Kit for typing detection of diarrheagenic Escherichia coli and application thereof

Abstract

The invention relates to the technical field of bacterial detection, and discloses a kit for detecting diarrheogenic Escherichia coli in a typing manner and application of the kit. According to the kit disclosed by the invention, by adopting a specific gene selection arrangement mode and matching with a specific primer probe sequence, the typing detection of five DEC can be realized only by two tubes and three channels, the result interpretation is relatively simple, the types of required fluorophores are relatively few, the detection quantity is low, and the kit is easier to deal with the screening of large-batch samples of foodborne disease outbreak events; moreover, in cooperation with specific target gene arrangement modes in the tube A and the tube B, detection primers and probe sequences of all target genes are specially designed, synchronous detection of genes of lt, stp, sth, ipaH and escV can be achieved in the tube A, synchronous detection of genes of stx1, stx2, aggR, astA and pic can be achieved in the tube B, and high detection specificity, sensitivity and accuracy are achieved.

Description

Technical Field

[0001] The present invention relates to the technical field of bacteria detection, in particular to a kit for typing and detecting diarrhea-causing Escherichia coli and application thereof. Background Art

[0002] Escherichia coli is a normal flora in the human body. Among them, there is a type of diarrheagenic Escherichia coli (DEC) that can cause human diseases by contaminating food, mainly causing diarrhea. It is divided into the following five types based on the characteristic genes it carries: enteroaggregative Escherichia coli (EAEC), enteropathogenic Escherichia coli (EPEC), enterotoxigenic Escherichia coli (ETEC), enterohemorrhagic Escherichia coli (EHEC) and enteroinrasive Escherichia coli (EIEC).

[0003] Real-time fluorescence quantitative PCR (qPCR) boasts high throughput, high sensitivity, high specificity, rapidity, and easy-to-interpret results. It can simultaneously detect multiple genes, shortening detection cycles and improving efficiency. Patent CN119639934A discloses a probe-primer combination and kit for the rapid detection of five diarrheagenic Escherichia coli strains. The kit uses a single-tube 12-channel method or a three-tube 4-channel method to detect 12 genes to determine the type of DEC in a sample. The results are complex to interpret, and the single-tube 12-channel method requires a wide variety of fluorescent groups. The three-tube 4-channel method requires a large number of samples for high-throughput testing, making it difficult to screen large quantities of samples. Summary of the Invention

[0004] To address the technical issues of existing methods for typing five types of diarrheagenic Escherichia coli, which often involve complex interpretation of results and require a wide variety of fluorescent groups or a large detection volume, the present invention provides a kit for typing and detecting diarrheagenic Escherichia coli and its use. Using the kit, five DEC typing tests can be performed with only two tubes and three channels. Results are relatively simple to interpret, and the kit requires fewer fluorescent groups and a lower detection volume, making it easier to screen large numbers of samples.

[0005] The specific technical solutions of the present invention are: In a first aspect, the present invention provides a kit for typing and detecting diarrhea-causing Escherichia coli, comprising a primer probe for tube A and a primer probe for tube B, each comprising the following primers and probes: In the table above, the fluorescent signals of fluorescent groups a, b, and c are different.

[0006] In the primer / probe names mentioned in the table above, the suffix "-F" indicates an upstream primer, the suffix "-R" indicates a downstream primer, and the suffix "-P" indicates a probe.

[0007] More than 97.0% of Escherichia coli are positive for the uidA gene, and foodborne disease samples are feces and anal swabs, which contain a large number of Escherichia coli. Therefore, the uidA gene must be positive, and the bfpB gene does not affect the EPEC determination. Therefore, in the present invention, uidA and bfpB can be omitted as detection target genes and do not affect the result determination of the five DEC typing tests.

[0008] In the present invention, by adopting a specific gene selection arrangement (specific target genes are arranged in tubes A and B, respectively, and the three fluorescent groups a, b, and c are respectively assigned to the detection probes of the specific target genes), and in combination with specific primer probe sequences, only two tubes and three channels are required to achieve typing detection of five types of DEC. The result interpretation is relatively simple, and the required types of fluorescent groups are relatively few (only three fluorescent groups are required), which makes it easy to avoid mutual interference between different fluorescent groups. At the same time, it also has the advantage of low detection volume, which makes it easier to cope with the screening of large quantities of samples in foodborne disease outbreaks.

[0009] Furthermore, the present invention is specially designed for the detection primers and probe sequences of each target gene in conjunction with the specific target gene arrangement in tubes A and B, thereby achieving high specificity and detection sensitivity. This minimizes mutual interference between different target genes in the same tube during detection and maintains similar amplification rates, thereby facilitating the simultaneous detection of lt, stp, sth, ipaH, and escV genes in tube A, and of stx1, stx2, aggR, astA, and pic genes in tube B, with high detection accuracy.

