CN112111584A - Method for rapidly detecting escherichia coli in water - Google Patents
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- CN112111584A CN112111584A CN201910538317.3A CN201910538317A CN112111584A CN 112111584 A CN112111584 A CN 112111584A CN 201910538317 A CN201910538317 A CN 201910538317A CN 112111584 A CN112111584 A CN 112111584A
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Abstract
The invention provides a primer group for detecting Escherichia coli in water, which comprises the following components: SEQ ID NO: 1 to 6. The primer group disclosed by the invention has the advantages of strong specificity, high accuracy, strong sensitivity, simplicity and convenience in operation, low cost and the like when being used for detecting the Escherichia coli in water, and is suitable for wide application.
Description
Technical Field
The invention relates to the field of water quality detection. Specifically, the invention relates to a method for rapidly detecting Escherichia coli in water. More specifically, the present invention relates to a primer set for detecting escherichia coli in water, a kit, and a method for detecting escherichia coli in water.
Background
The detection and control of water quality in the process of safe utilization of water resources is an important work, wherein pathogenic microorganisms are one of the most concerned problems in water quality detection. Pathogenic microorganisms in the water body mainly come from human and animal excreta, infect humans through ways of diet, respiration, skin contact and the like, and finally cause intestinal diseases or respiratory diseases. The pollution of pathogenic microorganisms in water poses a potential threat to human health, so that the detection and control of the pollution are necessary.
The morbidity and mortality caused by pathogenic bacteria in water is high, and the disease is a controllable hazard. Therefore, effective detection of pathogenic bacteria in water is an important measure for prevention and control of related disease outbreaks. The current water quality standard in China mainly adopts an indicator microorganism coliform group to evaluate the harm degree of pathogenic microorganisms. The coliform group is a common indicator microorganism, and bacteria of this group are generally considered to include Escherichia coli, Citrobacter, Klebsiella aerogenes, Enterobacter cloacae, and the like. Among them, Escherichia coli is the main bacterium in the Escherichia coli group. Currently, two methods for coliform enumeration are the MPN method and the plate enumeration method in the national standard (GB 4789.3-2016). Both methods require bacterial culture and have the defects of complex operation, time consumption, labor waste and the like.
Therefore, the detection method for Escherichia coli in water is still under study.
Disclosure of Invention
The present invention aims to solve at least to some extent at least one of the technical problems of the prior art. The invention provides a primer group, a kit and a method for detecting escherichia coli in water, and the primer group, the kit and the method have the advantages of strong specificity, high accuracy, strong sensitivity, simple and convenient operation, low cost and the like, and are suitable for wide application.
In one aspect of the present invention, the present invention provides a primer set for detecting Escherichia coli in water. According to an embodiment of the invention, the primer set comprises: SEQ ID NO: 1 to 6. The inventors have studied the total DNA sequence of Escherichia coli and found that beta-glucuronidases (beta-glucuronidases) are commonly present in Escherichia coli in sewage and that the presence or absence of Escherichia coli in water can be accurately determined by genetic testing of the beta-glucuronidases. Further, the following 3 pairs of primers were obtained by analyzing the total DNA of Escherichia coli in sewage, and β -glucuronidase was specifically amplified using the primers. Therefore, the primer set provided by the embodiment of the invention can be used for specifically detecting whether the Escherichia coli exists in water, has the advantages of strong detection result accuracy, high sensitivity, simplicity and convenience in operation, low cost and the like, and is suitable for wide application.
TABLE 1 primer sequences
In another aspect of the present invention, the present invention provides a method for detecting Escherichia coli in water. According to an embodiment of the invention, the method comprises: extracting total DNA in water; amplifying the total DNA and the ultrapure water respectively by utilizing the primer group so as to obtain a sample product and a control product; comparing the sample product to a control product to determine the presence or absence of Escherichia coli in the water. As described above, the primer can specifically amplify beta-glucuronidase on Escherichia coli, and can further specifically amplify beta-glucuronidase. Therefore, the method provided by the embodiment of the invention has the advantages of strong detection result accuracy, high sensitivity, simplicity and convenience in operation, low cost and the like, and is suitable for wide application.
