Constant-temperature rapid detection method for multiple nucleic acids of diarrheogenic escherichia coli
By establishing a rapid isothermal detection method for diarrhea-causing Escherichia coli using multiplex nucleic acids and recombinant enzymes and fluorescence signals, the problem of complex and time-consuming detection steps has been solved, achieving rapid and simple detection of diarrhea-causing Escherichia coli, suitable for both laboratory and field use.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- CHENGDU RES BASE OF GIANT PANDA BREEDING
- Filing Date
- 2026-02-11
- Publication Date
- 2026-04-17
AI Technical Summary
Existing technologies for detecting diarrhea-causing Escherichia coli involve complex and time-consuming steps, making it difficult to achieve rapid and accurate detection.
A rapid isothermal detection method for diarrhea-causing Escherichia coli was established. By designing 4- and 3-fold isothermal rapid detection systems targeting genes such as escV, bfpB, aggR, and invE, isothermal amplification was performed using recombinase, single-strand binding protein, and DNA polymerase, combined with fluorescence signal detection.
It enables rapid and convenient detection of diarrhea-causing Escherichia coli, shortens the detection time to 20 minutes, reduces dependence on professional instruments, is suitable for laboratory and field testing, and improves the accuracy and sensitivity of the detection.
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Figure CN121874375A_ABST
Abstract
Description
Technical Field
[0001] This invention belongs to the field of rapid microbial detection technology, specifically relating to a rapid isothermal detection method for multiplex nucleic acids of diarrhea-causing Escherichia coli. Background Technology
[0002] Escherichia coli is a Gram-negative bacterium and a common opportunistic pathogen in the animal intestines. Escherichia coli associated with intestinal diseases are collectively referred to as diarrhea-causing Escherichia coli. 致泻性大肠杆菌 Enteropathogenic Escherichia coli (DEC) generally includes five types: enteroaggregative Escherichia coli (EAEC), enteropathogenic Escherichia coli (EPEC), enterohemorrhagic Escherichia coli (EHEC), enterotoxigenic Escherichia coli (ETEC), and enteroinvasive Escherichia coli (EIEC). Diarrheal-causing Escherichia coli is one of the main pathogens causing intestinal diseases in animals, primarily characterized by diarrhea. Severe cases can lead to death. These pathogens are highly pathogenic and contagious, posing a significant threat to wildlife populations.
[0003] Rapid and accurate detection and monitoring are crucial for effective disease control. Currently, the detection of diarrheal Escherichia coli is achieved through PCR, which involves sample loading, PCR, and electrophoresis, a time-consuming process. Multi-enzyme isothermal rapid nucleic acid amplification technology primarily relies on recombinases, single-stranded binding proteins (SSBs), and DNA polymerases to achieve rapid nucleic acid amplification. The basic principle is as follows: at room temperature, the recombinase and primers form a complex; with the help of accessory proteins and SSBs, it invades the double-stranded DNA template. The primer binds to the homologous complementary region to form a D-loop region, while the recombinase disintegrates from the complex; the polymerase binds to the 3' end of the primer and initiates DNA synthesis, exponentially amplifying the target region on the template. This process cycles rapidly and efficiently, thus completing the ultra-rapid amplification of the target fragment. The fluorescent reagent in the isothermal rapid nucleic acid detection system adds specific exonucleases to the system, and a specific molecular probe designed according to the template is added to the reaction system. After the probe binds to the complementary region of the amplified fragment, the exonuclease cleaves specific sites on the probe, separating the luminescent and quenching groups, thereby releasing a fluorescent signal. Fluorescence detection equipment can be used to achieve real-time fluorescence detection of the target fragment amplification process. Summary of the Invention
[0004] In view of the above-mentioned prior art, the present invention provides a rapid isothermal detection method for multiplex nucleic acids of diarrheal Escherichia coli, which solves the problems of complex detection steps and long time consumption in the prior art, and provides a new method for the detection of diarrheal Escherichia coli.
