Primer combination and kit for detecting serotypes of Escherichia coli O8, O9 and O89

Abstract

The present invention provides a primer composition and a kit for detecting serotypes of Escherichia coli O8, O9 and O89, and belongs to the field of biotechnology and molecular biology. The primer composition for detecting serotypes of Escherichia coli O8, O9 and O89 comprises a primer pair A, a primer pair B and a primer pair C, wherein the primer pair A comprises primer 1 and primer 2, the primer pair B comprises primer 3 and primer 4, the primer pair C comprises primer 5 and primer 6, and the nucleotide sequences of the primers 1-6 are shown in SEQ ID NO: 1-6 in sequence. The kit of the present invention is simple to operate, has strong specificity and high sensitivity, and can simultaneously detect the primer composition and the kit of serotypes of Escherichia coli O8, O9 and O89.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology and molecular biology, and particularly relates to a primer composition and a kit for detecting serotypes of Escherichia coli O8, O9 and O89. Background Art

[0002] Pathogenic Escherichia coli is one of the common bacteria in the aquaculture industry, which not only has a serious impact on the aquaculture industry, but also poses a certain threat to public health and safety. Diarrhea caused by enteric pathogenic Escherichia coli and pneumonia caused by extraintestinal pathogenic Escherichia coli seriously affect the survival rate and breeding efficiency of animals. Coupled with the increasing prevalence and severity of bacterial resistance, bacterial diseases represented by Escherichia coli cause great economic losses to the aquaculture industry every year. Compared with the pig and poultry industries, the research and corresponding detection technology of sheep-derived Escherichia coli are obviously lagging behind.

[0003] Escherichia coli antigens are complex, mainly including bacterial antigens (O), capsule antigens (K), flagellar antigens (H) and pili antigens (F), among which O antigen is the most commonly used typing antigen and is related to the pathogenicity of Escherichia coli. The classic method for identifying Escherichia coli serotypes is to use agglutination test, but this method requires standard serum of each serotype strain, and the results are greatly affected by serum quality and subjective judgment, prone to false negatives and false positives, low accuracy, and not conducive to clinical diagnosis. In contrast, the PCR method has higher accuracy and specificity, and is more suitable for the identification of strain serotypes. Studies have shown that there are certain differences in the dominant serotypes of Escherichia coli infected in different animals, and the existing methods are not suitable for the identification of all animal-derived Escherichia coli. Through the study of sheep-derived Escherichia coli collected from farms and slaughterhouses, it was found that O8, O9, and O89 serotypes of Escherichia coli are popular dominant serotypes. However, the prior art lacks primer compositions and kits that can detect O8, O9, and O89 with high sensitivity and high specificity. Summary of the invention

[0004] The purpose of the present invention is to provide a primer combination and a kit which are simple to operate, have high specificity and high sensitivity, and can simultaneously detect serotypes O8, O9 and O89 of Escherichia coli.

[0005] The purpose of the present invention is achieved by the following technical solutions:

[0006] A primer composition for detecting serotypes of Escherichia coli O8, O9 and O89, comprising primer pair A, primer pair B and primer pair C, wherein the primer pair A comprises primer 1 and primer 2, the primer pair B comprises primer 3 and primer 4, the primer pair C comprises primer 5 and primer 6, and the nucleotide sequences of the primers 1-6 are shown in SEQ ID NOs: 1-6, respectively.

[0007] In the present invention, the concentration ratio of Primer 1, Primer 2, Primer 3, Primer 4, Primer 5 and Primer 6 is 2:2:5:5:5:5.

[0008] The present invention also provides a kit for detecting serotypes of Escherichia coli O8, O9 and O89, comprising primer 1, primer 2, primer 3, primer 4, primer 5 and primer 6, wherein the nucleotide sequences of primers 1-6 are shown in SEQ ID NOs: 1-6, respectively.

[0009] In the present invention, the kit further comprises 2×Rapid Taq Master Mix and O8, O9, O89 positive standards, wherein the 2×Rapid Taq Master Mix can be replaced by other PCR buffers having the same function.

[0010] In the present invention, the concentration of primer 1 and primer 2 is 4 μmol / L, and the concentration of primers 3-6 is 10 μmol / L.

