order to explanation the present invention, and be not used in qualification the present invention.
Real-time fluorescence quantitative PCR all is widely used in fields such as basic scientific research, clinical diagnosis, disease research and medicament research and development.Its ultimate principle is: in the PCR reaction system, introduce a kind of fluorescence chemical material, along with the carrying out of PCR reaction, the PCR reaction product constantly accumulates, and fluorescence signal intensity also equal proportion increases.Every through a circulation; Collect a fluorescence signal intensity, so just can obtain an amplified fluorescence graphic representation through the variation of fluorescence intensity variation monitoring product amount; There is linear relationship between the logarithmic value of PCR product amount and the starting template amount, can carries out quantitative analysis.
The embodiment of the invention provides a kind of adhesivity intestinal bacteria detection kit; Comprise DNA extraction liquid, PCR reaction solution; Said PCR reaction solution comprises first reaction solution, second reaction solution and paraffin; Comprise adhesivity intestinal bacteria first primer, adhesivity intestinal bacteria second primer, adhesivity intestinal bacteria probe in said first reaction solution; Said adhesivity intestinal bacteria first primer sequence is shown in SEQ ID NO:1, and adhesivity intestinal bacteria second primer sequence is shown in SEQ ID NO:2, and adhesivity intestinal bacteria probe sequence is shown in SEQ ID NO:3.
Particularly, embodiment of the invention adhesivity intestinal bacteria detection kit comprising DNA extraction liquid, PCR reaction solution, can be carried out the colibacillary detection of adhesivity simultaneously; Also comprise adhesivity intestinal bacteria negative control in this test kit, positive control.
The specification of test kit:
The specification of negative control is 1 pipe, 40 μ l/ pipe, and the specification of negative control is 1 pipe, and 40 μ l/ pipe, DNA extraction liquid are 5ml/ pipe (1 pipe), and the PCR reaction solution is 8 pipe/bars * 6, and 20 μ l/ manage.
This negative control is not for containing the Tris-EDTA damping fluid (0.01M pH8.0) of adhesivity bacillus coli gene; Also comprise adhesivity intestinal bacteria positive control, this positive control uses Tris-EDTA damping fluid (0.01M pH8.0) the dilution freezing preservation in back for using the positive template contrast article of adhesivity intestinal bacteria and DNA extraction preparation.Self-checking when this feminine gender and positive control are used to detect prevents to detect the appearance of error.See also Fig. 1 and Fig. 2, the pcr amplification collection of illustrative plates of positive control in Fig. 1 visualizingre agent box, the pcr amplification collection of illustrative plates of negative control in Fig. 2 visualizingre agent box.
Particularly, the concrete composition of this DNA extraction liquid (being used to extract DNA) is: Tris-EDTA damping fluid (0.01M pH8.0) contains 0.01%NP-40.
Particularly, this PCR reaction solution comprises first reaction, second reaction solution and paraffin, and this first reaction solution and second reaction solution are realized separating through paraffin; In the PCR reaction process; Because temperature is at 95 ℃, paraffin melts, and first reaction solution and second reaction solution mix; The PCR reaction is able to carry out, and the particular content of this paraffin is seen patent (200910190112.7).This PCR reaction reagent is deposited in the common centrifuge tube, and this first reaction solution is positioned at the lower end, and in the middle of paraffin was positioned at, this second reaction solution was positioned at above the paraffin.
Particularly, this first reaction solution comprises damping fluid, magnesium chloride, and dNTPs, adhesivity intestinal bacteria first primer, adhesivity intestinal bacteria second primer, adhesivity intestinal bacteria probe, and, the sterilization ultrapure water, the TV of first reaction solution is 18 microlitre/pipes.