According to an embodiment of the present invention, the above method for detecting escherichia coli in water may further have the following additional technical features:
according to the embodiment of the invention, the amplification is carried out by adopting a loop-mediated isothermal amplification mode. Compared with the conventional PCR, the loop-mediated isothermal amplification technology does not need the processes of thermal denaturation, temperature circulation, electrophoresis, ultraviolet observation and the like of a template, and reaction products can be detected simply and inexpensively, such as identification through turbidity observation, gel electrophoresis and a fluorescent dye method.
According to an embodiment of the invention, the alignment is performed by: staining the sample and control products with SYBR Green I dye; the sample product is colored green after staining and the control product is colored orange-red after staining, which is an indication of the presence of escherichia coli in the water; the sample and control products stained orange-red, which is an indication of the absence of escherichia coli in the water. Thus, the presence or absence of Escherichia coli in water can be accurately and conveniently determined.
According to the embodiment of the invention, the amplification is carried out on a fluorescent quantitative PCR instrument, and the content of Escherichia coli in water is determined by analyzing the amplification result. Therefore, the method has the advantages of high accuracy and high sensitivity.
According to an embodiment of the present invention, the amplification reaction system is as follows: WarmStart LAMP 2. mu.l premixed solution 12.5. mu.l, LAMP 50. mu.l fluorescent dye 0.5. mu.l, LAMP primer 2.5. mu.l, template 2. mu.l, ultrapure water 7.5. mu.l, reaction temperature 65 ℃ and time 45 min. Thus, the beta-glucuronidase gene can be specifically amplified to determine the presence or absence of Escherichia coli in water.
The invention establishes a method for detecting Escherichia coli in water, which reduces the time required by detection and can obtain a detection result in about one hour; meanwhile, the experiment can be carried out in a water bath kettle, the requirement on instruments and equipment is low, and the cost is greatly reduced. In conclusion, the method is a high-efficiency and low-cost method for detecting Escherichia coli in water, and can be used for evaluating the water quality safety.
In still another aspect of the present invention, the present invention provides a kit for detecting Escherichia coli in water. According to an embodiment of the invention, the kit comprises: the primer set described above. As described above, the primer set according to the embodiment of the present invention can specifically detect the presence or absence of Escherichia coli in water, and has the advantages of high accuracy of detection result, high sensitivity, easy operation, low cost, and the like, and is suitable for wide application.
Additional aspects and advantages of the invention will be set forth in part in the description which follows and, in part, will be obvious from the description, or may be learned by practice of the invention.
Drawings
The above and/or additional aspects and advantages of the present invention will become apparent and readily appreciated from the following description of the embodiments, taken in conjunction with the accompanying drawings of which:
FIG. 1 shows a diagram of isothermal PCR efficiency fluorescence detection according to one embodiment of the present invention;
FIG. 2 shows a SYBR Green I staining profile according to one embodiment of the present invention, wherein 1 is a control group, 2 is a 200-copy positive plasmid template, and 3 is a 20-copy positive plasmid template;
FIG. 3 shows a fluorescence detection map of isothermal PCR efficiency according to another embodiment of the present invention;
FIG. 4 shows a SYBR Green I staining profile according to one embodiment of the present invention, wherein 1 is a control group and 2 is an experimental group.
Detailed Description
The scheme of the invention will be explained with reference to the examples. It will be appreciated by those skilled in the art that the following examples are illustrative of the invention only and should not be taken as limiting the scope of the invention. The examples, where specific techniques or conditions are not indicated, are to be construed according to the techniques or conditions described in the literature in the art or according to the product specifications. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products commercially available.
Example 1
The following method is adopted to detect the amplification efficiency of the primer sequences shown in SEQ ID NO 1-6, and the specific method is as follows:
(1) construction of plasmid containing detection target gene fragment: the 6 primers are used for carrying out conventional PCR amplification on the beta-glucuronidase gene respectively, and the target gene fragment is connected into a plasmid vector.
(2) Transformation and amplification extraction of plasmids: the plasmids are transformed into competent cells respectively, and single clones are obtained by screening. Subsequently, the single-clone strains were inoculated in LB medium and cultured overnight at 37 ℃ and 220rpm/min, respectively. Plasmids were extracted using a plasmid extraction kit and their concentrations were determined.
