A CPA primer combination and method for detecting shiga toxin-producing escherichia coli

Abstract

The application discloses a cross primer nucleic acid isothermal amplification primer combination and method for detecting shiga toxin-producing escherichia coli. The primer combination is used for detecting 10 gene subtypes of stx1a, stx1c, stx1d, stx2a, stx2b, stx2c, stx2d, stx2e, stx2f and stx2g, and comprises primer group I with the nucleotide sequence of SEQ ID NO:1-5, primer group II with the nucleotide sequence of SEQ ID NO:6-10, and primer group III with the nucleotide sequence of SEQ ID NO:11-20. The method provided by the application is simple in operation, good in specificity, high in sensitivity, can complete nucleic acid detection within 50 min, provides a rapid screening result for a regulatory department to detect food pathogenic bacteria in major activity guarantee and emergency events. Meanwhile, the application firstly designs and screens the primer combination to realize detection of all gene subtypes of STEC, and in combination with a nucleic acid detection test strip and an integrated detection device, the whole process can be realized without instrument dependence and visual result judgment, and the requirement of on-site detection is greatly met.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology detection, and specifically relates to a CPA primer combination, kit, and method for detecting Shiga toxin-producing Escherichia coli. Background Technology

[0002] Shiga toxin-producing Escherichia coli (STEC) is an important group of foodborne pathogens that has emerged in recent years, causing infections and epidemics in humans in many countries. Although O157:H7 is the typical serotype of STEC, the global increase in non-O157 STEC infections in recent years underscores the importance of investigating non-O157 STEC. STEC can cause diarrhea and hemorrhagic colitis, with 2% to 7% of patients developing thrombotic thrombocytopenic purpura and hemolytic uremic syndrome, and in severe cases, even death. Therefore, foodborne illnesses caused by STEC have become a serious public health safety issue, seriously threatening people's health. Thus, rapid and accurate detection of STEC is a prerequisite for effective prevention and control of infection.

[0003] Currently, the main molecular target for detecting STEC is the stx gene encoding Shiga toxin. Both the National Standardization Organization and the U.S. Department of Agriculture use real-time quantitative PCR (qPCR) for initial screening of STEC in food. However, qPCR relies on expensive, sophisticated instruments, resulting in high testing costs and requiring highly skilled personnel, making it unsuitable for on-site testing. Crossing-primer amplification (CPA) is my country's only independently developed isothermal amplification technology. It can rapidly, specifically, and sensitively amplify target sequences under isothermal conditions without requiring expensive equipment, significantly reducing testing costs. It can be directly applied to on-site food safety assurance and emergency testing. Furthermore, compared to other isothermal amplification technologies, CPA, by labeling the detection primers, allows for direct visualization of the results via colloidal gold chromatography after amplification, making it more suitable for on-site testing.

[0004] Mutations and recombinations of stx gene subtypes are quite common, with 10 genotypes reported so far. These include three stx1 subtypes (stx1a, stx1c, and stx1d) and seven stx2 subtypes (stx2a, stx2b, stx2c, stx2d, stx2e, stx2g, and stx2f). The nucleotide sequences of different subtypes vary significantly, making it difficult to amplify all subtypes simultaneously using a single universal primer. For example, STEC carrying stx1a can cause HUS (hyperinflammatory syndrome), while strains carrying stx1c only cause mild diarrhea or no obvious symptoms. STEC carrying stx2a, stx2c, and stx2d can cause severe clinical symptoms, while STEC carrying stx2b, stx2e, and stx2f are milder and do not cause severe clinical symptoms. Foodborne STEC bacteria at concentrations below 100 CFU can cause illness in humans. There are over 400 serotypes of STEC. Among the non-O157:H7 serotype Shiga toxin-producing Escherichia coli (Non-O157 STEC), six strains are considered to have the highest detection rate and are the most harmful: O26 (22%), O111 (16%), O103 (12%), O121 (8%), O45 (7%), and O145 (5%). Data released by the US Centers for Disease Control and Prevention (CDC) shows that in 2016, there were 5,441 confirmed STEC infection events in the United States, with an estimated 260,000 patients and 30 deaths. Internationally, real-time polymerase chain reaction (real-time PCR) is the primary method used for detection. The International Organization for Standardization (ISO) and the United States Department of Agriculture (USDA) also employ this technology for initial screening of enrichment cultures suspected of containing STEC. However, real-time PCR requires constant temperature fluctuations between 50 and 100°C, placing high demands on the relevant testing equipment. This makes it impractical for portable applications and difficult to meet the needs of grassroots laboratories and rapid on-site testing. Summary of the Invention

[0005] To address the aforementioned technical problems, one objective of this invention is to provide a CPA primer set for detecting Shiga toxin-producing Escherichia coli, comprising primer set II or primer set III.