This damping fluid is 10 * Buffer, does not contain mg ion (precious biotechnology (Dalian) ltd) in this damping fluid.The volume of this damping fluid is 2～4 microlitre/pipes;
The concentration of this this magnesium chloride is 25mM, and the volume of this magnesium chloride is 2～4 microlitre/pipes;
The concentration of this dNTPs is 2.5mM, and volume is 2～4 microlitre/pipes;
The concentration of this adhesivity intestinal bacteria first primer is 50 μ M; Volume is 0.1～0.2 microlitre/pipe; 0.15 microlitre/pipe for example, the nucleotide sequence of this adhesivity intestinal bacteria first primer is specially shown in SEQ ID NO:1: 5 '-CA TGTGAGAATG ATATGAAAT-3 '-;
The concentration of this adhesivity intestinal bacteria second primer is 50 μ M, and volume is 0.1～0.2 microlitre/pipe, 0.15 microlitre/pipe for example, and the nucleotide sequence of this adhesivity intestinal bacteria second primer is specially shown in SEQ ID NO:2:
5’-TACAATTAGATGTATATAGTA-3’；
The concentration of this adhesivity intestinal bacteria probe is 50 μ M; Volume is 0.1～0.2 microlitre/pipe; 0.15 microlitre/pipe for example, the nucleotide sequence of this adhesivity intestinal bacteria probe is specially shown in SEQ ID NO:3: 5 '-(FAM) ATTAACAAT ATCAGAATAC ATC (DABCYL)-3 ';
The amount of this sterilization ultrapure water is 5.4～11.7 microlitre/pipes, and the TV of realizing first reaction solution is 18 microlitre/pipes.
Particularly, this second reaction solution comprises Taq enzyme, developer and sterilization ultrapure water, and the TV of this second reaction solution is 2 microlitre/pipes.
The volume of this Taq enzyme is 0.15-0.3 microlitre/pipe, and the volume of developer is 0.01-0.02 microlitre/pipe, and the amount of this sterilization ultrapure water is 1.84～1.68 microlitre/pipes, this developer for example, bromjophenol blue.Make the volume of second reaction solution satisfy 2 microlitre/pipes.
Particularly, the volume of this paraffin is 20 microlitre/pipes.
Embodiment of the invention adhesivity intestinal bacteria detection kit can detect the adhesivity intestinal bacteria; Need not increase bacterium for a long time to testing sample cultivates; Adding simultaneously has the primer of detection and probe; Compare with traditional culture method have fast, advantage such as sensitive, safety, can be applicable to the colibacillary detection of adhesivity.
The embodiment of the invention further provides a kind of adhesivity intestinal bacteria detection method, comprises the steps:
Step S01 increases bacterium and cultivates and sample disposal:
Provide adhesivity colibacillary DNA sample;
Step S02, real-time fluorescence PCR:
Carry out real-time fluorescence quantitative PCR and detect, use above-mentioned adhesivity intestinal bacteria detection kit in this fluorescence quantitative PCR detection step.
Particularly, step S01 operates under aseptic condition.
Particularly, among the step S01, gather sample, possibly contain the adhesivity intestinal bacteria in this sample, also possibly not contain, need the detection method of the embodiment of the invention to detect definite.Therefore, among this step S01, possibly contain the colibacillary DNA of adhesivity in the colibacillary DNA sample of adhesivity, also possibly not contain.
Cultivate carrying out increasing in 3 hours bacterium in the 37 ℃ of thermostatic chambers of sample placement after gathering; Carrying out following dna cleavage processing then:
Get the 1ml enrichment liquid and add DNA extraction liquid 50 μ L, aspirate abundant mixing sample repeatedly 3 times.
Particularly; Among the step S02; Get resulting supernatant among the step S01; Carry out the quantitative fluorescent PCR analysis, not restriction of employed quantitative fluorescent PCR equipment in this step, ABI series, Bio-Rad serial (ICycler/MJ Opticon 2), Stratagene MX series, Roche LightCycler, Cepheid SmartCycler, Corbett Rotor-Gene, the rich day series in Hangzhou.
The condition of PCR reaction is among the step S02:
50-55℃：2-5min；
94-95℃：2-3min；
94-95 ℃: 5-10S, 55-60 ℃: 40-80S; (40 circulation).
In the embodiment of the invention adhesivity intestinal bacteria detection method, each detection all is provided with positive control and negative control in the test kit, occurs to prevent the pollution in the testing process.
