CN116904623A - Primer group for detecting escherichia coli and detection method - Google Patents
Primer group for detecting escherichia coli and detection method Download PDFInfo
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- C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
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Abstract
The invention relates to the technical field of biology, in particular to a primer group for detecting escherichia coli and a detection method. The primer group for detecting escherichia coli comprises the nucleotide sequences of the primers shown in SEQ ID NO. 1-SEQ ID NO. 5; the primer group for detecting the escherichia coli provided by the invention is combined with the LAMP technology to detect the escherichia coli, has high sensitivity and good specificity, can realize simple preliminary screening of the escherichia coli within 1 hour, and does not need enrichment so as to meet the requirement of instant detection of the escherichia coli.
Description
Technical Field
The invention relates to the technical field of biology, in particular to a primer group for detecting escherichia coli and a detection method.
Background
Escherichia coli, also called Escherichia coli, is a conditional pathogenic bacterium, and under certain conditions, may cause gastrointestinal tract infection, urinary tract infection and other local tissue and organ infection of human and various animals. The strain is widely used in the nature, closely contacts with human beings, and easily causes food pollution to cause misfeeding of human beings, thereby causing a plurality of serious food safety accidents and clinical infection accidents. Therefore, how to rapidly, accurately and conveniently detect food-borne pathogenic microorganisms is very important to the supervision of food safety problems.
Currently, common detection methods for escherichia coli in foods include traditional culture methods, biochemical detection methods, immunological methods, molecular biological methods and the like. In the detection methods, the whole detection time of the traditional culture method and the biochemical detection method is long, and the detection methods are easily influenced by the morphology, the number and the variety of bacterial colonies of the mixed bacteria, so that false negative results appear; immunological methods, such as enzyme-linked immunosorbent assay, have long detection cycle and complicated steps, various non-relevant antigens are easy to cause cross reaction, and enterotoxin protein agglutination and the like generated in the food heating process can also cause false positive or false negative of detection results; molecular biology methods, such as multiplex PCR techniques, real-time fluorescent quantitative PCR methods, etc., are also susceptible to various factors such as sample components. These conventional methods for detecting Escherichia coli either require a long-term process, skilled operators and expensive laboratory equipment, or suffer from instability, low sensitivity, poor accuracy, and the like. The operation is complex, the detection period is long, the accuracy is low, and the adverse factors lead to difficulty in meeting the requirement of on-site rapid detection in sudden public health events (such as food poisoning); meanwhile, in application scenes of home food safety detection, food processing production sampling inspection, food safety monitoring in the cold chain transportation process and the like which are not suitable for laboratory detection, a novel rapid, accurate and portable detection method is needed to monitor escherichia coli in real time.
In recent years, a rapid detection method of Escherichia coli has been rapidly studied. Quick detection test paper for Escherichia coli, such as a test piece of Escherichia coli from 3M company, has been proposed at home and abroad, and the nutrition containing crystal violet neutral red bile salt (VRB), a gelling agent and a tetrazolium salt indicator, which are necessary for conventional test of Escherichia coli, are concentrated in the test piece to achieve the effect of quick detection, but the whole detection process still needs 24-48 hours of reaction time. The immune analysis detection method mainly uses enzyme-linked immune technology, and uses antigen (or antibody) to adsorb on solid carrier to retain activity, adds enzyme-labeled antibody, then makes immune enzyme staining on carrier, after the substrate is developed, and can determine pathogenic bacteria content by analyzing coloured product quantity. The molecular biological detection method, such as a multiplex PCR method, a real-time fluorescence quantitative PCR method and the like, has high accuracy and specificity, but the required instrument and equipment are complex, the operation requirement is high, and the rapid detection cannot be performed rapidly and conveniently; the LAMP isothermal amplification method is also developed and applied to a certain extent, and has the advantages of short amplification time and high sensitivity compared with the PCR technology, but the false positive of the LAMP product is difficult to distinguish, and the detection method of the LAMP amplification product is still fluorescent detection or turbidity detection, so that the LAMP amplification technology cannot be popularized and applied to a large extent due to large volume of required instruments and equipment, high price and high operation requirement.
The existing detection methods for escherichia coli all need the step of increasing bacteria, and the separation culture method needs to increase bacteria for many times in a plurality of days, so that the detection methods cannot be used in scenes needing instant detection, such as a school enterprise institution canteen, family detection, cold chain transportation spot inspection and the like.
Disclosure of Invention
In view of the above drawbacks of the prior art, an object of the present invention is to provide a primer set and a detection method for detecting escherichia coli, so as to solve the technical problems of complicated detection, low sensitivity and long detection time of escherichia coli in the prior art.