[0006] Primer set II, used to detect whether the analyte contains the stx1 subtype STEC, includes:

[0007] The nucleotide sequence of the stripping primer stx1-1 is shown in SEQ ID NO. 6;

[0008] The nucleotide sequence of the stripping primer stx1-2 is shown in SEQ ID NO.7;

[0009] The nucleotide sequence of the cross primer stx1-3 is shown in SEQ ID NO. 8;

[0010] The nucleotide sequence of the detection primer stx1-4 is shown in SEQ ID NO.9;

[0011] The nucleotide sequence of the detection primer stx1-5 is shown in SEQ ID NO.10;

[0012] Primer set III, used to detect whether the analyte contains the stx2 subtype STEC, includes:

[0013] The nucleotide sequence of the stripping primer stx2-1 is shown in SEQ ID NO.11;

[0014] The nucleotide sequence of the stripping primer stx2-2 is shown in SEQ ID NO.12;

[0015] The nucleotide sequence of the stripping primer stx2-3 is shown in SEQ ID NO.13;

[0016] The nucleotide sequence of the stripping primer stx2-4 is shown in SEQ ID NO.14;

[0017] The nucleotide sequence of the cross primer stx2-5 is shown in SEQ ID NO.15;

[0018] The nucleotide sequence of the cross primer stx2-6 is shown in SEQ ID NO.16;

[0019] The nucleotide sequence of the detection primer stx2-7 is shown in SEQ ID NO.17;

[0020] The nucleotide sequence of the detection primer stx2-8 is shown in SEQ ID NO.18;

[0021] The nucleotide sequence of the detection primer stx2-9 is shown in SEQ ID NO.19;

[0022] The nucleotide sequence of the detection primer stx2-10 is shown in SEQ ID NO.20;

[0023] The 5' end of the detection primers stx1-4 / stx2-7 / stx2-8 is labeled with a fluorescein isothiocyanate group, and the 5' end of the detection primers stx1-5 / stx2-9 / stx2-10 is labeled with a biotin fluorescein group.

[0024] It should be noted that the primers involved in this invention are crucial. Primer design not only needs to meet the requirements of conventional PCR primers, but also requires preliminary primer design based on the characteristics of the CPA method and the target sequence itself. Multiple primers must not interfere with each other, and subsequent rigorous screening is necessary. The base sequence differences between STEC gene subtypes are significant, especially stx2f, making primer design extremely challenging. In the initial stage of this invention, 30 primer combinations were designed for bioinformatics analysis and preliminary screening, followed by specificity and sensitivity tests to ensure the reliability of the detection method. Finally, three primer combinations were selected to optimize the detection performance of this patented method.

[0025] Another object of the present invention is to provide a reagent containing the aforementioned CPA primer combination, wherein:

[0026] The CPA reaction system containing primer set II is as follows:

[0027] Component preservation concentration and dosage

[0028]

[0029]

[0030] The CPA reaction system containing primer set III is as follows:

[0031] Component preservation concentration and dosage

[0032]

[0033] The Bst enzyme buffer consists of 200–500 mM Tris-HCl, 100–300 mM (NH4)2SO4, 100–300 mM KCl, 20 mM MgSO4, and 1%–5% Triton X-100 by mass. The pH value is 8.0–8.8 at 25°C.

[0034] Another object of the present invention is to provide a method for detecting Shiga toxin-producing Escherichia coli, comprising the following steps:

[0035] Step 1: Extract DNA from the sample to be tested;

[0036] Step 2: Prepare a reaction system containing primer set I, with the following component proportions:

[0037] Component preservation concentration and dosage

[0038]

[0039] The Bst enzyme buffer consists of 200–500 mM Tris-HCl, 100–300 mM (NH4)2SO4, 100–300 mM KCl, 20 mM MgSO4, and 1%–5% Triton X-100 by mass. The pH value is 8.0–8.8 at 25°C.

[0040] Primer set I is used to detect whether the DNA of the sample to be tested contains Escherichia coli;

[0041] Step 3: Mix the reaction system thoroughly and carry out the amplification reaction under constant temperature conditions; the reaction conditions are 60-67℃ for 30-50 minutes.