[0010] Preferably, the kit further comprises:

[0011] The primers and probes for detecting the uidA and bfpB genes can be used to subsequently confirm the genetic makeup of pure bacteria isolated and cultured. Specifically, after initially screening a sample using primers and probes for the lt, stp, sth, ipaH, escV, stx1, stx2, aggR, astA, and pic genes to determine the DEC type present, pure bacteria are isolated and cultured. These pure bacteria are then rechecked using primers and probes for the lt, stp, sth, ipaH, escV, stx1, stx2, aggR, astA, pic, uidA, and bfpB genes to verify whether they are the DEC type identified in the initial screening. This rechecking process can further improve the accuracy of the test results.

[0012] Preferably, the two ends of the probe uidA are respectively labeled with a fluorescent group d and a quenching group, and the fluorescent group d is different from the fluorescent groups a, b and c; the two ends of the probe bfpB-P are respectively labeled with a fluorescent group b and a quenching group; the kit further comprises: target gene Probe Name sequence Probe end labeling astA astA-P' SEQ ID NO:27 Fluorescent group a, quenching group stx2 stx2-P' SEQ ID NO:21 Fluorescent group b, quenching group stp stp-P' SEQ ID NO:6 Fluorescent group b, quenching group sth sth-P' SEQ ID NO:9 Fluorescent group c, quencher group

[0013] In the present invention, based on the primer probes for each A tube and the primer probe for each B tube, by additionally adding astA-P', stx2-P', stp-P' and sth-P' shown in the above table, a separate detection kit for five DECs can be formed, which facilitates the subsequent review of pure bacteria obtained by isolation and culture, without the need for additional identification of other genes or the use of other separate DEC detection kits.

[0014] When retesting for pure bacteria, primers and probes corresponding to the aggR, pic, astA, and uidA genes can be mixed (with the probe for astA using astA-P') for EAEC retesting; primers and probes corresponding to the escV, bfpB, and uidA genes can be mixed for EPEC retesting; primers and probes corresponding to the stx1, stx2, escV, and uidA genes can be mixed (with the probe for stx2 using stx2-P') for EHEC retesting; primers and probes corresponding to the lt, stp, sth, and uidA genes can be mixed (with the probes for stp and sth using stp-P' and sth-P', respectively) for ETEC retesting; and primers and probes corresponding to the ipaH and uidA genes can be mixed for EIEC retesting. During the retesting of each DEC, the corresponding characteristic genes can be detected simultaneously, minimizing interference and improving detection sensitivity and accuracy.

[0015] Preferably, the fluorescent groups a, b, c and d are independently selected from FAM, VIC, ROX and CY5.

[0016] Preferably, the quenching group is BHQ1.

[0017] Preferably, the kit further comprises a nucleic acid amplification reaction solution.

[0018] In a second aspect, the present invention provides a use of the kit for non-diagnostic detection of five types of diarrheagenic Escherichia coli, comprising the following steps: S1: Extract nucleic acid from the sample to be tested to obtain a nucleic acid sample; S2: Mix all the primers and probes in tube A and perform qPCR on the nucleic acid sample; S3: Mix all B tubes with primers and probes and perform qPCR on the nucleic acid sample; S4: Interpret the results of S1 and S2 according to the following table:

[0019] In the above table, “+” indicates a positive test result and “-” indicates a negative test result.

[0020] AstA and pic are drift genes. Therefore, in the result interpretation method of the present invention, when astA and pic are detected simultaneously with other characteristic genes except aggR, they are not used as the basis for determining EAEC.

[0021] Preferably, in S2 to S4, when the CT value measured in qPCR is ≤35 and there is an obvious amplification curve, it is judged as positive, otherwise it is judged as negative.

[0022] In a third aspect, the present invention provides a method for detecting five types of diarrhea-causing Escherichia coli for non-diagnostic purposes, comprising the following steps: S1: Extract nucleic acid from the sample to be tested to obtain a nucleic acid sample; S2: Mix all the primers and probes in tube A and perform qPCR on the nucleic acid sample; S3: Mix all B tubes with primers and probes and perform qPCR on the nucleic acid sample; S4: Interpret the results of S1 and S2 according to the following table: S5: Review the diarrhea-causing Escherichia coli identified in S4.

[0023] Preferably, in S2 to S4, when the CT value measured in qPCR is ≤35 and there is an obvious amplification curve, it is judged as positive, otherwise it is judged as negative.

[0024] Preferably, the specific process of S5 includes: isolating and culturing the diarrheagenic Escherichia coli in the sample to be tested, extracting nucleic acid to obtain a target bacterial nucleic acid sample; performing qPCR on the target bacterial nucleic acid sample according to the following method to confirm the type of diarrheagenic Escherichia coli contained in the sample to be tested:

[0025] The specific meanings of the above table are as follows: (1) When S4 determines that the sample to be tested contains enteroaggregative Escherichia coli, qPCR is performed on the target bacterial nucleic acid sample using astA-F, astA-R, astA-P', aggR-F, aggR-R, aggR-P, pic-F, pic-R, pic-P, uidA-F, uidA-R, and uidA-P; when the qPCR results show that at least one of the aggR, pic, and astA genes is positive, and the uidA gene is positive, it is confirmed that the sample to be tested contains enteroaggregative Escherichia coli; (2) When S4 determines that the sample to be tested contains enteropathogenic Escherichia coli, qPCR is performed on the target bacterial nucleic acid sample using escV-F, escV-R, escV-P, bfpB-F, bfpB-R, bfpB-P, uidA-F, uidA-R, and uidA-P; when the qPCR results show that both escV and uidA genes are positive (bfpB gene can be positive or negative), it is confirmed that the sample to be tested contains enteropathogenic Escherichia coli; (3) When S4 determines that the sample to be tested contains enterohemorrhagic Escherichia coli, qPCR is performed on the target bacterial nucleic acid sample using stx1-F, stx1-R, stx1-P, stx2-F, stx2-R, stx2-P', escV-F, escV-R, escV-P, uidA-F, uidA-R, and uidA-P; when the qPCR results show that at least one of the stx1 and stx2 genes is positive and the uidA gene is positive (the escV gene can be positive or negative), it is confirmed that the sample to be tested contains enterohemorrhagic Escherichia coli; (4) When S4 determines that the sample to be tested contains enterotoxigenic Escherichia coli, qPCR is performed on the target bacterial nucleic acid sample using lt-F, lt-R, lt-P, stp-F, stp-R, stp-P', sth-F, sth-R, sth-P', uidA-F, uidA-R, and uidA-P; when the qPCR results show that at least one of the lt, stp, and sth genes is positive, and the uidA gene is positive, it is confirmed that the sample to be tested contains enterotoxigenic Escherichia coli; (5) When S4 determines that the sample to be tested contains enteroinvasive Escherichia coli, qPCR is performed on the target bacterial nucleic acid sample using ipaH-F, ipaH-R, ipaH-P, uidA-F, uidA-R, and uidA-P. When the qPCR results show that both the ipaH and uidA genes are positive, it is confirmed that the sample to be tested contains enteroinvasive Escherichia coli.

[0026] Preferably, in S5, when the qPCR results show that both the ipaH and uidA genes are positive, Shigella sonnei and Shigella flexneri are excluded based on the colony growth on the SS plate and the lactose fermentation test, and then confirmed as enteroinvasive Escherichia coli.

[0027] Preferably, in S5, when the CT value measured in qPCR is ≤35 and there is an obvious amplification curve, it is judged as positive, otherwise it is judged as negative.

[0028] Compared with the prior art, the present invention has the following advantages: (1) In the kit of the present invention, by adopting a specific gene selection arrangement and combining with specific primer probe sequences, only two tubes and three channels are needed to achieve typing detection of five DECs. The result interpretation is relatively simple, and the required types of fluorescent groups are relatively few, the detection amount is low, and it is easier to cope with the screening of large quantities of samples in foodborne disease outbreaks.

[0029] (2) In the kit of the present invention, the detection primers and probe sequences of each target gene are specially designed in conjunction with the specific target gene arrangement in tube A and tube B, which can achieve simultaneous detection of lt, stp, sth, ipaH and escV genes in tube A, and simultaneous detection of stx1, stx2, aggR, astA and pic genes in tube B, with high detection specificity, sensitivity and accuracy; in addition, the use of the primers and probe sequences of the present invention can also achieve simultaneous detection of different characteristic genes in the same DEC during the review process, so that the review detection results have high accuracy.

[0030] (3) The kit of the present invention can be used to perform preliminary screening of the sample to be tested to determine the type of DEC present in the sample to be tested. It can also be used to recheck the pure bacteria isolated and cultured from the sample to be tested to verify whether the preliminary screening results are accurate, thereby ensuring the accuracy of the five DEC typing test results. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 The ETEC standard strain CICC10667 was used as the reproducibility test with a 0.5 McFarland standard (approximately 1×10 8 ~2×10 8CFU / mL) 10-fold gradient dilution, where A~E are the dilutions of 10 -1 , 10 -2 , 10 -3 , 10 -4 , 10 -5 .

[0032] Figure 2 Shown are the amplification curves of the uidA and ipaH genes of Shigella sonnei and Shigella flexneri.

[0033] Figure 3 The standard curve obtained for the lt gene in the sensitivity test.

[0034] Figure 4 Figure 1 is the amplification curve of lt gene at different plasmid copy concentrations in the sensitivity test. Among them, A~H are the amplification curves of lt gene at different plasmid copy concentrations of 5×10 7 copies / μL, 5×10 6 copies / μL, 5×10 5 copies / μL, 5×10 4 copies / μL, 5×10 3 copies / μL, 5×10 2 copies / μL, 5×10copies / μL, 5copies / μL. DETAILED DESCRIPTION

[0035] The present invention will be further described below with reference to the embodiments.

[0036] Overall embodiment First, the present invention relates to a kit for typing and detecting diarrhea-causing Escherichia coli, comprising a primer probe for tube A and a primer probe for tube B, which respectively comprise the following primers and probes: In the table above, the fluorescent signals of fluorescent groups a, b, and c are different.

