CN118240950A - LAMP-based escherichia coli detection kit and rapid detection method - Google Patents
LAMP-based escherichia coli detection kit and rapid detection method Download PDFInfo
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Abstract
The invention discloses an LAMP-based escherichia coli detection kit and a rapid detection method, wherein the kit comprises a kit specific to escherichia coli O157: the LAMP amplification specificity F3, B3, FIP and BIP primer groups for detecting the H7 specific gene rfbe are designed, a fast detection method of the escherichia coli based on the LAMP amplification is established by designing the primer groups through the specific gene, the advantages of rapidness, simplicity, high specificity and independence on expensive instruments and equipment are achieved, and experiments show that the LAMP amplification detection limit of the escherichia coli is 10 1 CFU/mL, and the method has high specificity and sensitivity.
Description
Technical Field
The invention relates to a detection method of escherichia coli, in particular to an LAMP-based escherichia coli detection kit and a rapid detection method.
Background
Coli O157: H7 is the most typical one of enterohemorrhagic E.coli (enterohemorrhage Escherichia coli, EHEC), EHEC O157: H7 is gram-negative bacteria, has no spores, has periflagella, most strains have capsules, has higher tolerance to low temperature, has acid resistance, and can cause infection by 50-100 bacteria. Infection with E.coli O157: H7 mainly causes diarrhea and hemorrhagic colitis in infants, and is complicated with hemolytic uremic syndrome and thrombotic thrombocytopenic purpura, leading to death in severe cases. Coli O157H 7 transmission paths are various, polluted raw meat and meat products, non-sterilized milk, polluted fruits and vegetables, polluted drinking water, farmland irrigation water and the like can cause infectious epidemic, wherein food-origin transmission is mainly used, and water-origin and contact transmission are also important transmission paths.
Coli O157 was found by sequence analysis: the specific gene rfbe gene of H7 and the rfbe gene of Escherichia coli O157H 7 are presumed to code mannitol synthase, the current PCR method for detecting Escherichia coli O157 is mostly based on rfbe as target gene and the immunological detection method is also established based on O antigen coded by the gene, wherein some detection methods, such as the detection basis of immunoassay, are based on the specific reaction of antigen and antibody, and the detection method has the advantages of high sensitivity and strong specificity, but is easy to generate false positive and false negative; for example, in the molecular biology detection method, the polymerase chain reaction (Polymerase Chain Reaction, PCR), reverse transcription-polymerase chain reaction (reverse transcription Polymerase Chain Reaction, RT-PCR), real-time fluorescent quantitative-polymerase chain reaction (real-time Polymerase Chain Reaction, real-time PCR) and the like are widely applied, but the PCR technology cannot always get rid of the limitations of dependence on precise instruments and equipment, high-cleanliness requirement operating conditions, overlong reaction time and the like.
Disclosure of Invention
The invention aims to solve the problems in the background art, and provides an LAMP-based escherichia coli detection kit and a rapid detection method, so as to solve the problems in the prior art.
The technical scheme adopted by the invention is as follows:
The LAMP-based escherichia coli detection kit comprises a pretreatment reagent and a detection reagent, wherein the detection reagent comprises a reagent specific to escherichia coli O157: LAMP amplification specificity F3, B3, FIP and BIP primer groups for detecting H7 specific gene rfbe genes, wherein the base sequences of the primers are as follows:
F3：CCAGTTAGAACAAGCTGATGA；
B3：GGCCTTGTTTCGATGAGTT；
FIP：CTTGTGGACTTGTACAAGACTGT-CGAAAACGTGAAATTGCTGA；
BIP：TTCACACTTATTGGATGGTCTCAAT-GGTGATTCCTTAATTCCTCTCT。
Further, the outer primers F3 and B3 in the F3, B3, FIP and BIP primer sets each contain 0.2. Mu. Mol/L, and the inner primers FIP and BIP each contain 1.6. Mu. Mol/L.
Further, the detection reagent also comprises 8U Bst DNA polymerase, 1.4mmol/L dNTPs, 6mmol/L MgSO 4, 1 xLAMP buffer, 1 xLAMP fluorescent dye, DNA template and ddH 2 O.
Further, the pretreatment reagent comprises one of 100. Mu.l of 50mM NaOH and 100. Mu.l of 50mM HCl, 100. Mu.l of 0.5% Triton X-100.