[0042] Step 4: After the isothermal amplification is complete, use a disposable nucleic acid test strip to detect the result, and observe the result after 5 minutes.

[0043] Result interpretation: If no red line appears, it indicates that the test has failed and needs to be repeated. If only one red line appears in the quality control area C, it means that the target gene was not detected in the sample. If two red lines appear, one test line and one quality control line, it means that the target gene was detected in the sample.

[0044] Step 5: If two red lines appear in Step 4, prepare the reaction system containing primer set II and the reaction system containing primer set III as described in claim 2.

[0045] Step 6: Thoroughly mix the reaction system containing primer group II and the reaction system containing primer group III, and carry out the amplification reaction under isothermal conditions; the reaction conditions are 60-67℃, 30-50 min.

[0046] Step 7: After the isothermal amplification is complete, use a disposable nucleic acid test strip to detect the result, and observe the result after 5 minutes;

[0047] Result interpretation: If no red line appears, it indicates that the test has failed and needs to be repeated. If only one red line appears in the quality control area C, it means that the target gene was not detected in the sample. If two red lines appear, one test line and one quality control line, it means that the target gene was detected in the sample, that is, Shiga toxin-producing Escherichia coli was found.

[0048] The final object of the present invention is to provide a kit comprising the reaction system containing primer set I as described in step two, and the reaction system mixed in step six.

[0049] Compared with the prior art, the beneficial effects of the present invention are:

[0050] This invention utilizes highly specific primer combinations for the uidA, stx1, and stx2 genes to achieve rapid detection of 10 genotypes of Shiga toxin-producing Escherichia coli. The multiple primers in each reaction recognize multiple specific regions of the STEC sequence, ensuring high amplification specificity. Direct detection of STEC bacterial suspensions achieves a sensitivity of up to 100 CFU / mL. After enrichment culture, 1 CFU / 25g of STEC contamination can be detected in artificially contaminated beef samples, achieving 100% accuracy compared to traditional methods. This invention, employing CPA nucleic acid detection technology and nucleic acid test strips, is simple, rapid, and provides visualized results, making it suitable for rapid on-site detection. In particular, the application of an integrated detection device eliminates instrument dependence, completing nucleic acid extraction, detection, and result analysis within 50 minutes, providing a reliable means for responding to major food security events and handling emergencies. Attached Figure Description

[0051] Figure 1 The results of the isothermal amplification sensitivity test of STEC cross-primers (the bacterial concentration of each target gene is 10 from left to right). 5 CFU / mL, 10 4 CFU / mL, 10 3 CFU / mL, 10 2 CFU / mL, 10 1 (CFU / mL);

[0052] Figure 2 Sensitivity test results for the STEC cross-primer amplification integrated detection device (bacterial concentration of each target gene from left to right is 10). 5 CFU / mL, 10 4 CFU / mL, 10 3 CFU / mL, 10 2 CFU / mL, 10 1 (CFU / mL);

[0053] Figure 3 The results of the isothermal amplification of the test bacteria shown in Table 2 using STEC cross primers are shown in Table 2 (the names of test strains 1#-22# are shown in Table 2, 1 represents the uidA gene, 2 represents the stx1 gene, and 3 represents the stx2 gene). Detailed Implementation

[0054] The present invention will be further described in detail below with reference to embodiments, but the embodiments of the present invention are not limited thereto. Unless otherwise specified, the reagents and equipment used in the present invention are conventional reagents and equipment in this technical field. Unless otherwise specified, the strains, reagents, etc. used in the present invention are all commercially available.

[0055] Example 1

[0056] CPA Primer Design and Screening

[0057] CPA is a novel isothermal nucleic acid amplification technique. Its primer design principles and rules differ from those of PCR, LAMP, and other amplification techniques; therefore, existing primer and probe sequences are not applicable. CPA primer design involves multiple primer pairs, and only through screening and comparison can the optimal amplification effect and specificity be obtained. This invention downloaded nucleic acid sequences of different subtypes of the uidA gene, stx1, and stx2 genes from the GeneBank database (www.ncbi.nlm.nih.gov / genbank), including three stx1 gene subtypes (stx1a, stx1c, stx1d) and seven stx2 gene subtypes (stx2a, stx2b, stx2c, stx2d, stx2e, stx2f, stx2g). Using MEGA6 software, conserved fragments of 200-300 bp were identified by comparing the above genes. With the assistance of professional software such as NCBI / Primer, Snapgene Viewer, and Priemer5, candidate primers and probes were selected for the chosen target sequences, ensuring low dG values ​​between primer pairs and minimal interference between primers. Through extensive sequence analysis, manual screening and optimization, and performance verification, three optimal primer sets were obtained. These were used to detect the uidA gene, three subtypes of the stx1 gene, and seven subtypes of the stx2 gene, respectively. Primer 4 in all three primer sets was labeled with a fluorescein isothiocyanate group at its 5' end, and primer 5 was labeled with a biotin fluorescein group at its 5' end, for interpreting nucleic acid test strip results. The sequences are as follows:

[0058] Table 1 Primer sequences for the detection of Shiga toxin-producing Escherichia coli (CPA)

[0059]

[0060]

[0061] The target gene stx2 contains seven genotypes: stx2a, stx2b, stx2c, stx2d, stx2e, and stx2g. The stx2 base sequences differ significantly, especially stx2f, making it difficult to detect all genotypes simultaneously using a single primer and probe set. Therefore, primer set III in this invention employs multiple primer pairs, each corresponding to a different genotype. Primers stx2-1, stx2-2, stx2-5, stx2-8, and stx2-10 primarily target six genotypes: stx2a, stx2b, stx2c, stx2d, stx2e, and stx2g. Primers stx2-3, stx2-4, stx2-6, stx2-7, and stx2-9 primarily target the stx2f genotype. Using all ten primers in combination allows for the simultaneous detection of all seven stx2 genotypes. A search revealed that existing patents and literature on STEC detection methods do not cover all genotypes, and in particular, do not address the detection of the stx2f genotype.

[0062] The STEC cross-primer isothermal amplification detection method includes the following steps:

[0063] Step 1. Rapid extraction of STEC genomic DNA. Take 1 mL of pure culture of Escherichia coli (accession number: CICC 24187), centrifuge at 12000 rpm for 3 min to collect the bacterial cells; add 100 μL of sterile ultrapure water to resuspend, boil at 100℃ for 5 min, centrifuge at 12000 rpm for 1 min, transfer the supernatant as a DNA template, and store at -20℃ for later use.

[0064] Step 2. Establishment of the cross-primer amplification method. Using the genomic DNA described above as a template, the optimal primer set I selected was used for CPA amplification to detect the presence of the uidA gene. The CPA reaction system is as follows:

[0065] Component preservation concentration and dosage

[0066]

[0067] The Bst enzyme buffer consists of 200–500 mM Tris-HCl, 100–300 mM (NH4)2SO4, 100–300 mM KCl, 20 mM MgSO4, and 1%–5% Triton X-100 by mass. The pH value is 8.0–8.8 at 25°C.

[0068] After the system is prepared, it is placed in a PCR instrument for reaction. The amplification conditions are: 60℃~67℃, 30min~50min.

[0069] After the reaction, take 10 μL of the amplification product and drop it onto the sample pad of a disposable nucleic acid test strip (provided by Hangzhou Youstar Biotechnology Co., Ltd.) that can detect nucleic acid samples labeled with both isothiocyanate fluorescent group and biotin fluorescent group. Insert the test strip into the cleaning solution for chromatography and wait 2-5 minutes to observe the results.

[0070] If no red line appears, the test has failed and needs to be repeated. If only one red line appears in control area C, the target gene is not detected in the sample. If two red lines appear, one test line and one control line, the target gene is detected in the sample. Then, using the genomic DNA as a template, select the optimal primer set II and primer set III for CPA amplification to detect whether the stx1 and stx2 genes are present. The CPA reaction system is as follows:

[0071] Component preservation concentration and dosage

[0072]

[0073] The CPA reaction system is as follows:

[0074] Component preservation concentration and dosage

[0075]

[0076] The Bst enzyme buffer consists of 200–500 mM Tris-HCl, 100–300 mM (NH4)2SO4, 100–300 mM KCl, 20 mM MgSO4, and 1%–5% Triton X-100 by mass. The pH value is 8.0–8.8 at 25°C.

[0077] After the system is prepared, it is placed in a PCR instrument for reaction. The amplification conditions are: 60℃~67℃, 30min~50min.

[0078] After the reaction, take 10 μL of the amplification product and drop it onto the sample pad of a disposable nucleic acid test strip (provided by Hangzhou Youstar Biotechnology Co., Ltd.) that can detect nucleic acid samples labeled with both isothiocyanate fluorescent group and biotin fluorescent group. Insert the test strip into the cleaning solution for chromatography and wait 2-5 minutes to observe the results.

