Isothermal amplification method for detecting escherichia coli, primer probe composition and application thereof

Abstract

The invention discloses an isothermal amplification method for detecting escherichia coli, a primer probe composition and application thereof. The primer probe composition provided by the invention is composed of a forward primer, a reverse primer and a probe, the forward primer has a nucleotide sequence as shown in SEQ ID No. 1, the reverse primer has a nucleotide sequence as shown in SEQ ID No. 2, and the probe has a nucleotide sequence as shown in SEQ ID No. 3. The invention also provides a method for detecting escherichia coli. The method for rapidly detecting escherichia coli is established by combining a recombinase polymerase isothermal amplification technology with a lateral chromatography technology, has the characteristics of simplicity in operation, high sensitivity, strong specificity, easiness in observation of reaction results, safety, no pollution and the like, and is very suitable for field detection and monitoring of food safety.

Description

Technical Field

[0001] The present invention relates to the detection of Escherichia coli, and specifically relates to a isothermal amplification method for detecting Escherichia coli, its primer-probe composition and uses, belonging to the technical field of food safety detection. Technical Background

[0002] In food safety issues, foodborne microorganisms have always ranked first. The diseases caused by foodborne microorganisms seriously endanger people's physical health and have become one of the most important worldwide health problems. Escherichia coli is usually called Escherichia coli and is one of the most common foodborne microorganisms in clinical practice at present. It is also often selected as an important research object in the fields of food hygiene and epidemiology. It was discovered by the German-Austrian pediatrician Escherich in 1885. For a long time, people always thought that it was a part of the normal intestinal flora and belonged to non-pathogenic bacteria. It was not until the mid-20th century that it was realized that some special serotypes of Escherichia coli are pathogenic to humans and animals, especially to infants and young livestock (poultry), often causing severe diarrhea and septicemia. Therefore, rapid and sensitive detection of Escherichia coli is an effective measure to ensure food safety and people's physical health.

[0003] At present, the methods for detecting Escherichia coli in food mainly include traditional culture methods, rapid detection methods and molecular detection methods. Among them, traditional culture detection is an internationally recognized method with high accuracy, but the culture process has a long cycle, is easily affected by environmental and human factors, and the biochemical experiments are cumbersome, and it cannot achieve rapid detection of batch products; the rapid detection method uses a fully automatic microbial biochemical identification and gene fingerprint identification instrument to identify Escherichia coli, which is convenient, fast, highly specific and sensitive, but there is a risk of false positives; the molecular detection method is a detection method based on conserved sequences such as 16S rRNA, 23S rRNA and the intergenic region sequence between 16S and 23S rRNA, using techniques such as PCR (polymerase chain reaction), product sequencing analysis and pulsed field gel electrophoresis. However, most of these detection techniques rely on subsequent electrophoresis processes, the operation process is relatively cumbersome, has high technical requirements for the detection personnel, and most require expensive instruments and equipment, so it is not suitable for on-site rapid detection. Therefore, there is an urgent need to develop a new rapid, simple and sensitive detection method for Escherichia coli.

[0004] Recombinase polymerase amplification (RPA) is a nucleic acid isothermal amplification technology developed by the British company TwistDx Inc in 2006. This technology can perform single-molecule nucleic acid detection at room temperature within 15 minutes, has very low requirements for hardware equipment, short reaction time, does not require complex sample processing, and the amplification products can be visually discriminated by lateral flow test strips, making it particularly suitable for in vitro diagnosis, veterinary medicine, food safety, biosafety, agriculture, etc. Summary of the Invention

[0005] The object of the present invention is to overcome the deficiencies of the prior art and provide a method for isothermal amplification for detecting Escherichia coli, its primer-probe composition and uses, and this method has the characteristics of high sensitivity, high specificity, visualization, simple operation and portability.