A fast detection method of colibacillus based on LAMP includes the following steps:
S1 sample pretreatment
Extracting a DNA template by a sample pretreatment method by using a pretreatment reagent;
screening of S2 LAMP amplification primers
Mixing the obtained DNA template with a detection reagent, wherein the reaction system is 20 mu L and comprises F3, B3, FIP, BIP primer groups, bst polymerase, dNTPs, mgSO 4, 10x LAMP buffer,LAMP fluorescent dye, DNA template, ddH 2 O, and putting the prepared LAMP reaction system into a qPCR instrument, setting the program to 65 ℃ for 40min, and screening out a proper amplification primer;
s3 LAMP amplification detection limit
Detecting the primer pair Escherichia coli O157: h7 detection limit, diluting escherichia coli into bacterial solutions with different concentrations by using distilled water, extracting bacterial solution DNA by using a pretreatment reagent, carrying out LA MP amplification detection by taking the DNA template as a target, and obtaining the detection limit of the primer by observing the generation of an amplification signal;
Further, the method further comprises the steps of culturing and counting escherichia coli to determine the concentration of bacterial liquid, inoculating escherichia coli strains into a sterilized LB culture medium, performing shaking culture on a shaking table at 37 ℃ and 200r/min for overnight, diluting the bacterial liquid with distilled water according to a 10-time gradient, taking 50 mu L of diluted bacterial liquid to be coated in the LB solid culture medium, placing the coated bacterial liquid into a constant temperature incubator, culturing overnight at 37 ℃, and recording the colony number of a flat plate.
Further, the sample pretreatment in the step S3 comprises a thermal cracking method by alkali addition, mixing well after adding 100. Mu.l of 50mM NaOH, heating on a metal bath at 95 ℃ for 10min, adding 100. Mu.l of 50mM HCl for neutralization, then centrifuging at 12000rpm/min at 25 ℃ for 3min, and collecting the supernatant as a DNA template.
Further, the sample pretreatment in the step S3 also comprises a Triton X-100+ thermal cracking method, 100 μl of 0.5% Triton X-100 is added and mixed uniformly, the mixture is placed on a metal bath and heated at 95 ℃ for 10min, then 12000 rpm/min and centrifuged at 25 ℃ for 3min, and the supernatant is collected as a DNA template.
Further, the detection limit of LAMP amplification of E.coli was 10 1 CFU/mL, which was obtained by pretreatment of the sample.
Compared with the prior art, the invention has the beneficial effects that:
The invention utilizes bioinformatics to determine specific genes of escherichia coli, and designs primers aiming at the genes, so that the rapid detection method of escherichia coli based on LAMP amplification is established, the advantages of rapidness, simplicity, high specificity and no dependence on expensive instruments and equipment are realized, experiments show that the detection effect of a DNA sample extracted by an alkali thermal cracking method is best, the Ct value is larger when the DNA sample extracted by a TritonX-100 thermal cracking method is detected, the detection time is longer than that of the alkali thermal cracking method, the detection limit of LAMP amplification of both methods reaches 10 1 CFU/mL, and the detection limit of the DNA sample extracted by a commercial bacterial genome DNA extraction kit is consistent with that of a bacterial liquid sample. The reagent used in the alkaline heating pyrolysis method has no obvious influence on the LAMP reaction system, does not need any concentration step when the bacterial liquid sample is processed, reduces the sample loss, greatly shortens the sample pretreatment time (10 min), and is obviously superior to a bacterial genome DNA extraction kit (90 min). When the LAMP amplification is used for detecting the DNA of the actual bacterial liquid sample extracted by the alkaline thermal cracking method, the sensitivity, the specificity and the total coincidence rate are high. The reagent required by the alkaline heating pyrolysis method for extracting the bacterial liquid sample DNA established by the research is simple, the detection cost is reduced without depending on a large instrument, the integrated on-site rapid detection can be realized, and the technical support is provided for developing on-site detection of food-borne pathogenic microorganisms.