[0079] If only one red line appears in the quality control area C, it means that the target gene was not detected in the sample. If two red lines appear, one detection line and one quality control line, it means that the target gene was detected in the sample. If no red line appears, it means that the test failed and needs to be repeated.

[0080] The experiment showed that the three target genes uidA, stx1, and stx2 were all positive in the standard strain of Escherichia coli (CICC 24187).

[0081] Through optimization of amplification conditions, the optimal amplification conditions for this invention were found to be 63°C for 45 minutes.

[0082] Step 3. The sensitivity of the STEC detection method provided in this embodiment was verified by bacterial cell detection. Fresh pure cultures of E. coli CICC 24187 were used to prepare OD=1.0 bacterial suspensions using a McFarland turbidimeter, and then serially diluted 10-fold with PBS to obtain 10... 8 CFU / mL-10 0 Genomic DNA was rapidly extracted from CFU / mL bacterial suspensions to assess the sensitivity of this method. Figure 1 It can be seen that the sensitivity of detecting STEC bacteria using primer sets I-III is 10. 2 At CFU / mL, the bacterial cells tested positive for all three genes at this concentration.

[0083] Example 2: STEC Cross-Primer Isothermal Amplification Integrated Detection Device

[0084] This embodiment provides an integrated detection device for cross-primer isothermal amplification;

[0085] The integrated detection device for cross-primer isothermal amplification was provided by Hangzhou Youstar Biotechnology Co., Ltd., as shown in patent CN202110418804.3. The device includes a power supply and switch, a base, a sandwich structure, a functional chamber, a top cover, reagent components, test strips, gaskets, and well caps.

[0086] The operation method includes the following steps:

[0087] Step 1: Assemble the integrated device. Insert the heating element and battery into the corresponding positions of the switch, install them into the base, and then cover the interlayer. Pre-coat the functional chamber with primer set I and primer sets II-III and the reaction reagents designed in Example 1 of this invention. Place the disposable nucleic acid test strip into the corresponding reading window position of the functional chamber and close the top cover. Add a pad to the sample loading well and close the well cover. The device is now complete.

[0088] Step 2: Rapid extraction of STEC genomic DNA. Take 1 mL of pure culture of Escherichia coli CICC 24187, centrifuge at 12000 rpm for 3 min to collect the bacterial cells; add 100 μL of sterile ultrapure water to resuspend, boil at 100℃ for 5 min, centrifuge at 12000 rpm for 1 min, transfer the supernatant as a DNA template, and store at -20℃ for later use.

[0089] Step 3: Amplification and Detection. Mix 50 μl of template DNA solution with 225 μL of reconstitution solution (reconstitution solution composition: 20–50 mM Tris-HCl, 10–30 mM (NH4)2SO4, 10–30 mM KCl, 2–6 mM MgSO4, 0.1%–0.5% Triton X-100 by mass percentage), and add to the sample well. Turn on the power switch and react for 45 min. Turn off the power, add washing buffer (physiological saline), and observe the results after 5 min. If only one red line appears in control zone C, it indicates that the target gene was not detected in the sample; if two red lines appear, one detection line and one control line, it indicates that the target gene was detected in the sample; if no red line appears, it indicates that the detection failed and needs to be repeated. This experiment shows that the three target genes uidA, stx1, and stx2 of the Escherichia coli standard strain CICC24187 were all detected positively.

[0090] Step 4: Verify the sensitivity of the STEC integrated detection device provided in this embodiment by bacterial cell detection. Fresh pure culture of E. coli CICC 21530 was used to prepare an OD=1.0 bacterial suspension using a McFarland turbidimeter, and then serially diluted 10-fold with PBS to obtain 10... 8 CFU / mL-10 0 Genomic DNA was rapidly extracted from CFU / mL bacterial suspensions to assess the sensitivity of this method. Figure 2 It can be seen that the sensitivity of detecting STEC bacteria using primer sets I-III is 10. 2 At CFU / mL, the bacterial cells tested positive for all three genes at this concentration.