[0005] To achieve the above objectives, the technical solution adopted by this invention is: to provide a rapid isothermal detection method for multiplex nucleic acids of diarrhea-causing Escherichia coli, targeting... escV , bfpB ,aggR and invE Gene has established a four-fold nucleic acid isothermal rapid detection system, targeting stx1 , stx2 and pic Gene has established a triple-layer nucleic acid isothermal rapid detection system, targeting 它 , 某物 and astA A triple-base isothermal rapid nucleic acid detection system was established; the following primer and probe sets were used in the detection system: For diarrheal Escherichia coli aggR Gene, upstream primer aggR The nucleotide sequence of F1 is shown in SEQ ID NO.1, and the downstream primer... aggR The nucleotide sequence of R3 is shown in SEQ ID NO.6. The probe... AggR The T nucleotide sequence is shown in SEQ ID NO. 7; For diarrheal Escherichia coli Pic Gene, upstream primer Pic The F3 nucleotide sequence is shown in SEQ ID NO.10, and the downstream primer... Pic The nucleotide sequence of R2 is shown in SEQ ID NO.12. The probe... Pic The T nucleotide sequence is shown in SEQ ID NO.14; For diarrheal Escherichia coli 某物 Gene, upstream primer 某物 The F2 nucleotide sequence is shown in SEQ ID NO.16, and the downstream primer... 某物 The nucleotide sequence of R1 is shown in SEQ ID NO.17. The probe... 某物 The T nucleotide sequence is shown in SEQ ID NO.20; For diarrheal Escherichia coli AstA Gene, upstream primer AstA The F1 nucleotide sequence is shown in SEQ ID NO.21, and the downstream primer... AstA The nucleotide sequence of R1 is shown in SEQ ID NO.22. The probe... AstA The T nucleotide sequence is shown in SEQ ID NO. 24; For diarrheal Escherichia coli InvE Gene, upstream primer InvE The F1 nucleotide sequence is shown in SEQ ID NO.25, and the downstream primer... InvE The nucleotide sequence of R2 is shown in SEQ ID NO.29. The probe... InvE The T nucleotide sequence is shown in SEQ ID NO. 31; For diarrheal Escherichia coli 它 Gene, upstream primer它 The F2 nucleotide sequence is shown in SEQ ID NO.33, and the downstream primer... 它 The nucleotide sequence of R2 is shown in SEQ ID NO.36. The probe... 它 The T nucleotide sequence is shown in SEQ ID NO.38; For diarrheal Escherichia coli EscV Gene, upstream primer EscV The F1 nucleotide sequence is shown in SEQ ID NO.39, and the downstream primer... EscV The nucleotide sequence of R2 is shown in SEQ ID NO.43. The probe... EscV The T nucleotide sequence is shown in SEQ ID NO. 45; For diarrheal Escherichia coli Stx1 Gene, upstream primer Stx1 The F2 nucleotide sequence is shown in SEQ ID NO.47, and the downstream primer... Stx1 The nucleotide sequence of R3 is shown in SEQ ID NO.51. The probe... Stx1 The T nucleotide sequence is shown in SEQ ID NO. 52; For diarrheal Escherichia coli Stx2 Gene, upstream primer Stx2 The F2 nucleotide sequence is shown in SEQ ID NO. 54, and the downstream primer... Stx2 The nucleotide sequence of R2 is shown in SEQ ID NO.57. The probe... Stx2 The T nucleotide sequence is shown in SEQ ID NO. 59; For diarrheal Escherichia coli BfpB Gene, upstream primer BfpB The F3 nucleotide sequence is shown in SEQ ID NO. 62, and the downstream primer... BfpB The nucleotide sequence of R3 is shown in SEQ ID NO. 65. The probe... BfpB The T nucleotide sequence is shown in SEQ ID NO. 66.
[0006] Based on the above technical solution, the present invention can be further improved as follows.