Drawings
Fig. 1: primer set 1 was used to amplify the LAMP amplification curves of E.coli at 10 7CFU/mL,106 CFU/mL and 10 5 CFU/mL;
Fig. 2: primer group 2 was used to amplify the graphs of E.coli LAMP with 10 7CFU/mL,106 CFU/mL and 10 5 CFU/mL;
Fig. 3: primer set 3 (invention) versus E.coli LAMP amplification plots of 10 7CFU/mL,106 CFU/mL and 10 5 CFU/mL;
Fig. 4: the LAMP amplification product verification chart of the primer group
Fig. 5: method a LAMP amplification curves;
fig. 6: a LAMP amplification contrast graph of method b and method a;
fig. 7: a LAMP amplification contrast graph of method c and method a;
fig. 8: a LAMP amplification contrast graph of the method d and the method a;
Fig. 9: a LAMP amplification contrast graph of the method e and the method a;
Fig. 10: a LAMP amplification contrast graph of method f and method a;
Fig. 11:10 LAMP amplification curve graph after 7 CFU/mL sample method b condition optimization;
fig. 12:10 LAMP amplification curve graph after 4 CFU/mL sample method b condition optimization;
fig. 13:10 LAMP amplification curve graph after 7 CFU/mL sample method c condition optimization;
fig. 14:10 LAMP amplification curve graph after 4 CFU/mL sample method c condition optimization;
Fig. 15: a LAMP amplification detection limit diagram of the method a;
fig. 16: the LAMP amplification detection limit diagram of the method b;
fig. 17: method c LAMP amplification detection limit map.
Detailed Description
In order to clearly illustrate the technical features of the present solution, the present application will be described in detail below with reference to the following detailed description and the accompanying drawings. In the following description, numerous specific details are set forth in order to provide a thorough understanding of the present application, but the present application may be practiced in other ways than those described herein, and therefore the scope of the present application is not limited to the specific embodiments disclosed below. Unless defined otherwise, all technical and scientific terms used hereinafter have the same meaning as commonly understood by one of ordinary skill in the art. The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the scope of the present application.
Example 1
An embodiment of the present invention provides: a fluorescent LAMP-based escherichia coli rapid detection kit,
The detection reagent comprises: the outer primers F3 and B3 in the F3, B3, FIP and BIP primer sets each contained 0.2. Mu.M, and the inner primers FIP and BIP each contained 1.6. Mu.M, 8U Bst DNA polymerase, 1.4mmol/L dNTPs, 6mmol/L MgSO 4, 1 xLAMP buffer, 1 xLAMP fluorescent dye, DNA template, ddH 2 O.
The pretreatment reagent comprises: 100. Mu.l of 50mM NaOH and 100. Mu.l of 50mM HCl, 100. Mu.l of one of 0.5% Triton X-100,
Wherein: for E.coli O157: amplification detection primer of H7 specific gene rfbe gene, LAMP amplification primer is designed by using primer design website LAMP PRIMER DESIGNING software PrimerExplorer (http:// primere xplorer.jp/e /), and comprises a pair of outer primers and a pair of inner primers, wherein the outer primers are F3 and B3, and the inner primers are FIP (F1 and F2) and BIP (B1 and B2); the sequence information is shown in Table 1.
Table 1 escherichia coli O157: h7 LAMP amplification primer sequence group Table
And screening the designed LAMP amplification primers of the escherichia coli, selecting primers with good amplification curve and quick peak time from the primers, and performing electrophoresis analysis on amplification products to verify the availability of the primers. The primer screening results are shown in FIGS. 1-3, and the E.coli concentrations in FIGS. 1-3 are 10 7CFU/mL,106 CFU/mL and 10 5 CFU/mL, respectively. The amplification effect of the 1 st group primer and the 2 nd group primer is poor, no amplification signal is generated by the 1 st group primer, the 2 nd group primer only generates amplification signals at the concentration of 10 7 CFU/mL and 10 6 CFU/mL, the cycle threshold (Ct) is larger, the 3 rd group primer generates amplification signals at the concentration of 3 samples, the Ct value is smaller, and non-specific amplification does not occur in a negative control group (ntc) which takes non-polluted enzyme-free water as an amplification template; the amplified products of the 3 rd primer set were then subjected to gel electrophoresis detection, and the results are shown in FIG. 4, lanes 1-4 of FIG. 4: marker,10 7 CFU/mL sample LAMP amplification product, 10 4 CFU/mL sample LAMP amplification product, negative control group (non-contaminated non-enzymatic water as amplification template). Since a large number of DNA mixtures with different lengths, different numbers of stem-loop structures and inverted repeat sequences can be generated after LAMP amplification, agarose gel electrophoresis presents trapezoid stripes with different lengths, and no stripe appears in a negative control group (ntc) which takes pollution-free enzyme-free water as an amplification template, which indicates that the 3 rd group primer does not generate nonspecific amplification and can be used for the LAMP detection kit amplification primer.