[0037] In some embodiments, the kit further comprises:

[0038] In some specific embodiments, the two ends of the probe uidA are respectively labeled with a fluorescent group d and a quencher group, and the fluorescent group d is different from the fluorescent groups a, b, and c; the two ends of the probe bfpB-P are respectively labeled with a fluorescent group b and a quencher group; the kit further comprises:

[0039] In some embodiments, the fluorescent groups a, b, c and d are independently selected from FAM, VIC, ROX and CY5.

[0040] In some embodiments, the quencher group is BHQ1.

[0041] In some specific embodiments, the kit further comprises a nucleic acid amplification reaction solution.

[0042] Second, the present invention relates to the use of the kit for non-diagnostic detection of five types of diarrheagenic Escherichia coli, comprising the following steps: S1: Extract nucleic acid from the sample to be tested to obtain a nucleic acid sample; S2: Mix all the primers and probes in tube A and perform qPCR on the nucleic acid sample; S3: Mix all B tubes with primers and probes and perform qPCR on the nucleic acid sample; S4: Interpret the results of S1 and S2 according to the following table:

[0043] In some specific embodiments, in S2 to S4, when the CT value measured in qPCR is ≤35 and there is an obvious amplification curve, it is judged as positive, otherwise it is judged as negative.

[0044] Thirdly, the present invention relates to the use of the kit for non-diagnostic detection of five types of diarrheagenic Escherichia coli, comprising the following steps: S1: Extract nucleic acid from the sample to be tested to obtain a nucleic acid sample; S2: Mix all the primers and probes in tube A and perform qPCR on the nucleic acid sample; S3: Mix all B tubes with primers and probes and perform qPCR on the nucleic acid sample; S4: Interpret the results of S1 and S2 according to the following table: S5: Review the diarrhea-causing Escherichia coli identified in S4.

[0045] In some specific embodiments, in S2 to S4, when the CT value measured in qPCR is ≤35 and there is an obvious amplification curve, it is judged as positive, otherwise it is judged as negative.

[0046] In some embodiments, the specific process of S5 includes: isolating and culturing the diarrheagenic Escherichia coli in the test sample, extracting nucleic acid to obtain a target bacterial nucleic acid sample; and performing qPCR on the target bacterial nucleic acid sample according to the following method to confirm the type of diarrheagenic Escherichia coli contained in the test sample:

[0047] In some embodiments, in S5, when the qPCR results show that both the ipaH and uidA genes are positive, Shigella sonnei and Shigella flexneri are excluded based on the colony growth on the SS plate and the lactose fermentation test, and then confirmed as enteroinvasive Escherichia coli.

[0048] In some specific embodiments, in S5, when the CT value measured in qPCR is ≤35 and there is an obvious amplification curve, it is judged as positive, otherwise it is judged as negative. Specific embodiments The present invention is described below by way of specific examples. It should be understood that these examples are intended only to illustrate the present invention and are not intended to limit the scope of the present invention. Without departing from the spirit and scope of the inventive concept, any changes and advantages that can be imagined by those skilled in the art are included in the present invention, and the scope of protection of the present invention is defined by the appended claims and any equivalents thereof.

[0050] Unless otherwise defined, all technical and scientific terms used herein have the same meanings as commonly understood by those skilled in the art to which this disclosure pertains. Unless otherwise specified, the raw materials and equipment used herein are conventional in the art and can be obtained from conventional commercial sources. The methods used herein are conventional in the art, unless otherwise specified.

[0051] Example 1: Preliminary screening of reaction system and reaction condition design 1.1 Preliminary screening of primer and probe sequences During the initial screening, 10 characteristic genes were detected, and the corresponding primer and probe sequences are shown in Table 1 .

[0052] Table 1. Preliminary screening primer and probe sequences

[0053] In the names of primers / probes in Table 1, the suffix "-F" indicates an upstream primer, the suffix "-R" indicates a downstream primer, and the suffix "-P" indicates a probe.

[0054] 1.2 Initial screening test method Follow the steps below to conduct a preliminary screening test on the sample to be tested: (1) The boiling method is used to extract DNA from the sample to be tested to obtain a nucleic acid sample.

[0055] (2) 0.5 μL of each upstream primer, downstream primer, and probe corresponding to each target gene in Table 1 were mixed to obtain 1.5 μL of primer-probe mixture for detecting the 10 target genes in Table 1, wherein the content of the upstream and downstream primers was 10 μmol / L, and the content of the probe was 5 μmol / L.

[0056] (3) Add 1.5 μL of primer-probe mixture for detecting lt gene, 1.5 μL of primer-probe mixture for detecting stp gene, 1.5 μL of primer-probe mixture for detecting sth gene, 1.5 μL of primer-probe mixture for detecting ipaH gene, 1.5 μL of primer-probe mixture for detecting escV gene, and nucleic acid amplification reaction solution Premix EXTaq to tube A. TM 12.5 μL of RR390A and 5.0 μL of nucleic acid sample were prepared into a total of 25 μL reaction system in tube A.