[0007] Furthermore, diarrhea-causing Escherichia coli include enteroaggregative Escherichia coli, enteropathogenic Escherichia coli, enterohemorrhagic Escherichia coli, enterotoxigenic Escherichia coli, and enteroinvasive Escherichia coli.
[0008] Furthermore, the rapid isothermal detection method for multiplex nucleic acids of diarrhea-causing Escherichia coli includes the following steps: extracting DNA from the sample to be tested, using the DNA of the sample to be tested as a template, performing isothermal amplification with recombinase, single-stranded binding protein and DNA polymerase, collecting fluorescence signals, and determining whether diarrhea-causing Escherichia coli is present in the sample to be tested.
[0009] Furthermore, the total volume of the isothermal amplification system was 50 µL. The quadruple nucleic acid isothermal rapid detection system included 29.4 µL of absorptive buffer, 2.5 µL of buffer B, 2.4 µL of probe, 5 µL of template, 8 µL of forward and reverse primers, and ddH2O was added to bring the volume to 50 µL. The triple nucleic acid isothermal rapid detection system included 29.4 µL of buffer A, 2.5 µL of buffer B, 1.8 µL of probe, 5 µL of template, 6 µL of forward and reverse primers, and ddH2O was added to bring the volume to 50 µL.
[0010] Furthermore, the concentrations of the upstream primer, downstream primer, and probe were all 10 µM, and the concentrations of each upstream primer and downstream primer were the same in the isothermal amplification system, as were the concentrations of each probe.
[0011] Furthermore, the reaction temperature was 42℃, fluorescence signals were collected every 30 s, and the reaction time was 20 min.
[0012] The beneficial effects of this invention are: this invention is based on standard strains from EAEC, EPEC, EHEC, ETEC and EIEC. aggR , astA , escV , stx1 , stx2 , bfpB , invE , 它 , pic , 某物 Primers were designed based on gene sequences to establish a rapid, isothermal, multiplex nucleic acid detection system for five diarrhea-causing Escherichia coli species, aiming to achieve rapid and convenient detection. After negative, positive, and sensitivity validation of the primer and probe set, a system targeting [specific pathogens] was successfully established. escV , bfpB , aggR , invE A quadruple nucleic acid isothermal rapid detection system for genes, targeting stx1 , stx2 , pic A triple nucleic acid isothermal rapid detection system for genes, targeting 它 , 某物 , astAThis rapid, isothermal detection system for triple nucleic acid typing can be used to detect diarrheal Escherichia coli EPEC, EHEC, ETEC, EAEC, and EIEC. Compared to traditional PCR, this method requires less time, completing the reaction in just 20 minutes, and the reaction is carried out at room temperature, reducing dependence on specialized equipment. This shortens the time required for laboratory bacterial testing and can also be applied to field testing. This method allows for typing of diarrheal Escherichia coli, enabling more accurate diagnosis and medication after infection with these five types of E. coli. Furthermore, the enzymes used in this technology react at room temperature, so primer and probe design does not need to consider melting temperature, which is more conducive to designing multiplex detection for pathogens and provides a new option for multiplex detection of other pathogens.