Example 2
A fast detection method of colibacillus based on LAMP includes the following steps:
S1 sample pretreatment
The sample pretreatment method was performed with 100. Mu.l of 50mM NaOH and 100. Mu.l of 50mM HCl pretreatment reagent to extract the DNA template;
s3 LAMP amplification primer design and test
The reaction system was 20. Mu.L, including F3, B3, FIP, BIP primer set, bst polymerase, dNTPs, mgSO 4, 10x LAMP buffer,LAMP fluorescent dye, DNA template, ddH 2 O, and LAMP reaction system was prepared and put into qPCR apparatus, setting the procedure at 65℃for 40min, and investigating the performance of the designed primers.
S3 LAMP amplification detection limit
Detecting the primer pair Escherichia coli O157: h7 detection limit, diluting Escherichia coli into bacterial solutions with different concentrations with distilled water, performing sample pretreatment, adding 100 μl of 50mM NaOH, mixing, heating at 95deg.C for 10min in a metal bath, adding 100 μl of 50mM HCl for neutralization, centrifuging at 12000rpm/min at 25deg.C for 3min, collecting supernatant as DNA template, performing LAMP amplification detection with the supernatant as template, and observing generation of amplification signals to obtain LAMP amplification detection limit of the different pretreatment methods.
Comparison of LAMP amplification results of E.coli genomic DNA extracted by different sample pretreatment methods and commercial kits
Screening of sample pretreatment methods
The steps of different sample pretreatment methods are shown in Table 2, and a high-concentration (10 7 CFU/mL) and low-concentration (10 4 CFU/mL) coliform bacteria liquid is selected as samples, and the method b is adopted respectively: alkali cracking and thermal cracking; method c: triton x-100+ thermal cracking; method d: lysozyme+sds; method e: SDS+proteinase K; method f: the 5 sample pretreatment methods of thermal cracking are carried out according to the method a: commercial bacterial genome DNA extraction kits are used as a control, and the LAMP amplification effect after pretreatment by different methods is compared.
TABLE 2 main steps of different sample pretreatment methods
And (3) adopting different sample pretreatment methods (method b to method f) to treat bacterial liquid samples, and taking a commercial kit (method a) to extract sample nucleic acid as a control to examine the LAMP amplification effect after the treatment of the different sample pretreatment methods. The LAMP amplification effect is shown in figures 5-10, and the results show that the LAMP amplification effects after treatment by the methods b, c and f are better than those of the commercial kit (method a), the Ct value is smaller, the detection time is shortened, but the experimental result shows that the LAMP amplification detection limit of the method f is higher (data not shown), the treatment steps are presumed to be too simple, and the LAMP amplification inhibition of residues after cell lysis is larger; the Ct value after the treatment of the method d is larger than that of a commercial kit; the amplification signal does not appear in the method e, which may be that the cell lysis effect of the method e is poor or that the added component has an influence on the LAMP reaction system. And finally, selecting the method b and the method c for pretreatment of the escherichia coli according to the comparison experiment result.
Condition optimization of sample pretreatment methods
The condition of the method b is optimized, an escherichia coli bacterial solution with high concentration (10 7 CFU/mL) and low concentration (10 4 CFU/mL) is selected as a sample, naOH with the concentration of 500mmol/L, 50mmol/L, 5mmol/L and 0mmol/L is selected to treat the bacterial solution sample, and then LAMP amplification is carried out. As shown in the results of figures 11-12, in the high-concentration bacterial liquid sample, the amplification efficiency of the amplification curve treated by 500mmol/L NaOH is lower, the Ct value is larger, and the Ct value of the amplification curve treated by 50mmol/L NaOH is minimum; the amplification result of the low-concentration bacterial liquid sample shows that no amplification signal appears after 500mmol/L NaOH treatment, which indicates that the LAMP amplification reaction is inhibited when the concentration of NaOH is too high, and Ct values after 50mmol/L and 5mmol/L NaOH treatment are relatively smaller, and finally 50mmol/L is selected as the optimal concentration.