[0091] Example 3: Specificity Detection of STEC Cross-Primer Isothermal Amplification

[0092] This embodiment uses the method provided in Example 1 to verify the specificity of the method. The specific method is as follows:

[0093] A total of 22 test strains were used in the experiment (Table 2). Among them, the 9 STEC strains numbered 1-9 were from the China National Institutes for Food and Drug Control, and were non-O157:H7 serotype Shiga toxin-producing Escherichia coli Non-O157 STEC (labeled as FC and ST strains). The other strains were from the strain preservation center. Of the first eight Non-O157 STEC strains (ST437, ST003, FC7783, ST426, FC7785, FC7662, FC7854, and FC7793), serotype and stx genotype information were determined according to the method published by Fan Yiling et al. in "Microfluidic Chip Technology for Recombinase Polymerase Amplification Detection of Shiga Toxin-Producing Escherichia coli". The corresponding serotypes were O111:H8, O45:H2, O8:K85, O121:H19, O118:H12, O177:HNT, O8:H9, and O2:H25, respectively. Fan Yiling et al. were also unable to identify and detect FC7789.

[0094] After DNA extraction, FC7789 was sequenced, and the nucleotide sequence was SEQ ID No. 21. The Stx genotype was Stx2f.

[0095] Table 2. Strains used in the experiment and results of real-time PCR and fluorescent RPA detection of the stx gene.

[0096]

[0097] Note: Information on FC and ST numbered bacterial species and related strains is provided by the China National Institutes for Food and Drug Control; / . Unknown, +. Positive, -. Negative.

[0098] Table 3 Information on strains used for specific detection

[0099]

[0100]

[0101] The above-mentioned microbial strains were inoculated into tryptic soy broth (TSB) and cultured overnight at 36°C. DNA was extracted, nucleic acid amplification was performed, and results were observed using the method provided in Example 2. The results showed that the identification of the target bacteria's uidA, stx1, and stx2 genes was consistent with the confirmed gene information in Table 3. The nucleic acid test results for non-target bacteria were all negative. Figure 3 As shown. This method can effectively detect all Shiga toxin-producing Escherichia coli stx gene subtypes.

[0102] Example 3: Comparison between the STEC cross-primer isothermal amplification integrated detection method and the national standard culture method

[0103] This embodiment verifies the method provided in Example 3 by detecting artificially contaminated samples. The specific steps are as follows:

[0104] Step 1. Take commercially available braised beef samples, sterilize and homogenize them, and divide them into 25 equal portions, each weighing approximately 25g. Randomly select one portion as a negative control, and divide the other samples into 3 groups, adding E. coli CICC 24187 bacterial suspension to achieve inoculation concentrations of 100 CFU / 25g, 10 CFU / 25g, and 1 CFU / 25g, respectively.

[0105] Step 2. Perform enrichment culture according to GB 4789.6-2016 "National Food Safety Standard - Microbiological Examination of Food - Examination of Diarrheal Escherichia coli". Take 1 mL of intestinal enrichment broth culture and centrifuge at 500 rpm for 1 min; transfer the supernatant to a new sterile centrifuge tube and centrifuge at 12000 rpm for 3 min; discard the supernatant, add 100 μL of ultrapure water to resuspend, boil at 100℃ for 5 min, centrifuge at 12000 rpm for 1 min, transfer the supernatant as a DNA template, and store at -20℃ for later use. Analyze the remaining broth culture according to national standard methods.

[0106] Step 3: Compare the results of the national standard method and the integrated detection method of cross-primer amplification to analyze the relative accuracy and detection level of the methods.

[0107] Table 4 Comparison of results from integrated detection using cross-primer isothermal amplification and national standard method.

[0108]

[0109] As shown in Table 4, the above two methods yielded positive results for samples with inoculation amounts of 100 CFU / 25g, 10 CFU / 25g, and 1 CFU / 25g contamination levels. The relative accuracy of the cross-primer amplification integrated detection method was 100%, with no false positives or false negatives observed, and the detection limit was 1 CFU / 25g.

[0110]

[0111]

[0112]

[0113]

[0114]