[0006] The present invention realizes the above-mentioned invention object through the following technical solutions:

[0007] A primer-probe composition for detecting Escherichia coli, which is composed of a forward primer, a reverse primer and a probe. The forward primer has the nucleotide sequence shown in SEQ ID No.1, the reverse primer has the nucleotide sequence shown in SEQ ID No.2, and the probe has the nucleotide sequence shown in SEQ ID No.3.

[0008] Preferably, the 5'-end of the reverse primer is labeled with biotin; the 5'-end of the probe is labeled with carboxyfluorescein FAM, and a tetrahydrofuran THF is added at 31 bases away from the carboxyfluorescent group, and the 3'-end is modified by C3spacer.

[0009] Preferably, the sequences of each primer and probe are as follows:

[0010] Forward primer: 5'-CACCGTTTGTGTGAACAACGAACTGAACTG-3'

[0011] Reverse primer: [5'-Biotin]-CTGTGACGCACAGTTCATAGAGATAACCTTC-3'

[0012] Probe: [5'-FAM]-GTGACGCATGTCGCGCAAGACTGTAACCACG-[THF]-GTCTGTTGACTGGCAGG-[3'-C3spacer]

[0013] The application of the above primer-probe composition in the detection of Escherichia coli in the non-disease diagnosis or treatment field.

[0014] Preferably, the non-disease diagnosis or treatment field is the food safety detection field.

[0015] A method for detecting Escherichia coli, comprising the following steps:

[0016] (1) RPA reaction system: 2.0 μL of 10 μmol / L forward primer, 2.0 μL of 10 μmol / L reverse primer, 0.6 μL of 10 μmol / L probe, 5.0 μL of template, 8.5 μL of ddH2O, 29.4 μL of Buffer A, 2.5 μL of magnesium acetate solution;

[0017] (2) RPA reaction amplification: Add the above reagents except the magnesium acetate solution to a 0.2 mL TwistAmp nfo reaction tube containing lyophilized enzyme powder in sequence, mix well, then add the magnesium acetate solution, mix well and quickly place it in a constant temperature water bath for incubation to carry out the PRA reaction;

[0018] (3) LFD detection of RPA products: Take 10 μL of the RPA amplification product and add it to 90 μL of diluent, mix well, then take 50 μL of the mixed solution and drop it onto a nucleic acid detection test strip for detection, develop color for 5 min, and determine the result according to the color development of the test strip.

[0019] Preferably, the PRA reaction conditions are: 42 °C, 20 min.

[0020] Preferably, when both the test line and the quality control line of the test strip show red, the determination result is positive; when the test line does not show color and the quality control line shows red, the determination result is negative; when the quality control line does not show color, the determination result is invalid.

[0021] The recombinase polymerase isothermal amplification method for detecting Escherichia coli of the present invention, the principle of which is to adopt the recombinase polymerase isothermal amplification technology to detect the conserved region of the specific gene of Escherichia coli, that is, the UidA gene of Escherichia coli, and this sequence can be used as one of the marker genes of Escherichia coli. The sample end of the lateral flow nucleic acid detection test strip carries colloidal gold particles labeled with an anti-FAM fluorescent group antibody, the test line on the NC membrane is coated with an anti-biotin antibody, and the quality control line is coated with an anti-anti-FAM antibody. After amplifying the target gene with a biotin-labeled primer and a FAM-labeled probe, the amplification solution is dropped into the sample end of the test strip, and a biotin antibody-nucleic acid-colloidal gold complex will be formed on the test line, showing a dark red band. Therefore, after the reaction, the presence or absence of Escherichia coli is judged by the color change of the test line on the lateral flow test strip.

[0022] Compared with the prior art, the advantages and positive effects of the present invention are as follows:

[0023] (1) Fast detection speed: The recombinase polymerase isothermal amplification method of the present invention for detecting Escherichia coli can complete amplification within 20 minutes at 42°C, and the entire detection process can be completed within 30 minutes. Compared with conventional PCR and real-time fluorescence quantitative PCR, which take several hours, the detection time is greatly shortened.