[0013] Furthermore, traditional multiplex qPCR primer design requires stricter requirements to ensure that multiple primers can work in coordination in the same system. Since the enzyme in this invention can react at room temperature, the design of primers and probes does not need to consider the differences in melting temperature between different primers, which is more conducive to designing complex multiplex reactions. It can develop technical methods suitable for multiplex detection of different pathogens, providing a new option for multiplex detection of pathogens that are difficult to be compatible. Attached Figure Description
[0014] Figure 1 for stx1 The results of the nucleic acid isothermal rapid detection primer and probe assay for the gene are negative. In this series, 1-3 represent: 1 indicates... stx1 F1-R1,2 is stx1 F2-R2, 3 is stx1 F3-R3; 4 is a positive control; Figure 2 for stx1 The results of positive verification (positive screening) of the primer and probe for rapid isothermal detection of the gene's nucleic acid, where 1 is... stx1 F1-R1,2 is stx1 F1-R2, 3 are stx1 F1-R3 and 4 are negative controls; Figure 3 for stx1 The positive verification (reverse screening) of primers and probes for rapid isothermal detection of gene nucleic acids, where 1 is... stx1 F1-R3, 2 is stx1 F2-R3, 3 is stx1 F3-R3, 4 is stx1 F1-R1, 5 is stx1 F2-R1, 6 is stx1 F3-R1, 7 served as negative controls; Figure 4The results are for sensitivity verification of isothermal rapid nucleic acid detection. In this table, 1 represents 10 ng / µL, 2 represents 1 ng / µL, 3 represents 0.1 ng / µL, 4 represents 0.01 ng / µL, and 5-7 represent: 5 represents 0.001 ng / µL, 6 represents 0.0001 ng / µL; 7 is the negative control. Figure 5 This is a result of a rapid isothermal detection of multiplex nucleic acids, where 1 is... escV F1-R2, 2 is bfpB F3-R3, 3 is aggR F1-R3, 4 is invE F1-R2; 5-8 were all negative controls; Figure 6 This is a result of a rapid isothermal detection of multiplex nucleic acids, where 1 is... stx1 F2-R3, 2 is [[ID=9 F2-R2, 3 is F3-R2; 4-6 were all negative controls; This is a result of a rapid isothermal detection of multiplex nucleic acids, where 1 is... F2-R2, 2 is F2-R1, 3 is F1-R1; 4-6 were all negative controls; The results of testing clinical samples using a multiplex nucleic acid isothermal detection method are shown, where 1 represents sample number 1. The test result is that sample number 2 is sample number 1. The test result showed that sample number 3 was sample number 2. Detection; 4-30 indicates: 4-20 is for the detection of the remaining virulence genes in samples 1-2, and 21-30 is the negative control; The results of PCR testing of clinical samples are shown, where M represents the DNA Marker and 1 represents sample number 1. The test result is that sample number 2 is the 2nd sample. For testing, + indicates a positive control, and - indicates a negative control; The results of PCR testing of clinical samples are shown, where M represents the DNA Marker and 1 represents sample number 1. The test result is that sample number 2 is the 2nd sample. For the test, + indicates a positive control; - indicates a negative control. Detailed Implementation
[0015] The specific embodiments of the present invention will be described in detail below with reference to examples.
[0016] Main reagents and materials for the experiment: (1) Strain Enterocytic Escherichia coli (EAEC), enteropathogenic Escherichia coli (EPEC), enterohemorrhagic Escherichia coli (EHEC), enterotoxigenic Escherichia coli (ETEC), and enteroinvasive Escherichia coli (EIEC) were all standard strains purchased from CICC China National Research Institute of Food Fermentation Industries Co., Ltd. and China Industrial Microbial Culture Collection Center.
[0017] (2) Main reagents and instruments Bacterial genomic DNA extraction kit, purchased from Tiangen Biotech Co., Ltd. Anpu Future fluorescent reagent kit (DNA type), purchased from Anpu Future Biotechnology Co., Ltd. High-speed refrigerated centrifuge, purchased from Eppendorf. Real-time PCR instrument, purchased from Bio-Rad.
[0018] (3) Primer design and synthesis According to EAEC, EPEC, EHEC, ETEC and EIEC standard strains , , , , , , , , , Primers were designed based on gene sequences. Three sets of upstream and downstream primers and probe primers were designed for each gene. The primer sequences are shown in Table 1. They were synthesized by Sangon Biotech (Shanghai) Co., Ltd.