Below in conjunction with specific embodiment above-mentioned adhesivity intestinal bacteria detection method is elaborated.
Embodiment one
Embodiment of the invention adhesivity intestinal bacteria detection method comprises the steps:
1, gather the colibacillary sample of adhesivity, this sample number is 20, contains the adhesivity intestinal bacteria in the some of them sample, and some samples do not have the adhesivity intestinal bacteria.Cultivate carrying out increasing in 3 hours bacterium in the 37 ℃ of thermostatic chambers of sample placement after gathering; Carrying out following dna cleavage processing then:
Get the 1ml enrichment liquid and add DNA extraction liquid 50 μ L, aspirate abundant mixing sample repeatedly 3 times, obtain the colibacillary DNA sample of adhesivity;
2, get above-mentioned PCR reaction solution, each component is in this PCR reaction solution:
First reaction solution
Second reaction solution
Taq enzyme 0.15 microlitre/pipe
Developer 0.01 microlitre/pipe
Sterilization ultrapure water 1.84 microlitre/pipes;
Under following condition, implement the quantitative fluorescent PCR reaction, and detect:
50-55℃：2-5min；
94-95℃：2-3min；
94-95 ℃: 5-10S, 55-60 ℃: 40-80S; (40 circulation).
Embodiment two
Embodiment of the invention adhesivity intestinal bacteria detection method comprises the steps:
1, gather the colibacillary sample of adhesivity, this sample number is 20, contains the adhesivity intestinal bacteria in the some of them sample, and some samples do not have the adhesivity intestinal bacteria.Cultivate carrying out increasing in 3 hours bacterium in the 37 ℃ of thermostatic chambers of sample placement after gathering; Carrying out following dna cleavage processing then:
Get the 1ml enrichment liquid and add DNA extraction liquid 50 μ L, aspirate abundant mixing sample repeatedly 3 times, obtain the colibacillary DNA sample of adhesivity;
2, get above-mentioned PCR reaction solution, each component is in this PCR reaction solution:
First reaction solution
Second reaction solution
Taq enzyme 0.2 microlitre/pipe
Developer 0.015 microlitre/pipe
Sterilization ultrapure water 1.785 microlitre/pipes;
Under following condition, implement the quantitative fluorescent PCR reaction, and detect:
50-55℃：2-5min；
94-95℃：2-3min；
94-95 ℃: 5-10S, 55-60 ℃: 40-80S; (40 circulation).
Embodiment three
Embodiment of the invention adhesivity intestinal bacteria detection method comprises the steps:
1, gather the colibacillary sample of adhesivity, this sample number is 20, contains the adhesivity intestinal bacteria in the some of them sample, and some samples do not have the adhesivity intestinal bacteria.Cultivate carrying out increasing in 3 hours bacterium in the 37 ℃ of thermostatic chambers of sample placement after gathering; Carrying out following dna cleavage processing then:
Get the 1ml enrichment liquid and add DNA extraction liquid 50 μ L, aspirate abundant mixing sample repeatedly 3 times, obtain the colibacillary DNA sample of adhesivity;
2, get above-mentioned PCR reaction solution, each component is in this PCR reaction solution:
First reaction solution
Second reaction solution
Taq enzyme 0.3 microlitre/pipe
Developer 0.02 microlitre/pipe
Sterilization ultrapure water 1.68 microlitre/pipes;
Under following condition, implement the quantitative fluorescent PCR reaction, and detect:
50-55℃：2-5min；
94-95℃：2-3min；
94-95 ℃: 5-10S, 55-60 ℃: 40-80S; (40 circulation).
Comparative Examples
This Comparative Examples adhesivity intestinal bacteria detection method comprises the steps:
1, collection contains the colibacillary sample of adhesivity, and the sample of this Comparative Examples is consistent with the sample of embodiment one, the sample after gathering is placed carried out increasing in 3 hours the bacterium cultivation in 37 ℃ of thermostatic chambers; Carrying out following dna cleavage processing then:
Get the 1ml enrichment liquid and add DNA extraction liquid 50 μ L, aspirate abundant mixing sample repeatedly 3 times, obtain the colibacillary DNA sample of adhesivity;
2, the international method of being said according to the front background technology detects.