[0011] The present invention also provides a method for detecting serotypes of Escherichia coli O8, O9, and O89 using the kit for non-diagnostic purposes, comprising the following steps:

[0012] (1) extracting nucleic acid from the sample to be tested;

[0013] (2) using the nucleic acid of the sample to be tested as a template and primers 1-6 to perform a triple PCR reaction;

[0014] (3) The amplified product is subjected to electrophoresis. If a 210 bp band appears in the amplified product, O8 serotype Escherichia coli is present in the sample to be tested; if a 411 bp band appears in the amplified product, O9 serotype Escherichia coli is present in the sample to be tested; if a 126 bp band appears in the amplified product, O89 serotype Escherichia coli is present in the sample to be tested.

[0015] In the present invention, the triple PCR reaction system is as follows: 10 μL of 2×Rapid Taq Master Mix, 1 μL of primers 1, 2, 3, 4, 5 and 6, 2 μL of nucleic acid of the sample to be tested, and deionized water to make up the volume to 20 μL.

[0016] In the present invention, the triple PCR reaction procedure is as follows: pre-denaturation at 94°C for 5 min; 94°C for 30 s, 54°C for 30 s, and 72°C for 20 s, for a total of 35 cycles; and finally extension at 72°C for 5 min.

[0017] Beneficial Effects

[0018] The primer combination provided by the present invention is used to simultaneously detect serotypes of Escherichia coli O8, O9, and O89, with strong specificity, high sensitivity, and simple operation, which significantly improves the detection efficiency, avoids the detection of only one serotype of Escherichia coli at a time, avoids pollution, is convenient and fast, and is convenient for the rapid identification of pathogenic Escherichia coli serotypes in mutton breeding and mutton production, and has broad application prospects. The kit of the present invention is used to detect other common serotypes of Escherichia coli (O15, O18, O22, O38, O86, O141, O157) and other common sheep bacteria (Shigella, Mannheimia hemolytica, Proteus mirabilis, Klebsiella pneumoniae, Pasteurella), and no reaction occurs; sensitivity tests show that the minimum nucleic acid detection amounts of the primer combination and the kit of the present invention for serotypes of Escherichia coli O8, O9, and O89 are 1pg / μL, 10pg / uL, and 1pg / uL, respectively. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 : Triple PCR amplification results of O8, O9 and O89, M: Trans2K plus DNA Marker; 1: PCR amplification product using nucleic acid of serotype O8 Escherichia coli as template; 2: PCR amplification product using nucleic acid of serotype O9 Escherichia coli as template; 3: PCR amplification product using nucleic acid of serotype O89 Escherichia coli as template; 4: Triple PCR amplification product using nucleic acid of three kinds of Escherichia coli mixture as template.

[0020] Figure 2 : Triple PCR amplification results using mixed bacterial nucleic acid as template, primer pairs A, B and different O89 primer pair combinations, 1: Triple PCR amplification products of primer pairs A, B, C; 2: Triple PCR amplification products of primer pairs A, B, J; 3: Triple PCR amplification products of primer pairs A, B, K; 4: Triple PCR amplification products of primer pairs A, B, L; 5: negative control; M: Trans2K plusDNA Marker.

[0021] Figure 3 : Optimization results of triple PCR primer concentrations of O8, O9, and O89, wherein lanes 1-5 are the triple PCR products when the concentrations of O8 primers are 4μmol / L, 6μmol / L, 8μmol / L, 10μmol / L, and 12μmol / L, respectively; 6: negative control; M: Trans2K plus DNA Marker.

[0022] Figure 4: Specific experimental results of the kit of the present invention, wherein M: Trans2K plus DNA Marker; 1: positive control; 2: negative control; 3-14: O15, O18, O22, O38, O86, O141, O157 serotypes of Escherichia coli, Shigella, Mannheimia haemolytica, Proteus mirabilis, Klebsiella pneumoniae, Pasteurella mitis, in sequence.