[0024] (2) Simple operation: The recombinase polymerase isothermal amplification method of the present invention for detecting Escherichia coli only needs to be kept at a constant temperature of 42°C to complete the experiment. This temperature is much lower than 60 - 95°C of fluorescence quantitative PCR and 63°C of loop-mediated isothermal amplification technology (LAMP), getting rid of the dependence on thermal cycling instruments and stable heat sources, and greatly expanding its scope of use.

[0025] (3) High sensitivity and strong specificity: The recombinase polymerase isothermal amplification method of the present invention for detecting Escherichia coli has high sensitivity, and the lowest detectable amount can reach 10 2 copies / μL; it has strong specificity and does not cross-react with other bacterial genera.

[0026] (4) Easy to promote: The recombinase polymerase isothermal amplification method of the present invention for detecting Escherichia coli is an isothermal amplification process without temperature changes, does not require expensive instruments and equipment, has low requirements for the professionalism of operators, can be applied to remote and resource-poor areas and on-site detection, and is expected to become a simple and conventional means for detecting Escherichia coli, which is of great significance for ensuring food safety. Description of the Drawings

[0027] Figure 1 It is a sensitivity test for the recombinase polymerase isothermal amplification method of detecting Escherichia coli.

[0028] Figure 2 It is a specificity test for the recombinase polymerase isothermal amplification method of detecting Escherichia coli. Detailed Embodiments

[0029] The present invention will be further elaborated below in conjunction with specific embodiments. It should be understood that these embodiments are only used to illustrate the present invention and not to limit the scope of the present invention. In addition, those skilled in the art may make various changes or modifications to the present invention within the scope defined by the appended claims, and these changes or modifications should also fall within the protection scope of the present invention.

[0030] Example 1: Design of RPA primers and probes

[0031] Through literature retrieval, the inventor analyzed and determined that the specific sequence in the UidA gene of Escherichia coli was used as the target gene in the present invention. According to the TwistAmp (https: / / www.twistdx.co.uk / en / support / manuals / twistamp-manuals) primer design operation manual, the RPA primers and probes were designed as shown in the following table.

[0032]

[0033]

[0034] Example 2: Establishment of a recombinase polymerase isothermal amplification method for detecting Escherichia coli

[0035] 1. RPA reaction system

[0036] 2.0 μL of 10 μmol / L forward primer, 2.0 μL of 10 μmol / L reverse primer, 0.6 μL of 10 μmol / L probe, 5.0 μL of template, 8.5 μL of ddH2O, 29.4 μL of Buffer A, 2.5 μL of magnesium acetate solution.

[0037] 2. RPA reaction amplification

[0038] Add the above reagents except the magnesium acetate solution to a 0.2 mL TwistAmp nfo reaction tube containing lyophilized enzyme powder in sequence, mix well, then add the magnesium acetate solution, mix well and quickly place it in a constant temperature water bath for incubation, and react at 42 °C for 20 min.

[0039] 3. LFD detection of RPA products

[0040] Take 10 μL of the RPA amplification product and add it to 90 μL of the dilution solution and mix well. Then take 50 μL of the mixed solution and drop it onto the nucleic acid test strip for detection. Color development for 5 min, and judge the result according to the color development of the test strip. If both the test line and the quality control line on the test strip show red, the judgment result is positive; if the test line does not show color and the quality control line shows red, the judgment result is negative; if the quality control line does not show color, the judgment result is invalid.

[0041] Example 3: Sensitivity evaluation of the recombinase polymerase isothermal amplification method for detecting Escherichia coli

[0042] Dilute the Escherichia coli positive plasmid by 10-fold serial dilution to 10 8 to 10 0A series of different concentrations such as copies / μL were taken, and 5.0 μL of each was detected by the recombinase polymerase isothermal amplification method established in Example 2 to determine the sensitivity of the recombinase polymerase isothermal amplification method for detecting Escherichia coli of the present invention. The DAN extraction solution was used as a negative control, and the results are shown in Figure 1 .