In one aspect, the present invention provides a primer set for detecting Escherichia coli, the primer set comprising the nucleotide sequence of the primer as follows:
SEQ ID NO.1：5’-TGGGCAGATGAACATGGAAT-3’；
SEQ ID NO.2：5’-GGTTCGTTGGCAATACTCCA-3’；
SEQ ID NO.3：
5’-TCTTTCGGCTTGTTGCCCGCTTTTCAGCTGTCGGCTTTAACCTC-3’；
SEQ ID NO.4：
5’-AAGAGGCAGTCAACGGGGAAACTTTTCTTGGGTGGTTTTTGTCACG-3’；
SEQ ID NO.5：5’-CAGCAGGCGCACTTACAGGC-3’。
wherein, the nucleotide sequence of the forward outer primer F3 is shown as SEQ ID NO. 1; the nucleotide sequence of the reverse outer primer B3 is shown as SEQ ID NO. 2; the nucleotide sequence of the forward inner primer FIP is shown as SEQ ID NO. 3; the nucleotide sequence of the reverse inner primer BIP is shown as SEQ ID NO.4, and the nucleotide sequence of the reverse loop primer LB is shown as SEQ ID NO. 5.
Specifically, the invention also provides application of the primer set, the primer set can be applied to preparation of an escherichia coli kit, or application of the primer set in detection of escherichia coli, application of the primer set in a microfluidic chip for detecting escherichia coli, and particularly application of the primer set in detection of escherichia coli by using a loop-mediated isothermal amplification-spectrum sensor-artificial intelligence technology platform.
In still another aspect, the present invention provides a method for detecting Escherichia coli, comprising: performing loop-mediated isothermal amplification on the sample to be detected by combining the LAMP detection technology with the primer group; the amplified products were analyzed.
Further, the reaction temperature of the loop-mediated isothermal amplification is 65 ℃ and the reaction time is 25-30 minutes.
Furthermore, the detection method is characterized in that before loop-mediated isothermal amplification is carried out, bacteria increase is carried out on the sample to be detected or not.
Further, the loop-mediated isothermal amplification reaction is performed in a microfluidic chip.
Further, the method for analyzing the amplified product comprises performing spectral analysis and/or chromogenic analysis on the LAMP reaction result.
The invention has the beneficial effects that the LAMP technology is combined with the primer group for detecting the escherichia coli to detect the escherichia coli, so that the sensitivity is high, the specificity is good, the simple preliminary screening of the escherichia coli can be realized within 1 hour, and the enrichment is not needed, so that the instant detection of the escherichia coli is satisfied.
Additional features and advantages of the invention will be set forth in the description which follows, and in part will be obvious from the description, or may be learned by practice of the invention. The objectives and other advantages of the invention will be realized and attained by the structure particularly pointed out in the written description and drawings.
In order to make the above objects, features and advantages of the present invention more comprehensible, preferred embodiments accompanied with figures are described in detail below.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings that are needed in the description of the embodiments or the prior art will be briefly described, and it is obvious that the drawings in the description below are some embodiments of the present invention, and other drawings can be obtained according to the drawings without inventive effort for a person skilled in the art.
FIG. 1 is a screening result of the LAMP reaction of 3 sets of primer groups designed for the uidA gene sequence in Escherichia coli at 30 minutes;
FIG. 2 shows the results of direct detection of Escherichia coli by LAMP reaction at various concentrations;
FIG. 3 shows the results of the specificity test of the present invention.
Detailed Description
For the purpose of making the objects, technical solutions and advantages of the embodiments of the present invention more apparent, the technical solutions of the present invention will be clearly and completely described below with reference to the accompanying drawings, and it is apparent that the described embodiments are some embodiments of the present invention, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
The E.coli standard bacterial liquid used in the examples of the present invention was ATCC25922 (Hirudo Highway Seabo Biotechnology Co., ltd. "Haabo organism") as the sampleThe concentration is about 10 6 CFU/mL. Salmonella (Sal) standard bacteria solution is ATCC14028 (Haibo organism), listeria Monocytogenes (LM) standard bacteria solution is ATCC19114 (Haibo organism), staphylococcus aureus standard bacteria solution ATCC6538 (Haibo organism), and the sample concentration of each standard bacteria solution is about 10 6 CFU/mL。
The 2 XLAP premix according to the embodiments of the present invention may be a commercially available NEB (U.S.) 2 XLAP premix, or may be self-contained and may contain MgSO 4 ，MgSO 4 The concentration of (2) is 6 mM-12 mM, buffer solution, dNTPs, KCl, (NH) 4 ) 2 SO 4 Bst DNA polymerase, phenol red, sterilized enzyme-free water, etc.