[0019] Table 1 Primer and probe sequences
[0020] The probe does not overlap with the specific primer recognition site, has a length of 44–53 nt, and avoids palindromic sequences, internal secondary structures, and consecutive repetitive bases. The probe has four modification sites: a dSpacer (tetrahydrofuran, THF) is labeled at the midpoint ≥26 nt from the 5' end as the exonuclease recognition site; an upstream fluorescent group (TexasRed, CY5, FAM, or VIC) is labeled upstream of the THF site, and a quencher group (BHQ1, BHQ2, or BHQ3) is labeled downstream, with a spacing of 1–3 nt between the two groups; the THF site is ≥15 nt from the 3' end, and a modification group (C3spacer) is labeled at the 3' end.
[0021] Example 1: Extraction of bacterial DNA The standard strains of EPEC, EHEC, ETEC, EAEC, and EIEC were inoculated into 5 mL of LB nutrient broth and cultured overnight at 37°C with shaking. Then, 1 mL of the bacterial suspension was taken and DNA was extracted according to the instructions of the DNA extraction kit.
[0022] Example 2: Primer and probe all-negative verification To determine 10 virulence genes ( , , , , , , , , , The primers and probes designed for the gene were negative and qualified. After the primer and probe synthesis was received, a mixing negative test was performed. Three sets of upstream and downstream primers (F1-R1, F2-R2, F3-R3) were randomly selected to be paired with probes for full negative verification. Prepare the reaction according to the instructions of the AMP Future Fluorescent Reagent Kit (DNA type). Add 29.4 µL of buffer A, 2 µL (10 µM) of upstream primer, 2 µL (10 µM) of downstream primer, 0.6 µL (10 µM) of probe primer, 5 µL of template (ddH2O is used as a substitute for negative verification), and 8.5 µL of ddH2O to the dry powder reaction tube. Finally, add 2.5 µL of buffer B and mix well. After mixing, quickly centrifuge the reaction solution to the bottom of the tube and then immediately place the reaction tube into the fluorescence detection device. The fluorescence detection program is as follows: constant temperature 42℃, collect fluorescence signal every 30 s, and reaction time is 20 min. If the fluorescence curves of the three sets of primers and probes are flat, it is determined that there is no negative abnormality (if the curve shows an abnormal tail, the corresponding primer and probe combination needs to be excluded).
[0023] The gene primer and probe all-negative verification results are as follows: As shown, the fluorescence curves of the three primer sets are flat, with no tailing, no peaks, and no Cq values, indicating that the negative test is qualified.
[0024] Example 3: Validation of Primer-Probe Positive Test (Positive Screening) After the all-negative verification in Example 2 was successful, primer screening was performed. First, the DNA of the target strain extracted by the method in Example 1 was used as a positive template for primer screening. One upstream primer (F1) was randomly fixed and combined with three downstream primers (R1, R2, R3) to obtain three sets of primer-probe combinations. The reaction procedure was the same as in Example 2. Primer-probe combinations with low Cq values, high relative fluorescence values, smooth overall amplification curves, and normal negative results were selected.
[0025] Genetic testing results such as As shown in the figure, the Cq value of primer pair 1 (F1-R1) is 5.7, primer pair 2 (F1-R2) has no Cq value, and primer pair 3 (F1-R3) has a Cq value of 5.49. Based on the Cq value, amplification curve, and relative fluorescence value, primer pair 3 is determined to be the optimal primer pair, followed by primer pair 1.
[0026] Example 4: Validation of positive primer-probe test (reverse screening) The optimal and second-optimal downstream primers, fixed in the forward screening, were selected and cross-combined with three upstream primers to obtain six sets of primer-probe combinations. The reaction procedure was the same as in Example 3, and primer-probe combinations with low Cq values, high relative fluorescence values, smooth overall amplification curves, and negative results were screened.
[0027] Genetic testing results such as As shown, the optimal downstream primer selection R3 was chosen as the second-best downstream primer, R1. In the figure, the Cq values of primer 1 (F1-R3) are 6.09, 2 (F2-R3) are 5.13, 3 (F3-R3) are 5.57, 4 (F1-R1) are 10.33, 5 (F1-R1) are 13, and 6 (F1-R1) are 5.54. Based on the Cq values, amplification curves, and relative fluorescence values, primers 2 (F2-R3) and 3 (F3-R3) are determined to be the optimal primer pairs.