[0057] (4) Add 1.5 μL of primer-probe mixture for detecting stx1 gene, 1.5 μL of primer-probe mixture for detecting stx2 gene, 1.5 μL of primer-probe mixture for detecting aggR gene, 1.5 μL of primer-probe mixture for detecting astA gene, 1.5 μL of primer-probe mixture for detecting pic gene, and nucleic acid amplification reaction solution Premix EX Taq TM 12.5 μL of RR390A and 5.0 μL of nucleic acid sample were prepared into a total of 25 μL reaction system in tube B.

[0058] (5) Perform qPCR on tubes A and B according to the following reaction procedure: ① 95°C for 20 seconds; ② 95°C for 1 second; ③ 60°C for 20 seconds to collect fluorescence; and cycle ② and ③ 40 times. The PCR instrument was set to FAST mode, and the detection channels were selected as FAM, VIC, and ROX.

[0059] 1.3 Methods for interpreting initial screening results Based on the qPCR results in tubes A and B, determine the type of DEC contained in the sample to be tested according to Table 2.

[0060] Table 2 Interpretation method of primary screening results

[0061] In Table 2, "+" indicates a positive test result and "-" indicates a negative test result. During the qPCR process, a positive result was determined when the measured CT value was ≤35 and there was a clear amplification curve; otherwise, it was determined to be negative.

[0062] Example 2: Initial screening results review reaction system and reaction condition design 2.1 Review of primer and probe sequences During the initial screening, 12 characteristic genes were detected, and the corresponding primer and probe sequences are shown in Table 3 .

[0063] Table 3 Preliminary screening primer and probe sequences

[0064] In the names of primers / probes in Table 3, the suffix "-F" indicates an upstream primer, the suffix "-R" indicates a downstream primer, and the suffix "-P" indicates a probe.

[0065] 2.2 Review of testing methods and result interpretation methods Follow the steps below to review the initial screening results in Example 1: (1) According to the national standard GB 4789.6, the sample to be tested was isolated and cultured to obtain a single DEC colony.

[0066] (2) Pick a single DEC colony and extract nucleic acid using the boiling method to obtain the target bacterial nucleic acid sample.

[0067] (3) 0.5 μL of each upstream primer, downstream primer, and probe corresponding to each target gene in Table 3 were mixed to obtain 1.5 μL of primer-probe mixture for detecting the 10 target genes in Table 1, wherein the content of the upstream and downstream primers was 10 μmol / L, and the content of the probe was 5 μmol / L.

[0068] (4) For different initial screening results, according to Table 4, the corresponding primer probes were selected for re-examination, that is: when the initial screening results showed that the sample to be tested contained EAEC, the primer probes for detecting astA, aggR, pic and uidA in Table 3 were used for re-examination; when the initial screening results showed that the sample to be tested contained EPEC, the primer probes for detecting bfpB, escV and uidA in Table 3 were used for re-examination; when the initial screening results showed that the sample to be tested contained EHEC, the primer probes for detecting stx1, stx2, escV and uidA in Table 3 were used for re-examination; when the initial screening results showed that the sample to be tested contained ETEC, the primer probes for detecting lt, stp, sth and uidA in Table 3 were used for re-examination; when the initial screening results showed that the sample to be tested contained EIEC, the primer probes for detecting ipaH and uidA in Table 3 were used for re-examination.

[0069] Table 4 Review of target genes and result interpretation methods

[0070] (5) The recheck reaction system is as follows: add 1.5×nμL of primer-probe mixture (n is the number of genes to be detected) and nucleic acid amplification reaction solution Premix EX Taq TM RR390A 12.5μL, nuclease-free water 6-1.5×(n-1)μL and target bacterial nucleic acid sample 5.0μL were prepared into a reaction system of 25μL.

[0071] (6) Perform qPCR according to the following procedure: ① 95°C for 20 s; ② 95°C for 1 s; ③ 60°C for 20 s to collect fluorescence; repeat steps ② and ③ 40 times. Select the FAST mode on the PCR instrument and select the detection channels according to Table 4.

[0072] (7) Based on the qPCR results, the DEC type was determined according to Table 4 to verify the accuracy of the initial screening results, namely: when at least one of the aggR, pic, and astA genes was positive and the uidA gene was positive, it was EAEC; when both the escV and uidA genes were positive (the bfpB gene could be positive or negative), it was EPEC; when at least one of the stx1 and stx2 genes was positive and the uidA gene was positive (the escV gene could be positive or negative), it was EHEC; when at least one of the lt, stp, and sth genes was positive and the uidA gene was positive, it was ETEC; when both the ipaH and uidA gene tests were positive, it was EIEC. During the qPCR process, a positive result was determined when the measured CT value was ≤35 and there was a clear amplification curve; otherwise, it was determined to be negative.

[0073] Example 3: Specificity test 3.1 Experimental strains The following 16 experimental bacterial strains were used: Salmonella typhimurium (ATCC50115), Shigella sonnei (ATCC5133), Shigella flexneri (ATCC12022), Staphylococcus aureus (ATCC26003), Vibrio parahaemolyticus (ATCC17802), Vibrio vulnificus (ATCC27526), ​​E. coli (ATCC44102), EAEC (CICC21530), EPEC (CICC24189), ETEC (CICC10667), ETEC (laboratory strain, stp gene positive), EIEC (CICC10662), EHEC (CICC24187), Vibrio cholerae nucleic acid (Inaba type, provided by Zhoushan Municipal Center for Disease Control and Prevention), Campylobacter jejuni (laboratory strain), Listeria monocytogenes (laboratory strain).