[0023] Figure 5 : Sensitivity test results of the kit of the present invention, M: Trans2K plus DNA Marker; 1-5: nucleic acid template concentrations are 1 ng / μL, 100 pg / μL, 10 pg / μL, 1 pg / μL, 0.1 pg / μL, respectively; 6: negative control. DETAILED DESCRIPTION

[0024] Example 1 Primer design and triple PCR test

[0025] 1. Primer Design and Synthesis

[0026] Through the analysis and comparison of the gene sequences of Escherichia coli serotypes O8, O9 and O89, a variety of target genes were selected from the genes of the three serotypes of Escherichia coli to design primers. The nucleic acids of Escherichia coli serotypes O8, O9 and O89 were used as templates, and the primer combinations that can accurately identify the three serotypes of Escherichia coli were screened by PCR test. First, using Primer Premier 5 software and MPprimer software, dozens of pairs of primers were designed for the gene sequences of Escherichia coli serotypes O8, O9 and O89, and the feasibility of different primer combinations was analyzed, in order to screen the best primer combination that can detect the three serotypes simultaneously. When designing multiple primers, in addition to considering the Tm value, GC content and whether the primers are specific for a single pair of primers, it is also ensured that these primer pairs have similar Tm values, and the size of each amplification product is limited to ensure that different amplification products can be clearly distinguished by the naked eye; then the specificity of each primer pair is analyzed to ensure that there is no non-specific amplification of the primer pair. Through a lot of creative work, we finally obtained primer pair A for O8 serotype, primer pair B for O9 serotype and primer pair C for O89 serotype, which can be used to simultaneously detect O8, O9 and O89 serotypes of Escherichia coli.

[0027] Primer pair A was designed for the Escherichia coli O8 gene sequence (GenBank accession number: CP076704), primer pair B was designed for the Escherichia coli O9 gene (GenBank accession number: CP019271), and primer pair C was designed for the Escherichia coli O89 gene (GenBank accession number: KJ755555).

[0028] Primer pair A for Escherichia coli serotype O8, including primer 1 and primer 2:

[0029] The nucleotide sequence of primer 1 (SEQ ID NO: 1): 5'-CAGGCACGCTATCAAACTAG-3',

[0030] Nucleotide sequence of primer 2 (SEQ ID NO: 2): 5'-TACGCTCTGACCTTTATCCAG-3'.

[0031] Primer pair B for E. coli serotype O9, including primer 3 and primer 4:

[0032] The nucleotide sequence of primer 3 (SEQ ID NO: 3): 5'-ACTTTCAGTCCCGTTACCAG-3',

[0033] The nucleotide sequence of primer 4 (SEQ ID NO: 4): 5'-TGAACAGCATCTGGACGATC-3'.

[0034] Primer pair C for E. coli serotype O89, including primers 5 and 6:

[0035] The nucleotide sequence of primer 5 (SEQ ID NO: 5): 5'-AGAAAGCAGCACAGTTACGG-3',

[0036] The nucleotide sequence of primer 6 (SEQ ID NO: 6): 5'-CCAGCCAAGAGTTTCAGTAGCG-3'.

[0037] 2. Triple PCR Assay

[0038] The Axygen DNA / RNA extraction kit was used to extract nucleic acids from the culture fluids of serotype O8 Escherichia coli, serotype O9 Escherichia coli, and serotype O89 Escherichia coli, respectively. The culture fluids of serotype O8 Escherichia coli, serotype O9 Escherichia coli, and serotype O89 Escherichia coli were mixed in equal volumes, and nucleic acids were extracted using the Axygen DNA / RNA extraction kit to obtain nucleic acids from the mixture of the three Escherichia coli.

[0039] PCR amplification was performed using the nucleic acid of E. coli serotype O8, E. coli serotype O9, and E. coli serotype O89 as templates, respectively, and primer pairs for each serotype of E. coli were used. The reaction system was as follows: 10 μL of 2× Rapid Taq Master Mix, 1 μL of upstream primer with a concentration of 10 μmol / L, 1 μL of downstream primer with a concentration of 10 μmol / L, and 2 μL of extracted nucleic acid were added to a 0.2 ml PCR reaction tube, and the total volume was made up to 20 μL with deionized water, and mixed.

[0040] The nucleic acid of the three E. coli mixtures was used as templates, and PCR amplification was performed using primer pairs A, B, and C. The reaction system was as follows: 10 μL of 2× Rapid Taq Master Mix, 1 μL of primer 1 with a concentration of 4 μmol / L, 1 μL of primer 2 with a concentration of 4 μmol / L, 1 μL of primer 3 with a concentration of 10 μmol / L, 1 μL of primer 4 with a concentration of 10 μmol / L, 1 μL of primer 5 with a concentration of 10 μmol / L, 1 μL of primer 6 with a concentration of 10 μmol / L, and 2 μL of the nucleic acid of the three E. coli mixtures were added to a 0.2 ml PCR reaction tube, and the total volume was made up to 20 μL with deionized water, and mixed.