[0028] The methods described in Examples 2-4 were respectively used to... , , , , , , , , The primers and probes for the gene are subjected to all-negative verification, positive test verification (positive screening), and positive test verification (reverse screening) to obtain the optimal primer pair.
[0029] The optimal primer pairs for the 10 virulence genes were selected respectively. (F1-R2) (F3-R3) (F1-R3) (F1-R2) (F2-R3) (F2-R2) (F3-R2) (F2-R2) (F2-R1) (F1-R1).
[0030] Example 5: Primer and probe sensitivity test Sensitivity experiments were conducted on the selected optimal primer-probe combinations to detect differences between concentrations, including relative fluorescence value, Cq value, and detection limit. The target strain DNA extracted using the method in Example 1 was used as the positive template. Template concentrations were selected as follows: 10, 1, 0.1, 0.01, 0.001, and 0.0001 ng / µL. The reaction was prepared according to the instructions of the Amp Future Fluorescent Kit (DNA type). 29.4 µL of buffer A, 2 µL (10 µM) of upstream primer, 2 µL (10 µM) of downstream primer, 0.6 µL (10 µM) of probe primer, 5 µL of template, and 8.5 µL of ddH2O were added to the dry powder reaction tube. Finally, 2.5 µL of buffer B was added and mixed thoroughly. After mixing, the reaction solution was rapidly centrifuged to the bottom of the tube, and then the reaction tube was immediately placed in a fluorescence detection device. The fluorescence detection program was: constant temperature 42℃, fluorescence signal collected every 30 s, and reaction time 20 min.
[0031] Genetic testing results such as As shown, the Cq value was 5.19 when the template concentration was 10 ng / µL, 6.49 when the template concentration was 1 ng / µL, 8.19 when the template concentration was 0.1 ng / µL, and 15.91 when the template concentration was 0.01 ng / µL. No Cq value was detected at template concentrations of 0.001 ng / µL and below. The sensitivity of the primers and probes was 0.01 ng / µL. The sensitivity detection results for 10 virulence genes are shown in Table 2.
[0032] Table 2 Sensitivity Detection Results
[0033] Example 6: Validation and Optimization of Rapid Isothermal Detection of Multiplex Nucleic Acids The optimal primer-probe combination was verified by multiplex nucleic acid isothermal rapid detection. The target strain DNA extracted by the method in Example 1 was used as a positive template. The reaction was prepared according to the instructions of the AMP Future fluorescent reagent kit (DNA type). The upstream and downstream primers were adjusted to 1 µL, and the amount of ddH2O was adjusted according to the number of multiple primers. The total system was then made up to 50 µL.
[0034] Ten virulence genes were combined to screen for a rapid multiplex nucleic acid isothermal detection system with low Cq values, high relative fluorescence values, and smooth overall amplification curves.
[0035] against , , , The established four-stage nucleic acid isothermal rapid detection system is shown in Table 3; [The text abruptly ends here, likely due to an incomplete sentence or a formatting error.] , , The established triple-stage nucleic acid isothermal rapid detection system is shown in Table 4; [The text abruptly ends here, likely due to an incomplete sentence or a formatting error.] , , The established triple nucleic acid isothermal rapid detection system is shown in Table 5; the above three systems can be used to detect Escherichia coli EPEC, EHEC, ETEC, EAEC and EIEC.
[0036] Table 3 Targeting , , , A four-fold nucleic acid isothermal rapid detection system for genes
[0037] Table 4 Targeting , , Triple nucleic acid isothermal rapid detection system for genes
[0038] Table 5 Targeting , , Triple nucleic acid isothermal rapid detection system for genes
[0039] The reaction temperature was 42℃, fluorescence signals were collected every 30 s, and the reaction time was 20 min.