[0043] It can be seen from Figure 1 that a positive reaction can be presented starting from 10 2 copies / μL. Therefore, it is determined that the lowest detectable amount of Escherichia coli by the recombinase polymerase isothermal amplification method for detecting Escherichia coli of the present invention is 10 2 copies / μL.

[0044] Example 4: Specificity evaluation of the recombinase polymerase isothermal amplification method for detecting Escherichia coli

[0045] For the specificity evaluation, the genomic DNAs of Salmonella typhimurium, Listeria monocytogenes, Staphylococcus aureus, Vibrio parahaemolyticus, Enterobacter sakazakii, and Shigella flexneri were used as controls to determine the specificity of the recombinase polymerase isothermal amplification method for detecting Escherichia coli of the present invention.

[0046] Using the genomic DNAs of Salmonella typhimurium, Listeria monocytogenes, Staphylococcus aureus, Vibrio parahaemolyticus, Enterobacter sakazakii, and Shigella flexneri as templates respectively, the recombinase polymerase isothermal amplification method established in Example 2 was used for detection. At the same time, the DAN extraction solution was set as a negative control, and the results are shown in Figure 2 .

[0047] It can be seen from Figure 2 that no bands appeared in the detection lines of the genomic DNA samples of Salmonella typhimurium, Listeria monocytogenes, Staphylococcus aureus, Vibrio parahaemolyticus, Enterobacter sakazakii, and Shigella flexneri, showing negative results, indicating that the recombinase polymerase isothermal amplification method for detecting Escherichia coli of the present invention has strong specificity for Escherichia coli.

Claims

1. A primer-probe composition for detecting Escherichia coli, characterized in that, The primer-probe composition consists of a forward primer, a reverse primer, and a probe. The forward primer has the nucleotide sequence shown in SEQ ID No.1, the reverse primer has the nucleotide sequence shown in SEQ ID No.2, and the probe has the nucleotide sequence shown in SEQ ID No.

3.

2. The primer-probe composition according to claim 1, wherein The 5'-end of the reverse primer is labeled with biotin; the 5'-end of the probe is labeled with carboxyfluorescein FAM, and a tetrahydrofuran THF is added at 31 bases away from the carboxyfluorescent group, and the 3'-end is modified with C3spacer.

3. Application of the primer-probe composition according to claim 1 in the detection of Escherichia coli in the field of non-disease diagnosis or treatment.

4. The application according to claim 3, wherein The field of non-disease diagnosis or treatment is the field of food safety detection.

5. A method for detecting Escherichia coli, characterized in that, It includes the following steps: (1) RPA reaction system: 2.0 μL of 10 μmol / L forward primer, 2.0 μL of 10 μmol / L reverse primer, 0.6 μL of 10 μmol / L probe, 5.0 μL of template, 8.5 μL of ddH2O, 29.4 μL of Buffer A, 2.5 μL of magnesium acetate solution; (2) RPA reaction amplification: Add the above reagents except the magnesium acetate solution to a 0.2 mL TwistAmp nfo reaction tube containing freeze-dried enzyme powder in sequence, mix well, then add the magnesium acetate solution, mix well and quickly place it in a constant temperature water bath for incubation to carry out the PRA reaction; (3) LFD detection of RPA products: Take 10 μL of the RPA amplification product and add it to 90 μL of diluent and mix evenly. Then take 50 μL of the mixed solution and drop it onto a nucleic acid test strip for detection, develop color for 5 min, and judge the result according to the color development of the test strip.

6. The method according to claim 5, wherein The PRA reaction conditions are: 42 °C, 20 min.

7. The method according to claim 5, characterized in that, When both the test line and the quality control line of the test strip show red, the judgment result is positive; when the test line does not show color and the quality control line shows red, the judgment result is negative; when the quality control line does not show color, the judgment result is invalid.

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