Primer sequences involved in embodiments of the present invention may be ordered from the division of biological engineering (Shanghai).
1. Primer design and screening
Downloading a conserved sequence on the uidA gene of beta-D-glucuronidase in Escherichia coli (E.coli) from NCBI as a primer design template; a plurality of LAMP primer sets were designed in the primer design website (http:// primrexPLorer. Jp/e /).
By the above method, the sequences of 3 LAMP primer groups were preliminarily designed for selection, and the nucleotide sequences of the primers of each group are shown in Table 1 below:
TABLE 1 primer design
Using a standard escherichia coli bacterial solution as a positive sample, sterilizing enzyme-free water (DW) as a negative sample, performing LAMP amplification experiments on the three groups of primers respectively by adopting the negative sample and the positive sample, observing color change of an LAMP reaction system, and photographing at 30 minutes, wherein the LAMP reaction system of the positive sample of the third group of primers starts to turn orange yellow after 30 minutes of constant temperature reaction, the LAMP reaction system of the positive sample of the first group of primers turns orange yellow after 15 minutes and turns yellow after 20 minutes, and the LAMP reaction system of the negative sample does not turn color after 30 minutes of constant temperature reaction, so that the first group of primers is the primer group with optimal LAMP reaction.
2. Reaction conditions
After several rounds of different reaction condition tests, the following conditions are finally selected as the final reagent proportion and reaction conditions:
(1) The concentration ratio of the primer:
TABLE 2
| Working concentration (mu M) | 10 Xprimer mix concentration (μM) | |
| FIP | 1.6 | 16 |
| BIP | 1.6 | 16 |
| F3 | 0.2 | 2 |
| B3 | 0.2 | 2 |
| LB | 0.4 | 4 |
(2) Preparing a 10 x primer mixture:
TABLE 3 Table 3
| Primer concentration (μM) | Primer name | 40 reactions (. Mu.L) were prepared |
| 100 | FIP | 8 |
| 100 | BIP | 8 |
| 10 | F3 | 10 |
| 10 | B3 | 10 |
| 100 | LB | 2 |
| Sterilized enzyme-free water | 12 | |
| Total volume (mu L) | 50 |
(3) Primer in LAMP reaction system, 2×LAMP premix and positive/negative sample ratio:
TABLE 4 Table 4
| 1 reaction (mu L) | |
| 2×LAMP premix | 12.5 |
| 10X primer mix | 2.5 |
| Positive/negative samples | 10 |
The positive sample may be a purchased standard escherichia coli broth, or a cultured escherichia coli broth dilution, or a single colony of escherichia coli culture or a dilution thereof, or escherichia coli inoculated into different food samples.
The negative sample may be sterilized enzyme-free water (DW), or sterilized pure milk.
(4) Reaction conditions
The amplification is carried out for 25 to 30 minutes at the constant temperature of 65 ℃ and has better detection result.
The heating can be performed by a constant temperature heating device (a water bath, a metal bath or a constant temperature oven).
3. Detection of Escherichia coli without the need for enrichment
The instant detection method is simple and easy to operate, can be completed within 30-60 minutes from sampling to outputting the result, and can be used for simple and convenient primary screening of the escherichia coli in liquid foods without increasing bacteria.
First step, preparation of Escherichia coli positive/negative samples
Concentration is set to 10 9 The CFU/ml escherichia coli bacterial liquid is sequentially diluted by 10 times of diluted sterile milk according to 1/10 times of gradient, and the obtained concentration is 10 respectively 6 CFU/ml，10 5 CFU/ml，10 4 CFU/ml，10 3 CFU/ml escherichia coli positive sample; sterile milk diluted 10-fold with sterilized enzyme-free water was used as a negative sample.
In the second step, a 10X primer mixture was prepared according to the method described in Table 3.
In the third step, 10. Mu.L of each of the positive/negative samples prepared in the first step was taken, and the LAMP reaction system for each reaction was prepared according to the method shown in Table 4.
Fourth, heating at 65 ℃ and reacting for 25-30 minutes.
As a result of the reaction, as shown in FIG. 2, escherichia coli incorporated in milk was clearly detected (color changed to yellow) at a limit of 10 4 CFU/ml (of 5 positive samples, 5 colors yellow); while no clear color change occurred in the negative samples (mauve in all 5 samples).
4. Specificity experiments
For detection of the specificity of the detection primer set of Escherichia coli, a cross-reaction test was performed using Salmonella (Sal) and Listeria Monocytogenes (LM) and Staphylococcus Aureus (SA), respectively.