See also following table, following table shows that the method for application implementation example one and Comparative Examples detects the result of 20 routine samples:
Can find out that from this table it is consistent that the method for the embodiment of the invention one and the method for Comparative Examples meet the side result.
See also Fig. 3, Fig. 4, Fig. 3 is that the embodiment of the invention one adhesivity intestinal bacteria detect positive sample pcr amplification figure in the method for testing; Fig. 4 is that the embodiment of the invention one adhesivity intestinal bacteria detect negatives pcr amplification figure in the method for testing.With knowing that the adhesivity intestinal bacteria detection method of the embodiment of the invention one can detect positive sample and negatives in the sample after Fig. 3 and Fig. 1, Fig. 4 and Fig. 2 contrast, detected result is correct.
The above is merely preferred embodiment of the present invention, not in order to restriction the present invention, all any modifications of within spirit of the present invention and principle, being done, is equal to and replaces and improvement etc., all should be included within protection scope of the present invention.
Claims (8)
1. adhesivity intestinal bacteria detection kit; Comprise DNA extraction liquid, PCR reaction solution; It is characterized in that; Said PCR reaction solution comprises first reaction solution, second reaction solution and paraffin, comprises adhesivity intestinal bacteria first primer, adhesivity intestinal bacteria second primer, adhesivity intestinal bacteria probe in said first reaction solution, and said adhesivity intestinal bacteria first primer sequence is shown in SEQ ID NO:1; Adhesivity intestinal bacteria second primer sequence is shown in SEQ ID NO:2, and adhesivity intestinal bacteria probe sequence is shown in SEQ ID NO:3.
2. adhesivity intestinal bacteria detection kit as claimed in claim 1 is characterized in that said first reaction solution and second reaction solution are separated through said paraffin.
3. adhesivity intestinal bacteria detection kit as claimed in claim 2 is characterized in that, said first reaction solution also comprises sterilization ultrapure water, damping fluid, magnesium chloride, dNTPs.
4. adhesivity intestinal bacteria detection kit as claimed in claim 2 is characterized in that, said second reaction solution comprises Taq enzyme, sterilization ultrapure water and developer.
5. like claim 3 or 4 described adhesivity intestinal bacteria detection kit, it is characterized in that each component volume of said first reaction solution is:
6. like claim 3 or 4 described adhesivity intestinal bacteria detection kit, it is characterized in that each component volume of said second reaction solution is:
Taq enzyme 0.15～0.3 microlitre/pipe
Developer 0.01～0.02 microlitre/pipe
Sterilization ultrapure water 1.84～1.68 microlitre/pipes.
7. an adhesivity intestinal bacteria detection method comprises the steps:
Provide adhesivity colibacillary DNA sample;
Carry out fluorescence quantitative PCR detection, use like each described adhesivity intestinal bacteria detection kit of claim 1-7 in the said fluorescence quantitative PCR detection step.
8. adhesivity intestinal bacteria detection method as claimed in claim 7 is characterized in that, said PCR reaction conditions is:
50-55℃：2-5min；
94-95℃：2-3min；
94-95℃：5-10S；
55-60℃：40-80S；
40 circulations.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN103484552A (en) * | 2013-10-09 | 2014-01-01 | 武汉康录生物技术有限公司 | Simplified Sanger gene sequencing method |
| CN106978506A (en) * | 2017-05-05 | 2017-07-25 | 武汉爱基百客生物科技有限公司 | A kind of fluorescence quantifying PCR method for detecting Escherichia coli |
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2012
- 2012-03-15 CN CN2012100680985A patent/CN102758004A/en active Pending
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| CN103484552A (en) * | 2013-10-09 | 2014-01-01 | 武汉康录生物技术有限公司 | Simplified Sanger gene sequencing method |
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Application publication date: 20121031 |