[0041] The conditions of each PCR reaction were as follows: pre-denaturation at 94°C for 5 min; 35 cycles of 94°C for 30 s, 54°C for 30 s, and 72°C for 20 s; and finally extension at 72°C for 5 min.

[0042] The PCR products were electrophoresed on 1.5% agarose gel.

[0043] from Figure 1 It can be seen that using the nucleic acids of O8, O9 and O89 serotypes of Escherichia coli as templates, bands of 210bp, 411bp and 126bp in size can be amplified respectively; using the nucleic acids of the three Escherichia coli mixture as a template, bands of 126bp, 210bp and 411bp in size can be amplified simultaneously, and the test results are all positive, proving that the primer combination can simultaneously detect O8, O9 and O89 serotypes of Escherichia coli.

[0044] During the screening process, it was found that many primer pairs can detect O8, O9 and O89 serotypes of E. coli separately, but cannot detect O8, O9 and O89 serotypes of E. coli simultaneously in the same system through triple PCR. Some examples of primers are as follows: Primer pairs A, D, E, and F for O8 serotype E. coli can all specifically amplify gene fragments of O8 serotype E. coli; Primer pairs B, G, H, and I for O9 serotype E. coli can all specifically amplify gene fragments of O9 serotype E. coli; Primer pairs C, J, K, and L for O89 serotype E. coli can all specifically amplify gene fragments of O89 serotype E. coli. However, except for primer pairs A, B, and C, which can detect three serotypes of E. coli simultaneously, combinations of other primer pairs (AL) cannot effectively detect O8, O9, and O89 serotypes of E. coli simultaneously in the same system through triple PCR. For example, using three kinds of E. coli mixed bacterial nucleic acid as template, under the premise of using primer pair A and B, combined with multiple sets of O89 primers (JL) for PCR amplification, it is impossible to obtain effective amplification of O89 ( Figure 2 ).

[0045] Primer pair D (targeting O8, product size 428 bp):

[0046] Upstream primer: 5'-CTGAGATGCTGGATAAAGG-3',

[0047] Downstream primer: 5′-ATAGGCGAAGATGGACTG-3′.

[0048] Primer pair E (targeting O8, product size 180 bp):

[0049] Upstream primer: 5'-GAGGTGATGAAGGCAAGA-3',

[0050] Downstream primer: 5′-GATCGGCAGACAGATTTT-3′.

[0051] Primer pair F (for O8, product size 208 bp):

[0052] Upstream primer: 5'-CAGGCACGCTATCAAACTAG-3',

[0053] Downstream primer: 5′-CGCTCTGACCTTTATCCAGC-3′.

[0054] Primer pair G (for O9, product size 326 bp):

[0055] Upstream primer: 5'-GTACGTTACTGAAGCTGCTG-3',

[0056] Downstream primer: 5′-GAGCGCTTCATCGACGATCA-3′.

[0057] Primer pair H (for O9, product size 452 bp):

[0058] Upstream primer: 5'-CTACCGTGGCTTTATTATTG-3',

[0059] Downstream primer: 5′-CGAGGGTGAACAGCATCT-3′.

[0060] Primer pair 1 (for O9, product size 355 bp):

[0061] Upstream primer: 5'-TTTGCCTACAGTATCTACCTCT-3',

[0062] Downstream primer: 5′-GAAGCAGGATATTAACGAACT-3′.

[0063] Primer pair J (targeting O89, product size 126 bp):

[0064] Upstream primer: 5'-CTTGAAAGATGTGGGTGTT-3',

[0065] Downstream primer: 5′-TTAACCGTAACTGTGCTGC-3′.

[0066] Primer pair K (targeting O89, product size 407 bp):

[0067] Upstream primer: 5'-CTGTTGGACATCGCTCTAGG-3',

[0068] Downstream primer: 5′-ATCATCTTTGGTTTACCGCCC-3′.

[0069] Primer pair L (targeting O89, product size 533 bp):

[0070] Upstream primer: 5'-TTGAATTTCCCATTTACGACGC-3',

[0071] Downstream primer: 5′-GAAGGAAGCATCGCCAGTA-3′.

[0072] Example 2 Establishment of triple PCR detection method

[0073] 1. Preparation of Positive Standards

[0074] The nucleic acids of E. coli types O8, O9 and O89 were extracted respectively and the concentration was adjusted to 3 ng / μL. Then, equal volumes of nucleic acids of the three serotypes of E. coli were mixed to obtain a nucleic acid mixture with 1 ng / μL of nucleic acid of each serotype.