[0115] sequence list <110> Hubei Provincial Institute of Food Quality and Safety Supervision and Inspection <120> A cross-primer combination and method for isothermal amplification of nucleic acid using cross-primers to detect Shiga toxin-producing Escherichia coli. <160> twenty one <170> SIPOSequenceListing 1.0 <210> 1 <211> 19 <212> DNA <213> Artificial Sequence <400> 1 aggcacagca catcaaaga 19 <210> 2 <211> 18 <212> DNA <213> Artificial Sequence <400> 2 agcgcgtgac aaaaacca 18 <210> 3 <211> 40 <212> DNA <213> Artificial Sequence <400> 3 ccgccagtgg cgcgaaatat tggagtattg ccaacgaacc 40 <210> 4 <211> 20 <212> DNA <213> Artificial Sequence <400> 4 ccgccagtgg cgcgaaatat 20 <210> 5 <211> 18 <212> DNA <213> Artificial Sequence <400> 5 caccttgcgg acgggtat 18 <210> 6 <211> 19 <212> DNA <213> Artificial Sequence <400> 6 ttagaaccag aggaagagc 19 <210> 7 <211> 18 <212> DNA <213> Artificial Sequence <400> 7 atccccgtac gactgatc 18 <210> 8 <211> 43 <212> DNA <213> Artificial Sequence <400> 8 ttcacatgtt acctttcctg gtacgctgta acgtggtata gct 43 <210> 9 <211> twenty two <212> DNA <213> Artificial Sequence <400> 9 ttcacatgtt acctttcctg gt 22 <210> 10 <211> twenty two <212> DNA <213> Artificial Sequence <400> 10 cggttacatt gtctggtgac ag 22 <210> 11 <211> 20 <212> DNA <213> Artificial Sequence <400> 11 gatgcatcca gagcagttct 20 <210> 12 <211> 19 <212> DNA <213> Artificial Sequence <400> 12 aggatattct ccccactct 19 <210> 13 <211> twenty one <212> DNA <213> Artificial Sequence <400> 13 cacatatttc agtgcctgat g 21 <210> 14 <211> 19 <212> DNA <213> Artificial Sequence <400> 14 tctgctatca ctgtgacaa 19 <210> 15 <211> 41 <212> DNA <213> Artificial Sequence <400> 15 tgtgtatacg atgacgccgg gaagcacatt gctgattcgc c 41 <210> 16 <211> 43 <212> DNA <213> Artificial Sequence <400> 16 tcattcactg gttggttcat atctgatagc tctggatgat gcg 43 <210> 17 <211> 25 <212> DNA <213> Artificial Sequence <400> 17 tcattcactg gttggttcat atctg <210> 18 <211> 19 <212> DNA <213> Artificial Sequence <400> 18 tgtgtatacg atgacgccg <210> 19 <211> 23 <212> DNA <213> Artificial Sequence <400> 19 taatggagtt cagaggacgt tcc <210> 20 <211> 20 <212> DNA <213> Artificial Sequence <400> 20 acgtggacct cactctgaac <210> 21 <211> 1386 <212> DNA <213> Artificial Sequence <400> 21 atgcgacata tattattaaa gctggtgttg tttttttgtg tttgcttgtc ttcagtatct tatgcagatg agtttactgt ggatttctct tcgcaaaaga gctatgttga ttcattgaat agtataggt cggcaatatc cactccgctt ggaatatat ctcagggtgg tatttctgtt tcagtaatta atcatgttcc aggtggaaac fathers tgaatgttag aggccttgag ccatatagcg agagatttaa ccacctccgt ttaataatgg aacggaataa cttatatgtt 300 gcaggcttta ttaatactga aacgaatacc ttcacagat tctccgattt ctcacatatt 360 tcagtgcctg atgtgataac tgtttccatg acgacggaca gcagttattc atcattacag 420 cgaatcgcag atctggaacg tacaggtg cagattgggc gtcattcact ggttggttca 480 tatctggatt tatggatt spacecgt tcatgaccc gcgcatc spacectag 540 ctgcgttttg tcacagtgat agcagaagct ctgcgattca gataca gcggggattc 600 cgaccggcgc tgtctgaggc atctccgct tatacaatga cggctcagga tgttgacctt 660 accctgaact ggggaagaat aagtaatgtt cttccagagt acagaggaga ggaggggta 720 agaatcggta ggatatcttt tatagtctt tctgcgattc tcggaagtgt tgcggtcatc 780 cttaattgcc actcaccgg aagttattca gttcgttccg tgagccaaa acagaaaaca 840 gatgccaga ttgttggaga caggggcggcc attaaagtaa ataatgtttt gtgggaagcg 900 atacaatcg ctgctttatt aaatcgcaag cctcagggtc ttactgaacc aaaccaataa 960 cagggggtga attgaagaa gatgattatt gcagttttat tcggtctctt ttctgctaat tccatggcgg cggattgtgc tgtaggaaaa attgagtttt ccaagtata tgaggataat acctttactg tgagggtgtc aggagaga tactggacga acagatgga tttgcagcca ttgttacaaa gtgctcagct gacagggatg actgtaacaa tcatatctaa tacctgcagt tcaggctcag gctttgccca ggtgaagttt aactgagaat ctacggttta tttatgcgcg tcttttgttt ctggacgcag atattattag tgttgtggat gctgattaa tttggtcagt 1320. gttttcgtta aagtcatata aatacagggg cgttcacgcc cctttttggt ctgtagttgg 1380 gtgaag 1386