[0074] 3.2 Specificity test method After culturing the 16 experimental strains using the corresponding culture medium, colonies were picked and DNA was extracted by the boiling method. Then, using the primers and probes corresponding to the target genes in Table 3 (the probes for the astA, stx2, stp, and sth genes were astA-P, stx2-P, stp-P, and sth-P in Table 2, respectively), qPCR was performed to detect whether the 12 target genes shown in Table 3 were present in the DNA extracted from the 16 experimental strains. During the qPCR detection process, the qPCR reaction system and procedure were as follows: (1) Reaction system: Add 1.5 μL of primer-probe mixture (mixed with 0.5 μL of upstream primer, downstream primer and probe of the target gene to be detected, with the content of upstream and downstream primers both at 10 μmol / L and the content of probe at 5 μmol / L) to each tube, and nucleic acid amplification reaction solution Premix EX Taq TM RR390A 12.5 μL, nuclease-free water 6 μL and plasmid dilution 5.0 μL were prepared to make a total reaction system of 25 μL.

[0075] (2) Reaction procedure: ① 95°C for 20 s; ② 95°C for 1 s; ③ 60°C for 20 s to collect fluorescence; ② and ③ steps were cycled 40 times. The PCR instrument was set to FAST mode.

[0076] 3.3 Specificity test results The results showed that typical amplification curves could be detected for EAEC, EPEC, ETEC, EIEC and EHEC, which were consistent with the situation in Table 4. Only the uidA gene amplification curve was detected for E. coli. The uidA and ipaH gene amplification curves were detected for Shigella sonnei and Shigella flexneri (e.g. Figure 2 ); the remaining strains (Salmonella, Listeria monocytogenes, Staphylococcus aureus, Campylobacter jejuni, Vibrio parahaemolyticus, Vibrio vulnificus and Vibrio cholerae) did not have DEC characteristic gene amplification curves that met the conditions shown in Table 4. The above results show that, except for Shigella sonnei and Shigella flexneri, the detection reagent of the present invention has no cross-reaction with other common intestinal pathogens, indicating that the primer probe set used in the present invention has good specificity; the EIEC phenotype is closely related to Shigella sonnei and Shigella flexneri, and therefore is not easy to distinguish by qPCR alone, but there are differences in their colony growth on SS plates and lactose fermentation test results. Therefore, when the initial screening and re-examination results show EIEC, it can be determined whether the sample to be tested contains EIEC or Shigella sonnei or Shigella flexneri by distinguishing the colony growth on SS plates and the lactose fermentation test (when no colorless translucent colonies appear on the SS plate and the lactose fermentation test is positive, it can be preliminarily determined to be EIEC).

[0077] Example 4: Repeatability and accuracy test 4.1 Experimental strains The following six experimental strains were used: EAEC (CICC21530), EPEC (CICC24189), ETEC (CICC10667), ETEC (laboratory strain, stp gene positive), EIEC (CICC10662), and EHEC (CICC24187).

[0078] 4.2 Primer-probe set detection repeatability test method Six experimental strains were inoculated into MAC medium and cultured at 36°C for 24 h. The strains were picked and transferred to TSB broth. The turbidity was adjusted to 0.5 McFarland standard (about 1×10 8 ~2×10 8 After 1 mL of broth was extracted, DNA was extracted by boiling method, and 10-fold gradient dilution was performed with nucleic acid diluent. -1 ~10 -7 Serial dilutions were performed using primers and probes corresponding to the target genes in Table 3 (the probes for astA, stx2, stp, and sth genes were astA-P, stx2-P, stp-P, and sth-P in Table 2, respectively). qPCR was performed to detect the presence of the 12 target genes shown in Table 3. During the qPCR detection process, the qPCR reaction system and procedure were as follows: (1) Reaction system: Add 1.5 μL of primer-probe mixture (mixed with 0.5 μL of upstream primer, downstream primer and probe of the target gene to be detected, with the content of upstream and downstream primers both at 10 μmol / L and the content of probe at 5 μmol / L) to each tube, and nucleic acid amplification reaction solution Premix EX Taq TM RR390A 12.5 μL, nuclease-free water 6 μL and plasmid dilution 5.0 μL were prepared to make a total reaction system of 25 μL.

[0079] (2) Reaction procedure: ① 95°C for 20 s; ② 95°C for 1 s; ③ 60°C for 20 s to collect fluorescence; ② and ③ steps were cycled 40 times. The PCR instrument was set to FAST mode.

[0080] Three parallel groups were set up for each dilution gradient, and the coefficient of variation (CV) of the Ct value at each dilution was calculated.