The condition of the method c is optimized, a high-concentration (10 7 CFU/mL) and low-concentration (10 4 CFU/mL) escherichia coli bacterial liquid is selected as a sample, the bacterial liquid samples are treated by Triton X-100 with the concentration of 2% (v/v), 1% (v/v), 0.5% (v/v) and 0.25% (v/v) in sequence, finally LAMP amplification comparison is carried out, the results are shown in fig. 13-14, the high-concentration bacterial liquid samples are treated by Triton X-100 with different concentrations, the LAMP amplification Ct values are not obviously different, the amplification results of the low-concentration bacterial liquid samples show that the Ct values of 2% (v/v) and 1% (v/v) Triton X-100 are larger, the Ct values of Triton X-100 after being treated by 0.5% (v/v) and the Ct values of Triton X-100 after being treated by 0.25% (v/v) are relatively smaller, and the Ct values of Triton X-100 are minimum, and finally the concentration of Triton X-100 with the concentration of 0.5% (v/v) is selected as the most suitable concentration.
Detection limit comparison of pretreatment method
The bacterial liquid sample with the original concentration is diluted by 10 times of gradient, pretreatment is carried out by different sample pretreatment methods, then LAMP amplification comparison is carried out, the LAMP amplification result is shown in figures 15-17, the detection limit of the sample after being treated by a commercial kit (method a) is 10 1 CFU/mL, the detection limit of the sample after being treated by the method b can also reach 10 1 CFU/mL, the Ct value of the bacterial liquid sample with the low concentration in the method c is larger, the detection time is longer, and the detection limit is 10 1 CFU/mL. And comparing the experimental results, wherein the method b is the optimal pretreatment method.
Experiments show that the escherichia coli DNA sample pretreated and extracted by adopting a lysozyme and SDS (sodium dodecyl sulfate) cracking method, a SDS and proteinase K cracking method and a thermal cracking method has influence on gene amplification, has poor detection effect, has larger Ct value of the DNA sample extracted by a Triton X-100 heating cracking method, has longer detection time, has the best detection effect of the DNA sample extracted by an alkali heating cracking method, and has detection limits of 10 1 CFU/mL for both the alkali and the Triton X-100 heating cracking methods, and is consistent with the detection limit of the bacterial liquid sample DNA extracted by a commercial bacterial genome DNA extraction kit. The reagent used in the alkaline heating pyrolysis method has no obvious influence on the LAMP reaction system, does not need any concentration step when the bacterial liquid sample is processed, reduces the sample loss, greatly shortens the sample pretreatment time (10 min), and is obviously superior to a bacterial genome DNA extraction kit (90 min). When the LAMP amplification is used for detecting the DNA of the actual bacterial liquid sample extracted by the alkaline thermal cracking method, the sensitivity, the specificity and the total coincidence rate are high.
Example 3
A fast detection method of colibacillus based on LAMP includes the following steps:
s1 E.coli culture and enumeration
Coli strain was inoculated into sterilized LB medium and shake cultured overnight at 37℃with 200r/min shaking bed. The bacterial liquid is diluted by distilled water according to a gradient of 10 times, 50 mu L of diluted bacterial liquid is coated in LB solid medium, and the bacterial liquid is placed in a constant temperature incubator for culture overnight at 37 ℃, and the colony number of the flat plate is recorded.
S2 sample pretreatment
Extracting a DNA template by a sample pretreatment method by using a pretreatment reagent;
s3 LAMP amplification primer design and test
The reaction system was 20. Mu.L, including F3, B3, FIP, BIP primer set, bst polymerase, dNTPs, mgSO 4, 10x LAMP buffer,LAMP fluorescent dye, DNA template, ddH 2 O, and LAMP reaction system was prepared and put into qPCR apparatus, setting the procedure at 65℃for 40min, and investigating the performance of the designed primers.
S3 LAMP amplification detection limit
Detecting the primer pair Escherichia coli O157: h7 detection limit, diluting Escherichia coli into bacterial solutions with different concentrations by using distilled water, carrying out sample pretreatment, adding 100 μl of 0.5% Triton X-100, uniformly mixing, placing on a metal bath, heating at 95 ℃ for 10min, then centrifuging at 12000rpm/min at 25 ℃ for 3min, collecting the supernatant as a template, carrying out LAMP amplification detection by using the supernatant as the template, and obtaining the LAMP amplification detection limit of the different pretreatment methods by observing the generation of amplification signals.
It will be evident to those skilled in the art that the invention is not limited to the details of the foregoing illustrative embodiments, and that the present invention may be embodied in other specific forms without departing from the spirit or essential characteristics thereof. The present embodiments are therefore to be considered in all respects as illustrative and not restrictive, the scope of the invention being indicated by the appended claims rather than by the foregoing description, and all changes which come within the meaning and range of equivalency of the claims are therefore intended to be embraced therein.