(3) And (3) detecting the amplification efficiency of the primers: the copy number of the plasmid was calculated according to the concentration and size of the plasmid, and was diluted to 200 copies/. mu.l and 20 copies/. mu.l as a template for detection of the amplification efficiency of the primer. The reaction system is prepared according to the following proportion: WarmStart LAMP 2. mu.l premixed solution 12.5. mu.l, LAMP 50. mu.l fluorescent dye 0.5. mu.l, LAMP primer 2.5. mu.l, template 2. mu.l, and ultrapure water 7.5. mu.l. LAMP reaction is carried out on a real-time fluorescence quantitative PCR instrument, the reaction temperature is 65 ℃, and the reaction time is 45 min. The fluorescence intensity in the reaction system reflects the LAMP amplification condition, and the real-time fluorescence quantitative PCR instrument detects the change of the fluorescence intensity in the reaction system in real time, thereby realizing the detection of the amplification efficiency of the primers.
The amplification results are shown in FIGS. 1 and 2, wherein, as can be seen from FIG. 2, the results obtained by using 20 and 200 copies amplification are not very different, and the target fragment can be efficiently amplified.
Example 2
In this example, Escherichia coli in sewage was measured according to the following method:
(1) extracting DNA from sewage: 50ml of sewage is put into a centrifuge tube for high-speed centrifugation for 20min, and the supernatant is discarded. The total DNA of the wastewater was extracted in 50. mu.l, and the concentration thereof was measured using NanoDrop 2000.
(2) Detection of escherichia coli in sewage by LAMP: the reaction system is prepared according to the following proportion: WarmStart LAMP 2X 12.5. mu.l of premix, LAMP fluorochrome (50X) 0.5. mu.l, LAMP primer 2.5. mu.l, template 2. mu.l, and ultrapure water 7.5. mu.l. LAMP reaction is carried out on a real-time fluorescence quantitative PCR instrument, the reaction temperature is 65 ℃, and the reaction time is 45 min. In this example, a control group (ultrapure water as a template) and an experimental group (sewage DNA as a template) were set.
(3) The data obtained on the fluorescent quantitative PCR instrument were analyzed, and the results are shown in FIG. 3.
The amplification products obtained from the experimental group and the control group are detected by using a SYBR Green I staining method, which comprises the following steps:
(a) preparing SYBR Green I working solution: SYBR Green I is prepared into working solution with the concentration of 100 multiplied, and the working solution is stored in dark.
(b) Detection of LAMP products: adding SYBR Green I working solution into the LAMP product according to the proportion of 1:10, and fully shaking and uniformly mixing.
(c) And (4) interpretation of results: the mixture of SYBR Green I and LAMP product was centrifuged, and the color was observed, with the positive being Green and the negative being orange-red. That is, if the experimental group is green and the control group is orange-red, it indicates that escherichia coli exists in the water; if both the experimental and control groups are orange-red, this is an indication that no Escherichia coli is present in the water. The results are shown in FIG. 4.
As can be seen, the fluorescent quantitative PCR detection result is consistent with the SYBR Green I staining detection result, which shows the accuracy of the detection result of the invention and can effectively realize the determination of the Escherichia coli in water.
In the description herein, references to the description of the term "one embodiment," "some embodiments," "an example," "a specific example," or "some examples," etc., mean that a particular feature, structure, material, or characteristic described in connection with the embodiment or example is included in at least one embodiment or example of the invention. In this specification, the schematic representations of the terms used above are not necessarily intended to refer to the same embodiment or example. Furthermore, the particular features, structures, materials, or characteristics described may be combined in any suitable manner in any one or more embodiments or examples. Furthermore, various embodiments or examples and features of different embodiments or examples described in this specification can be combined and combined by one skilled in the art without contradiction.
Although embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting the present invention, and that variations, modifications, substitutions and alterations can be made to the above embodiments by those of ordinary skill in the art within the scope of the present invention.