[0040] Multiple detection results such as As shown, all virulence genes were detected, among which, The Cq value is 3.28. The Cq value is 19.65. The Cq value is 5.73. The Cq value is 19.41. The Cq value is 3.42. The Cq value is 17.95. The Cq value is 5.15. The Cq value is 14.4. The Cq value is 8.13. The Cq value is 5.53.
[0041] Example 7: Clinical application results of rapid isothermal detection of multiplex nucleic acids The optimized multiplex primer-probe combination was validated using clinical samples. Twenty giant panda fecal samples were selected for clinical sample DNA extraction and E. coli typing. The detection methods used were conventional PCR and a multiplex nucleic acid isothermal rapid detection method (the method in Example 6). The concordance rate between the multiplex nucleic acid isothermal rapid detection and PCR detection was determined based on the results.
[0042] Results of multiplex nucleic acid isothermal detection (partial) As shown, the virulence gene was detected in sample number 1. and The corresponding Cq values were 3.49 and 1.96, and the virulence gene was detected in sample 2. The corresponding Cq value is 4.98. The PCR test results are shown below. , 10 As shown, the virulence gene was detected in sample number 1. and The virulence gene was detected in sample number 2. The results showed that the results of multiplex nucleic acid isothermal rapid detection were consistent with those of PCR detection, with a concordance rate of 100%.
[0043] In summary, based on the standard strains of EAEC, EPEC, EHEC, ETEC, and EIEC... , , , , , , , , , Primers were designed based on gene sequences, and a novel isothermal rapid nucleic acid detection technology was used to establish a multiplex detection system for rapid and convenient testing. Compared to traditional PCR, this method requires less time and operates at room temperature. It can shorten the time required for laboratory bacterial testing and can also be applied to field testing. This method was used to type diarrheal Escherichia coli, enabling more accurate diagnosis and medication after infection with five different types of diarrheal Escherichia coli.
[0044] Although specific embodiments of the present invention have been described in detail with reference to examples, they should not be construed as limiting the scope of protection of this patent. Various modifications and variations that can be made by those skilled in the art without inventive effort within the scope described in the claims are still within the scope of protection of this patent.
Claims
1. A rapid isothermal detection method for multiplex nucleic acids of diarrhea-causing Escherichia coli, characterized in that: against escV , bfpB , aggR and invE Gene has established a four-fold nucleic acid isothermal rapid detection system, targeting stx1 , stx2 and pic Gene has established a triple-layer nucleic acid isothermal rapid detection system, targeting It , sth and astA A triple-base isothermal rapid nucleic acid detection system was established; the following primer and probe sets were used in the detection system: For diarrheal Escherichia coli aggR Gene, upstream primer aggR The nucleotide sequence of F1 is shown in SEQ ID NO.1, and the downstream primer... aggR The nucleotide sequence of R3 is shown in SEQ ID NO.
6. The probe... AggR The T nucleotide sequence is shown in SEQ ID NO. 7; For diarrheal Escherichia coli Pic Gene, upstream primer Pic The F3 nucleotide sequence is shown in SEQ ID NO.10, and the downstream primer... Pic The nucleotide sequence of R2 is shown in SEQ ID NO.
12. The probe... Pic The T nucleotide sequence is shown in SEQ ID NO.14; For diarrheal Escherichia coli Sth Gene, upstream primer Sth The F2 nucleotide sequence is shown in SEQ ID NO.16, and the downstream primer... Sth The nucleotide sequence of R1 is shown in SEQ ID NO.
17. The probe... Sth The T nucleotide sequence is shown in SEQ ID NO.20; For diarrheal Escherichia coli AstA Gene, upstream primer AstA The F1 nucleotide sequence is shown in SEQ ID NO.21, and the downstream primer... AstA The nucleotide sequence of R1 is shown in SEQ ID NO.
22. The probe... AstA The T nucleotide sequence is shown in SEQ ID NO. 24; For diarrheal Escherichia coli InvE Gene, upstream primer InvE The F1 nucleotide sequence is shown in SEQ ID NO.25, and the downstream primer... InvE The nucleotide sequence of R2 is shown in SEQ ID NO.