Claims

1. A kit containing a combination of CPA primers for detecting Shiga toxin-producing Escherichia coli, characterized in that, It includes three sets of CPA primers: primer set I for detecting uidA genotype Shiga toxin-producing Escherichia coli, primer set II for detecting stx1 subtype Shiga toxin-producing Escherichia coli, and primer set III for detecting stx2 subtype Shiga toxin-producing Escherichia coli. The primer set I includes: stripping primer uidA-1, whose nucleotide sequence is shown in SEQ ID NO.1; stripping primer uidA-2, whose nucleotide sequence is shown in SEQ ID NO.2; cross primer uidA-3, whose nucleotide sequence is shown in SEQ ID NO.3; detection primer uidA-4, whose nucleotide sequence is shown in SEQ ID NO.4; and detection primer uidA-5, whose nucleotide sequence is shown in SEQ ID NO.

5. Primer set II includes: stripping primer stx1-1, the nucleotide sequence of which is shown in SEQ ID NO.6; stripping primer stx1-2, the nucleotide sequence of which is shown in SEQ ID NO.7; cross primer stx1-3, the nucleotide sequence of which is shown in SEQ ID NO.8; detection primer stx1-4, the nucleotide sequence of which is shown in SEQ ID NO.9; and detection primer stx1-5, the nucleotide sequence of which is shown in SEQ ID NO.

10. Primer set III includes: stripping primer stx2-1, whose nucleotide sequence is shown in SEQ ID NO.11; stripping primer stx2-2, whose nucleotide sequence is shown in SEQ ID NO.12; stripping primer stx2-3, whose nucleotide sequence is shown in SEQ ID NO.13; stripping primer stx2-4, whose nucleotide sequence is shown in SEQ ID NO.14; cross primer stx2-5, whose nucleotide sequence is shown in SEQ ID NO.15; cross primer stx2-6, whose nucleotide sequence is shown in SEQ ID NO.16; detection primer stx2-7, whose nucleotide sequence is shown in SEQ ID NO.17; detection primer stx2-8, whose nucleotide sequence is shown in SEQ ID NO.18; detection primer stx2-9, whose nucleotide sequence is shown in SEQ ID NO.19; and detection primer stx2-10, whose nucleotide sequence is shown in SEQ ID NO.

20. The 5' ends of detection primers stx1-4, stx2-7, and stx2-8 are labeled with fluorescein isothiocyanate groups, while the 5' ends of detection primers stx1-5, stx2-9, and stx2-10 are labeled with biotin fluorescein groups.

2. The reagent kit according to claim 1, characterized in that, The concentration of each CPA detection primer in the kit is 10 μmol / L.

3. A method for detecting Shiga toxin-producing Escherichia coli for non-disease diagnostic purposes using the kit according to any one of claims 1 to 2, characterized in that, Includes the following steps: Step 1: Extract DNA from the sample to be tested; Step 2: Prepare the reaction system using primer set I from the kit and add the DNA sample; Step 3: Thoroughly mix the reaction system and carry out the amplification reaction under constant temperature conditions; Step four: Detect the amplification products using disposable nucleic acid test strips and observe the results; Result interpretation: If no red line appears, it indicates that the test has failed and needs to be repeated; if only one red line appears in the quality control area C, it means that the target gene Escherichia coli was not detected in the sample. If two red lines appear, namely a test line and a control line, it means that the target gene Escherichia coli has been detected in the sample. Continue with the following steps. Step 5: Prepare reaction systems using primer set II and primer set III from the kit, and add the DNA sample; Step 6: Thoroughly mix the reaction system containing primer set II and the reaction system containing primer set III, and carry out the amplification reaction under isothermal conditions; Step 7: Detect the amplification products using disposable nucleic acid test strips and observe the results; Result interpretation: If no red line appears, it indicates that the test has failed and needs to be repeated; if only one red line appears in the quality control area C, it means that the target gene was not detected in the sample to be tested. If two red lines appear, namely a test line and a control line, it means that the target gene has been detected in the sample, that is, Shiga toxin-producing Escherichia coli has been found.

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