[0075] 2. Optimization of Primer Concentration

[0076] In the fixed PCR reaction system, the concentrations of primers 3, 4, 5, and 6 for O9 and O89 were 10 μmol / L, and the concentrations of primers 1 and 2 for O8 were equal and five concentration gradients (4 μmol / L, 6 μmol / L, 8 μmol / L, 10 μmol / L, and 12 μmol / L) were set to perform triple PCR reactions. The reaction system was as follows: 10 μL of 2× Rapid Taq Master Mix, 1 μL of primer 1, 1 μL of primer 2, 1 μL of primer 3 with a concentration of 10 μmol / L, 1 μL of primer 4 with a concentration of 10 μmol / L, 1 μL of primer 5 with a concentration of 10 μmol / L, 1 μL of primer 6 with a concentration of 10 μmol / L, and 2 μL of E. coli nucleic acid mixture of O8, O9, and O89 (each serotype nucleic acid was 1 ng / μL), and the total volume was filled to 20 μL with deionized water and mixed. The reaction conditions were: 94℃ pre-denaturation for 5 min; 94℃ for 30 s, 54℃ for 30 s, 72℃ for 20 s, for a total of 35 cycles; and finally 72℃ extension for 5 min. The PCR products were electrophoresed on a 1.5% agarose gel. Figure 3 It can be seen that when the concentrations of primers 1 and 2 were both 4 μmol / L, the three bands O8, O9, and O89 were clear and uniform in brightness, which was the optimal reaction condition.

[0077] 3. Optimization of annealing temperature

[0078] According to the optimal reaction conditions determined in Title 2 of this Example, only the annealing temperature during PCR amplification was set to 58°C, 56°C, 54°C, 52°C, and 50°C, respectively, to investigate the effect of annealing temperature. The results showed that there was no significant difference between the temperatures of 50-56°C, and the optimal annealing temperature was finally determined to be 54°C.

[0079] Example 3 Kit assembly and sample testing

[0080] 1. Kit Assembly

[0081] The triple PCR detection kit for detecting serotypes of Escherichia coli O8, O9 and O89 (abbreviated as the kit of the present invention) includes the following reagents:

[0082] (1) Primers

[0083] The primers include primer 1, primer 2, primer 3, primer 4, primer 5 and primer 6 (the sequences are the same as those in Example 1), the concentration of primer 1 is 4 μmol / L, the concentration of primer 2 is 4 μmol / L, and the concentrations of primer 3, primer 4, primer 5 and primer 6 are all 10 μmol / L.

[0084] (2) 2× Rapid Taq Master Mix

[0085] 2×Rapid Taq Master Mix was purchased from Nanjing Novozyme Biotechnology Co., Ltd.

[0086] (3) Positive standard

[0087] E. coli serotypes O8, O9 and O89 were inoculated into LB liquid culture medium and cultured overnight at 37°C for 16 hours. 200 μL of bacterial solution was taken from each strain and the nucleic acid of the sample to be tested was extracted according to the instructions of the Axygen DNA / RNA extraction kit. The nucleic acids of each serotype of E. coli were mixed to prepare a positive standard. The nucleic acid concentration of each serotype of E. coli in the positive standard was 10 ng / μL.

[0088] 2. Specific method for detecting samples using the kit of the present invention

[0089] Pick a single colony of Escherichia coli after separation and purification, extract nucleic acid using Axygen DNA / RNA extraction kit, and use the kit of the present invention to perform triple PCR reaction. The reaction system is: add 10μL of 2×RapidTaq Master Mix, 1μL of primers 1, 2, 3, 4, 5 and 6, 2μL of extracted nucleic acid of the sample to be tested in a 0.2ml PCR reaction tube, add deionized water to the total volume to 20μL, and mix well. The reaction conditions are: 94℃ pre-denaturation for 5min; 94℃30s, 54℃30s, 72℃20s, a total of 35 cycles; finally 72℃ extension for 5min. In the positive control, the nucleic acid of the sample to be tested is replaced by the positive standard, and the other components, volume and reaction procedures in the reaction system remain unchanged. In the negative control, the nucleic acid of the sample to be tested is replaced by deionized water, and the other components, volume and reaction procedures in the reaction system remain unchanged. Take the PCR product for electrophoresis on 1.5% agarose gel.