Claims (10)
1. The LAMP-based escherichia coli detection kit is characterized by comprising a pretreatment reagent and a detection reagent, wherein the detection reagent comprises a reagent specific to escherichia coli O157: LAMP amplification specificity F3, B3, FIP and BIP primer groups for detecting H7 specific gene rfbe genes, wherein the base sequences of the primers are as follows:
F3：CCAGTTAGAACAAGCTGATGA；
B3：GGCCTTGTTTCGATGAGTT；
FIP：CTTGTGGACTTGTACAAGACTGT-CGAAAACGTGAAATTGCTGA；
BIP：TTCACACTTATTGGATGGTCTCAAT-GGTGATTCCTTAATTCCTCTCT。
2. The LAMP-based E.coli detection kit according to claim 1, wherein the outer primers F3 and B3 in the F3, B3, FIP and BIP primer sets each contain 0.2. Mu. Mol/L, and the inner primers FIP and BIP each contain 1.6. Mu. Mol/L.
3. The LAMP-based E.coli detection kit according to claim 1, wherein the detection reagent further comprises 8U Bst DNA polymerase, 1.4mmol/L dNTPs, 6mmol/LMgSO 4, 1x LAMP buffer, 1x LAMP fluorescent dye, DNA template, ddH 2 O, and a negative control is set by replacing the DNA template with non-contaminating enzyme-free water.
4. The LAMP-based E.coli detection kit according to claim 1, wherein the pretreatment reagent comprises 100. Mu.l of 50mM NaOH and 100. Mu.l of 50mM HCl.
5. The LAMP-based E.coli detection kit according to claim 1, wherein the pretreatment reagent comprises 100. Mu.l of 0.5% Triton X-100.
6. The LAMP-based escherichia coli rapid detection method is characterized by comprising the following steps of:
S1 sample pretreatment
Extracting a DNA template by a sample pretreatment method by using a pretreatment reagent;
screening of S2 LAMP amplification primers
Mixing the obtained DNA template with a detection reagent, wherein the reaction system is 20 mu L and comprises F3, B3, FIP, BIP primer groups, bst polymerase, dNTPs, mgSO 4, 10x LAMP buffer,LAMP fluorescent dye, DNA template, ddH 2 O, and putting the prepared LAMP reaction system into a qPCR instrument, setting the program to 65 ℃ for 40min, and screening out a proper amplification primer;
s3 LAMP amplification detection limit
Detecting the primer pair Escherichia coli O157: h7 detection limit, diluting colibacillus into bacterial solutions with different concentrations by distilled water, extracting bacterial solution DNA by adopting a pretreatment reagent, carrying out LA MP amplification detection by taking the DNA template as a target, and obtaining the detection limit of the primer by observing the generation of an amplification signal.
7. The rapid detection method of Escherichia coli based on LAMP of claim 4, further comprising culturing and counting Escherichia coli to determine concentration of bacterial liquid, inoculating Escherichia coli strain into sterilized LB medium, shake culturing overnight at 37deg.C at 200r/min, diluting bacterial liquid with distilled water at 10-fold gradient, coating 50 μl of diluted bacterial liquid in LB solid medium, culturing overnight at 37deg.C, and recording colony count of flat plates.
8. The rapid detection method of LAMP-based escherichia coli as defined in claim 4, wherein the sample pretreatment in the step S1 comprises adding 100. Mu.l of 50mM NaOH, uniformly mixing, heating on a metal bath at 95 ℃ for 10min, adding 100. Mu.l of 50mM HCl for neutralization, and centrifuging at 12000rpm/min at 25 ℃ for 3min, and collecting the supernatant as a DNA template.
9. The rapid detection method of LAMP-based escherichia coli as set forth in claim 4, wherein the sample pretreatment in the step S1 further comprises adding 100. Mu.l of 0.5% Triton X-100, mixing, heating on a metal bath at 95℃for 10min, followed by centrifugation at 12000rpm/min at 25℃for 3min, and collecting the supernatant as a DNA template.
10. The rapid detection method of Escherichia coli based on LAMP according to claim 7 or 8, wherein the detection limit of LAMP amplification of the DNA template obtained by pretreatment of the sample is 10 1 CFU/mL.
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