First step, preparation of Escherichia coli positive/negative samples for cross test
Concentration is set to 10 6 The escherichia coli bacterial liquid of CFU/ml is taken as a positive sample, and the concentration of each bacterial liquid is 10 6 The mixed solution of the salmonella standard bacterial solution, the listeria monocytogenes standard bacterial solution and the staphylococcus aureus standard bacterial solution of CFU/ml is used as a negative sample;
in the second step, a 10X primer mixture was prepared according to the method described in Table 3.
In the third step, 10. Mu.L of each of the positive/negative samples prepared in the first step was taken, and the LAMP reaction system for each reaction was prepared by the method shown in Table 4.
Fourth, heating at 65 ℃ and reacting for 25-30 minutes.
As shown in FIG. 3, it was found that Escherichia coli was clearly detected (color changed to yellow), and the reaction was not affected by other food-borne pathogens such as Salmonella, listeria monocytogenes, and Staphylococcus aureus, and that no clear color change, i.e., no clear LAMP reaction, was observed in each of the negative samples containing Salmonella, listeria monocytogenes, and Staphylococcus aureus.
The sensitivity and specificity of the LAMP detection of Escherichia coli were calculated preliminarily from the experimental results of FIGS. 2 and 3, and 20 of 20 positive samples were counted as clear positive results (one positive sample had a change in color of orange yellow, and a pink color of negative sample had formed a more clear change, and could be judged as positive samples) from the experimental results of FIGS. 2 and 3, so the sensitivity was calculated as:according to the experimental result statistics of fig. 3, 20 of the negative samples of 20 other strains exhibited clear negative results, so the specificity was calculated as:
the detection method can also detect the escherichia coli through a loop-mediated isothermal amplification-spectrum Sensor-artificial intelligence (LAMP-Spectral Sensor-AI) technology platform, can directly use diluted liquid food samples (such as milk), inject the diluted liquid food samples into an LAMP reaction reagent integrated in a reaction cavity of a microfluidic chip for amplification, analyze amplification products through a spectrum Sensor, and comprehensively judge the collected spectrum signals through an AI technology, thereby rapidly judging whether the samples are polluted by the escherichia coli. By confining the sample to be tested to the microfluidic environment of the device, the risk of sample contamination is reduced and the sample volume and reagents required for detection are minimized, thereby further reducing the overall cost of screening and detection. The detection technology can be used for the instant detection of food, the detection is carried out without bacteria increase, the result can be obtained within 30-60 minutes, the detection technology has the advantages of rapidness, accuracy, simplicity, convenience, pollution prevention and the like, and the detection of rapid, accurate and automatic operation in household, transportation and sudden public health events is of great significance to the establishment of a food safety monitoring system.
With the above-described preferred embodiments according to the present invention as an illustration, the above-described descriptions can be used by persons skilled in the relevant art to make various changes and modifications without departing from the scope of the technical idea of the present invention. The technical scope of the present invention is not limited to the description, but must be determined according to the scope of claims.
Claims (9)
1. A primer set for detecting escherichia coli, characterized in that the primer set comprises the nucleotide sequence of a primer as follows:
SEQ ID NO.1：5’-TGGGCAGATGAACATGGAAT-3’；
SEQ ID NO.2：5’-GGTTCGTTGGCAATACTCCA-3’；
SEQ ID NO.3：
5’-TCTTTCGGCTTGTTGCCCGCTTTTCAGCTGTCGGCTTTAACCTC-3’；
SEQ ID NO.4：
5’-AAGAGGCAGTCAACGGGGAAACTTTTCTTGGGTGGTTTTTGTCACG-3’；
SEQ ID NO.5：5’-CAGCAGGCGCACTTACAGGC-3’。
2. the use of the primer set according to claim 1 for preparing an escherichia coli kit.
3. The use of the primer set according to claim 1 for detecting Escherichia coli.
4. The use of the primer set according to claim 1 in a microfluidic chip for detecting escherichia coli.
5. A method for detecting escherichia coli, comprising:
performing loop-mediated isothermal amplification on a sample to be detected by combining the LAMP detection technology with the primer group according to claim 1;
the amplified products were analyzed.
6. The method for detecting Escherichia coli according to claim 5,
the reaction temperature of the loop-mediated isothermal amplification is 65 ℃ and the reaction time is 25-30 minutes.
7. The method for detecting Escherichia coli according to claim 5,
before loop-mediated isothermal amplification, the sample to be detected is subjected to or not subjected to enrichment.
8. The method for detecting Escherichia coli according to claim 6,
the loop-mediated isothermal amplification reaction is performed in a microfluidic chip.
9. The method for detecting Escherichia coli according to claim 5,
the method for analyzing the amplified product comprises spectral analysis and/or chromogenic analysis of the LAMP reaction result.
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