[0081] 4.3 Results of primer-probe set detection repeatability test The qPCR test results at each dilution showed that EAEC (CICC21530) was positive for astA, aggR, pic, and uidA; EPEC (CICC24189) was positive for bfpB, escV, and uidA; and ETEC (CICC10667) was positive for lt, sth, and uidA. The amplification curves of the lt gene at each dilution were shown in Figure 2. Figure 1; ETEC (laboratory strain) was stp and uidA positive; EIEC (CICC10662) was ipaH and uidA positive; EHEC (CICC24187) was stx1, stx2, escV and uidA positive.

[0082] The Ct values ​​and CV values ​​measured at various dilutions are shown in Tables 5 and 6.

[0083] Table 5 10 -1 ~10 -3 Repeatability test results under dilution gradient

[0084] Table 6 10 -4 ~10 -6 Repeatability test results under dilution gradient

[0085] The dilution reaches 10 -6 After 10 ℃, the characteristic genes could not be stably detected, so the dilution of 10 is not listed in Tables 5 and 6. -7 data; for the convenience of calculating the dilution 10 -6 The CV value at that time was assigned a CT value of 40.0 for a negative result.

[0086] It can be seen from Table 5 and Table 6 that: -1 ~10 -5 When the 12 characteristic genes of DEC were detected in parallel, the CT value was less than 35, and the CV value was less than 1.0%, indicating that the primer probe set used in the present invention can achieve good repeatability and detection accuracy.

[0087] 4.4 Test method for accuracy of primary screening system Six experimental strains were inoculated into MAC medium and cultured at 36°C for 24 h. The strains were picked and transferred to TSB broth. The turbidity was adjusted to 0.5 McFarland standard (about 1×10 8 ~2×10 8 After 1 mL of broth was extracted, DNA was extracted by boiling method, and 10-fold gradient dilution was performed with nucleic acid diluent. -1 ~10 -7 The gradient dilutions were subjected to qPCR detection according to the method in Example 1. Three parallel groups were set up for each dilution gradient, and the coefficient of variation (CV) of the Ct value at each dilution was calculated.

[0088] 4.5 Results of the accuracy test of the primary screening system The qPCR test results at each dilution showed that: when detecting EAEC (CICC21530), the VIC and ROX fluorescence channels in tube B showed positive, and the other fluorescence channels in tubes A and B showed negative; when detecting EPEC (CICC24189), the ROX fluorescence channel in tube A showed positive, and the other fluorescence channels in tubes A and B showed negative; when detecting ETEC (CICC10667) and ETEC (laboratory strain), the FAM fluorescence channel in tube A showed positive, and the other fluorescence channels in tubes A and B showed negative; when detecting EIEC (CICC10662), the VIC fluorescence channel in tube A showed positive, and the other fluorescence channels in tubes A and B showed negative; when detecting EHEC (CICC24187), the ROX fluorescence channel in tube A and the FAM fluorescence channel in tube B showed positive, and the other fluorescence channels in tubes A and B showed negative. This shows that the primary screening method in Example 1 has higher accuracy.

[0089] 4.6 Review system accuracy test method Six experimental strains were inoculated into MAC medium and cultured at 36°C for 24 h. The strains were picked and transferred to TSB broth. The turbidity was adjusted to 0.5 McFarland standard (about 1×10 8 ~2×10 8 After 1 mL of broth was extracted, DNA was extracted by boiling method, and 10-fold gradient dilution was performed with nucleic acid diluent. -1 ~10 -7 The gradient dilutions were subjected to qPCR detection according to the method in Example 2. Three parallel groups were set up for each dilution gradient, and the coefficient of variation (CV) of the Ct value at each dilution was calculated.

[0090] 4.7 Results of the qPCR test for accuracy of the recheck system: EAEC (CICC21530) was positive for astA, aggR, pic, and uidA; EPEC (CICC24189) was positive for bfpB, escV, and uidA; ETEC (CICC10667) was positive for lt, sth, and uidA; ETEC (laboratory strain) was positive for stp and uidA; EIEC (CICC10662) was positive for ipaH and uidA; and EHEC (CICC24187) was positive for stx1, stx2, escV, and uidA. This indicates that the recheck method in Example 2 has high accuracy.

[0091] Example 5: Sensitivity test 5.1 Sensitivity test method Shanghai Jierui Bioengineering Co., Ltd. was commissioned to construct plasmids containing 12 characteristic genes, namely lt, stp, sth, ipaH, escV, stx1, stx2, aggR, astA, pic, uidA, and bfpB. The plasmids were prepared with nucleic acid diluent to a copy concentration of 5 to 5 × 10 7 A series of plasmid dilutions with a concentration of 100 copies / μL were prepared and qPCR detection was performed using the primers and probes corresponding to the target genes in Table 3 (the probes for the astA, stx2, stp, and sth genes were astA-P, stx2-P, stp-P, and sth-P in Table 2, respectively). During the qPCR detection process, the qPCR reaction system and procedure were as follows: (1) Reaction system: Add 1.5 μL of primer-probe mixture (mixed with 0.5 μL of upstream primer, downstream primer and probe of the target gene to be detected, with the content of upstream and downstream primers both at 10 μmol / L and the content of probe at 5 μmol / L) to each tube, and nucleic acid amplification reaction solution Premix EX Taq TM RR390A 12.5 μL, nuclease-free water 6 μL and plasmid dilution 5.0 μL were prepared to make a total reaction system of 25 μL.