SEQUENCE LISTING
<110> Qinghua university
<120> method for rapidly detecting Escherichia coli in water
<130> PIDC3192058
<160> 6
<170> PatentIn version 3.5
<210> 1
<211> 19
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<220>
<223> SEQ ID NO：1
<400> 1
ggatccatcg cagcgtaat 19
<210> 2
<211> 18
<212> DNA
<213> Artificial Sequence
<220>
<223> SEQ ID NO：2
<400> 2
ccacttgcaa agtcccgc 18
<210> 3
<211> 38
<212> DNA
<213> Artificial Sequence
<220>
<223> SEQ ID NO：3
<400> 3
gtcttgcgcg acatgcgtca ctctacacca cgccgaac 38
<210> 4
<211> 40
<212> DNA
<213> Artificial Sequence
<220>
<223> SEQ ID NO：4
<400> 4
gtctgttgac tggcaggtgg tgcaaccacc tgttgatccg 40
<210> 5
<211> 20
<212> DNA
<213> Artificial Sequence
<220>
<223> SEQ ID NO：5
<400> 5
<210> 6
<211> 19
<212> DNA
<213> Artificial Sequence
<220>
<223> SEQ ID NO：6
<400> 6
ggccaatggt gatgtcagc 19
Claims (7)
1. A primer set for detecting Escherichia coli in water, comprising:
SEQ ID NO: 1 to 6.
2. A method for detecting Escherichia coli in water, comprising:
extracting total DNA in water;
amplifying the total DNA and ultrapure water using the primer set of claim 1, respectively, to obtain a sample product and a control product;
comparing the sample product to a control product to determine the presence or absence of Escherichia coli in the water.
3. The method of claim 2, wherein the amplification is performed using loop-mediated isothermal amplification.
4. The method of claim 2, wherein the alignment is performed by:
staining the sample and control products with SYBR Green I dye;
the sample product is colored green after staining and the control product is colored orange-red after staining, which is an indication of the presence of escherichia coli in the water; the sample and control products stained orange-red, which is an indication of the absence of escherichia coli in the water.
5. The method of claim 3, wherein the amplification is performed on a fluorescent quantitative PCR instrument and the amplification result is analyzed to determine the Escherichia coli content in the water.
6. The method according to claim 3, wherein the amplification reaction system is as follows:
WarmStart LAMP 2. mu.l premixed solution 12.5. mu.l, LAMP 50. mu.l fluorescent dye 0.5. mu.l, LAMP primer 2.5. mu.l, template 2. mu.l, ultrapure water 7.5. mu.l, reaction temperature 65 ℃ and time 45 min.
7. A kit for detecting Escherichia coli in water, comprising: the primer set of claim 1.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113564267A (en) * | 2021-06-07 | 2021-10-29 | 广东粤港供水有限公司 | Application of gusA gene in detection of concentration of Escherichia coli in drinking water, detection reagent, detection method and detection device |
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| WO2012073053A1 (en) * | 2010-11-30 | 2012-06-07 | Diagon Kft. | Procedure for nucleic acid-based molecular diagnostic determination of bacterial germ counts and kit for this purpose |
| CN104818333A (en) * | 2015-05-08 | 2015-08-05 | 博奥生物集团有限公司 | System of detecting pathogenic microorganisms related to respiratory tract infection and LAMP primer specially used therefor |
| US20160251702A1 (en) * | 2015-02-26 | 2016-09-01 | IDGenomics, Inc. | Process and Kit for Predicting Antibiotic Resistance and Susceptibility of Bacteria |
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2019
- 2019-06-20 CN CN201910538317.3A patent/CN112111584A/en active Pending
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| CN101570783A (en) * | 2009-03-20 | 2009-11-04 | 郑秋月 | Detection kit and detection method for 9 species of pathogenic organisms in marine products |
| WO2012073053A1 (en) * | 2010-11-30 | 2012-06-07 | Diagon Kft. | Procedure for nucleic acid-based molecular diagnostic determination of bacterial germ counts and kit for this purpose |
| US20130324436A1 (en) * | 2010-11-30 | 2013-12-05 | Diagon Kft | Procedure for nucleic acid-based diagnostic determination of bacterial germ counts and kit for this purpose |
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| Title |
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| CHONNIKAN TONGPHROMA等: "Rapid and sensitive enumeration of total coliforms and Escherichia coli in water and foods by most-probable-number loop-mediated isothermal amplification (MPN-LAMP) method", 《SCIENCEASIA》 * |
| 谭贵良等: "《现代分子生物学及组学技术在食品安全检测中的应用》", 30 June 2014, 中山大学出版社 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113564267A (en) * | 2021-06-07 | 2021-10-29 | 广东粤港供水有限公司 | Application of gusA gene in detection of concentration of Escherichia coli in drinking water, detection reagent, detection method and detection device |
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Application publication date: 20201222 |