[0090] Result determination: When three bands of 126bp, 210bp and 411bp appear in the electrophoresis diagram of the positive control, and there is no amplified band in the negative control, it means that the experimental results are reliable. If a band of 210bp appears in the nucleic acid amplification product of the sample to be tested, the test result is positive for O8 serotype, and O8 serotype Escherichia coli exists in the sample to be tested; if a band of 411bp appears in the amplification product, the test result is positive for O9 serotype, that is, O9 serotype Escherichia coli exists in the sample to be tested; if a band of 126bp appears in the amplification product, the sample to be tested is positive for O89 serotype, that is, O89 serotype Escherichia coli exists in the sample to be tested; if there is no amplification product, the test result is negative, that is, O8, O9 and O89 Escherichia coli do not exist in the sample to be tested.

[0091] Example 4 Specificity

[0092] Detect the specificity of the kit of the present invention. The kit of the present invention was used to detect Escherichia coli serotypes O15, O18, O22, O38, O86, O141, and O157 and other common bacterial pathogens of sheep (Shigella, Mannheimia haemolytica, Proteus mirabilis, Klebsiella pneumoniae, and Pasteurella mitis). In the positive control, the positive standard was used to replace the nucleic acid of the sample to be tested, and in the negative control, the deionized water was used to replace the nucleic acid of the sample to be tested. The results are shown in FIG. Figure 4 The kit of the present invention can amplify three bands of 210 bp, 411 bp and 126 bp for the nucleic acid of the mixture of O8, O9 and O89 types of Escherichia coli, and no bands are amplified for other pathogens, indicating that the kit of the present invention has good specificity for O8, O9 and O89 types of Escherichia coli.

[0093] Example 5 Sensitivity

[0094] Extract the nucleic acids of E. coli types O8, O9 and O89 respectively, and adjust the concentration to 3ng / μL, then mix the nucleic acids of the three serotypes of E. coli in equal volumes to obtain a nucleic acid mixture in which the nucleic acid of each serotype is 1ng / μL. Dilute the above 1ng / μL nucleic acid mixture 10 times in a gradient manner until the nucleic acid of each serotype of E. coli is 0.1pg / μL, and then use the kit of the present invention to detect them respectively to examine the sensitivity of the kit of the present invention to E. coli types O8, O9 and O89. Take 2μL of each dilution as a template for detection, observe the positive bands, and calculate the sensitivity based on the highest dilution of the template amount used to produce the positive bands. The results are as follows: Figure 5 It can be seen that the minimum detection amount of the kit of the present invention for O8 and O89 is 1 pg / μL, and the minimum detection amount for O9 is 10 pg / μL.

[0095] Example 5 Stability test of the kit

[0096] The same batch of the kit of the present invention was used to detect Escherichia coli serotypes O8, O9 and O89, and the results were repeated three times. The PCR products were electrophoresed on a 1.5% agarose gel, and the corresponding target bands were amplified, and the brightness of the target bands was consistent. The test results of this example show that the kit of the present invention has good stability.

[0097] Example 6: Using the kit of the present invention to detect clinical samples

[0098] The kit of the present invention was used to detect 114 strains of Escherichia coli collected from 2015 to 2021. In addition, a single PCR was used with primer pair A as a primer to detect whether the sample contained O8 type Escherichia coli (the primers in the reaction system only contained primer pair A, and other components and reaction procedures were the same as the kit of the present invention); primer pair B was used as a primer, and PCR was used to detect whether the sample contained O9 type Escherichia coli (the primers in the reaction system only contained primer pair B, and other components and reaction procedures were the same as the kit of the present invention); primer pair C was used as a primer, and PCR was used to detect whether the sample contained O89 type Escherichia coli (the primers in the reaction system only contained primer pair C, and other components and reaction procedures were the same as the kit of the present invention).

[0099] The test results of the kit of the present invention showed that 21 of the 114 strains of E. coli were serotype O8, 24 were serotype O9, and 10 were serotype O89. The test results of the kit of the present invention were consistent with the single O8, O9 and O89 serotype PCR test results by 97.2%, 95.3% and 96.4%, respectively.