[0092] (2) Reaction procedure: ① 95°C for 20 s; ② 95°C for 1 s; ③ 60°C for 20 s to collect fluorescence; ② and ③ steps were cycled 40 times. The PCR instrument was set to FAST mode.

[0093] Three parallel groups were set for each copy concentration, and the coefficient of variation (CV) of the Ct value of each copy concentration was calculated. A standard curve was drawn based on the corresponding relationship between the copy concentration and the Ct value, and the correlation coefficient r was calculated. 2 Values ​​and effective amplification Eff (%) values.

[0094] 5.2 Sensitivity test results The results of the sensitivity test are shown in Table 7; the amplification curve and standard curve of the lt gene are shown in Figure 3 and Figure 4 The results showed that in the detection of each characteristic gene, 5 to 5×10 7 The linearity was good in the range of copies / μL, and the correlation coefficient r 2 The values ​​were 0.994-0.998, the effective amplification Eff(%) values ​​were 100.13-108.96, the CV values ​​at each copy concentration were all <1.0%, and the minimum detection limit of each characteristic gene was 5 copies / μL.

[0095] Table 7 Sensitivity test results characteristic genes Standard curve <![CDATA[r 2 Value]]> Eff value (%) aggR -3.319X+38.19 0.997 100.13 astA -3.272X+36.64 0.994 102.13 pic -3.271X+35.43 0.996 102.17 escV -3.286X+37.24 0.995 101.50 BfP -3.289X+37.57 0.995 101.39 stx1 -3.299X+36.67 0.997 100.97 stx2 -3.305X+36.98 0.997 100.69 lt -3.29X+37.02 0.998 101.35 sth -3.274X+35.93 0.996 102.05 stp -3.282X+36.23 0.997 101.68 ipaH -3.187X+35.77 0.997 105.95 uidA -3.124X+34.59 0.994 108.96

[0096] Unless otherwise specified, the raw materials and equipment used in the present invention are commonly used in the art; the methods used in the present invention are conventional methods in the art unless otherwise specified.

[0097] The above description is only a preferred embodiment of the present invention and does not limit the present invention in any way. Any simple modification, change and equivalent transformation made to the above embodiment based on the technical essence of the present invention still fall within the scope of protection of the technical solution of the present invention.

Claims

1. A kit for typing and detecting diarrhea-causing Escherichia coli, characterized in that: Includes primer probe for tube A and primer probe for tube B, which respectively include the following primers and probes: In the table above, the fluorescent signals of fluorescent groups a, b, and c are different.

2. The kit according to claim 1, wherein Also includes:

3. The kit according to claim 2, wherein The two ends of the probe uidA are respectively labeled with a fluorescent group d and a quenching group, and the fluorescent group d is different from the fluorescent groups a, b and c; the two ends of the probe bfpB-P are respectively labeled with a fluorescent group b and a quenching group; the kit also include: 。 4. The kit according to claim 3, wherein The fluorescent groups a, b, c and d are independently selected from FAM, VIC, ROX and CY5.

5. The kit according to claim 1 or 3, characterized in that The quenching group is BHQ1.

6. The kit according to claim 1, wherein Also included is a nucleic acid amplification reaction solution.

7. Use of the kit according to any one of claims 1 to 6 for non-diagnostic detection of five types of diarrheagenic Escherichia coli, characterized in that: The following steps are involved: S1: Extract nucleic acid from the sample to be tested to obtain a nucleic acid sample; S2: Mix all the primers and probes in tube A and perform qPCR on the nucleic acid sample; S3: Mix all B tubes with primers and probes and perform qPCR on the nucleic acid sample; S4: Interpret the results of S1 and S2 according to the following table:

8. Use of the kit according to claim 3 or 4 for non-diagnostic detection of five types of diarrheagenic Escherichia coli, characterized in that: The following steps are involved: S1: Extract nucleic acid from the sample to be tested to obtain a nucleic acid sample; S2: Mix all the primers and probes in tube A and perform qPCR on the nucleic acid sample; S3: Mix all B tubes with primers and probes and perform qPCR on the nucleic acid sample; S4: Interpret the results of S1 and S2 according to the following table: S5: Review the diarrhea-causing Escherichia coli identified in S4.

9. The use according to claim 8, characterized in that The specific process of S5 includes: isolating and culturing the diarrheagenic Escherichia coli in the sample to be tested, extracting nucleic acid to obtain a target bacterial nucleic acid sample; performing qPCR on the target bacterial nucleic acid sample according to the following method to confirm the type of diarrheagenic Escherichia coli contained in the sample to be tested:

10. The use according to claim 9, characterized in that In S5, when the qPCR results showed that both the ipaH and uidA genes were positive, Shigella sonnei and Shigella flexneri were excluded based on the colony growth on the SS plate and the lactose fermentation test, and then it was confirmed as enteroinvasive Escherichia coli.

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