29. The probe... InvE The T nucleotide sequence is shown in SEQ ID NO. 31; For diarrheal Escherichia coli It Gene, upstream primer It The F2 nucleotide sequence is shown in SEQ ID NO.33, and the downstream primer... It The nucleotide sequence of R2 is shown in SEQ ID NO.
36. The probe... It The T nucleotide sequence is shown in SEQ ID NO.38; For diarrheal Escherichia coli EscV Gene, upstream primer EscV The F1 nucleotide sequence is shown in SEQ ID NO.39, and the downstream primer... EscV The nucleotide sequence of R2 is shown in SEQ ID NO.
43. The probe... EscV The T nucleotide sequence is shown in SEQ ID NO. 45; For diarrheal Escherichia coli Stx1 Gene, upstream primer Stx1 The F2 nucleotide sequence is shown in SEQ ID NO.47, and the downstream primer... Stx1 The nucleotide sequence of R3 is shown in SEQ ID NO.
51. The probe... Stx1 The T nucleotide sequence is shown in SEQ ID NO. 52; For diarrheal Escherichia coli Stx2 Gene, upstream primer Stx2 The F2 nucleotide sequence is shown in SEQ ID NO. 54, and the downstream primer... Stx2 The nucleotide sequence of R2 is shown in SEQ ID NO.
57. The probe... Stx2 The T nucleotide sequence is shown in SEQ ID NO. 59; For diarrheal Escherichia coli BfpB Gene, upstream primer BfpB The F3 nucleotide sequence is shown in SEQ ID NO. 62, and the downstream primer... BfpB The nucleotide sequence of R3 is shown in SEQ ID NO.
65. The probe... BfpB The T nucleotide sequence is shown in SEQ ID NO.
66.
2. The method for rapid isothermal detection of multiplex nucleic acids of diarrhea-causing Escherichia coli according to claim 1, characterized in that: The diarrhea-causing Escherichia coli include enteroaggregative Escherichia coli, enteropathogenic Escherichia coli, enterohemorrhagic Escherichia coli, enterotoxigenic Escherichia coli, and enteroinvasive Escherichia coli.
3. The method for rapid isothermal detection of multiplex nucleic acids of diarrhea-causing Escherichia coli according to claim 2, characterized in that, Includes the following steps: DNA was extracted from the sample to be tested. Using the DNA as a template, recombinase, single-stranded binding protein and DNA polymerase were used for isothermal amplification. Fluorescence signals were collected to determine whether diarrhea-causing Escherichia coli was present in the sample.
4. The method for rapid isothermal detection of multiplex nucleic acids of diarrhea-causing Escherichia coli according to claim 3, characterized in that: The total volume of the isothermal amplification system was 50 µL. The quadruple nucleic acid isothermal rapid detection system included 29.4 µL of buffer A, 2.5 µL of buffer B, 2.4 µL of probe, 5 µL of template, 8 µL of forward and reverse primers, and ddH2O was added to bring the volume to 50 µL. The triple nucleic acid isothermal rapid detection system included 29.4 µL of buffer A, 2.5 µL of buffer B, 1.8 µL of probe, 5 µL of template, 6 µL of forward and reverse primers, and ddH2O was added to bring the volume to 50 µL.
5. The method for rapid isothermal detection of multiplex nucleic acids of diarrhea-causing Escherichia coli according to claim 4, characterized in that: The concentrations of the upstream primer, downstream primer, and probe were all 10 µM. The concentrations of each upstream primer and downstream primer were the same in the isothermal amplification system, and the concentrations of each probe were the same.
6. The method for rapid isothermal detection of multiplex nucleic acids of diarrhea-causing Escherichia coli according to claim 5, characterized in that: The reaction temperature was 42℃, fluorescence signals were collected every 30 s, and the reaction time was 20 min.