[0100] Nucleic acid was extracted from several clinical samples and tested using the kit of the present invention. After separation by agarose gel electrophoresis, the PCR product was positive. DNA fragments were recovered according to the instructions of the microgel recovery kit (Axygen Company), sequenced and analyzed by BLAST comparison on the NCBI website, confirming that they were O8, O9 and O89 gene sequences. This shows that the triple PCR detection method established by the present invention can be used for the rapid and simultaneous detection of O8, O9 and O89 serotypes of Escherichia coli in clinical samples. The experimental results of this example show that the detection accuracy of the kit of the present invention is high. SEQUENCE LISTING <110> Jiangsu Academy of Agricultural Sciences Jiangsu University Dongying Animal Husbandry and Veterinary Station <120> Primer combination and kit for detecting serotypes of Escherichia coli O8, O9 and O89 <130> 20220322 <160> 6 <170> PatentIn version 3.3 <210> 1 <211> 20 <212> DNA <213> artificial <220> <223> Primer 1 <400> 1 caggcacgct atcaaactag 20 <210> 2 <211> twenty one <212> DNA <213> artificial <220> <223> Primer 2 <400> 2 tacgctctga cctttatcca g 21 <210> 3 <211> 20 <212> DNA <213> artificial <220> <223> Primer 3 <400> 3 actttcagtc ccgttaccag 20 <210> 4 <211> 20 <212> DNA <213> artificial <220> <223> Primer 4 <400> 4 tgaacagcat ctggacgatc 20 <210> 5 <211> 20 <212> DNA <213> artificial <220> <223> Primer 5 <400> 5 agaaagcagc acagttacgg 20 <210> 6 <211> twenty two <212> DNA <213> artificial <220> <223> Primer 6 <400> 6 ccagccaaga gtttcagtag cg 22

Claims

1. A triple PCR primer composition for detecting serotypes of Escherichia coli O8, O9, and O89, characterized in that: The primer composition includes primer pair A, primer pair B and primer pair C, wherein primer pair A includes primer 1 and primer 2, primer pair B includes primer 3 and primer 4, and primer pair C includes primer 5 and primer 6. The nucleotide sequences of primers 1-6 are shown in SEQ ID NO: 1-6, respectively.

2. The triple PCR primer composition for detecting serotypes O8, O9, and O89 of Escherichia coli according to claim 1, characterized in that: The concentration ratio of primer 1, primer 2, primer 3, primer 4, primer 5 and primer 6 is 2:2:5:5:5:

5.

3. A kit for detecting O8, O9, and O89 serotypes of Escherichia coli, characterized in that it includes a triple PCR primer composition, wherein the triple PCR primer composition includes primer 1, primer 2, primer 3, primer 4, primer 5, and primer 6, and the nucleotide sequences of primers 1-6 are shown in SEQ ID NOs: 1-6, respectively.

4. The kit for detecting serotypes O8, O9 and O89 of Escherichia coli according to claim 3, characterized in that The kit also includes 2×Rapid Taq Master Mix and O8, O9, and O89 serotype positive standards.

5. The kit for detecting serotypes O8, O9 and O89 of Escherichia coli according to claim 4, characterized in that The concentrations of the primers 1 and 2 were both 4 μmol / L, and the concentrations of the primers 3-6 were all 10 μmol / L.

6. A method for detecting serotypes O8, O9, and O89 of Escherichia coli using the kit of claim 3, wherein the method is for non-diagnostic purposes and is characterized in that The steps include: (1) Extracting nucleic acid from the sample to be tested; (2) Using the nucleic acid of the sample to be tested as a template and primers 1-6, a triple PCR reaction was performed; (3) The amplified product is subjected to electrophoresis. If a 210 bp band appears in the amplified product, O8 serotype Escherichia coli is present in the sample to be tested; if a 411 bp band appears in the amplified product, O9 serotype Escherichia coli is present in the sample to be tested; if a 126 bp band appears in the amplified product, O89 serotype Escherichia coli is present in the sample to be tested.

7. The method according to claim 6, characterized in that The triple PCR reaction system was as follows: 10 μL of 2× Rapid TaqMaster Mix, 1 μL each of primers 1, 2, 3, 4, 5, and 6, 2 μL of nucleic acid of the sample to be tested, and deionized water was added to a volume of 20 μL.

8. The method according to claim 7, characterized in that The program of the triple PCR reaction was as follows: pre-denaturation at 94 °C for 5 min; 35 cycles of 94 °C for 30 s, 54 °C for 30 s, and 72 °C for 20 s; and finally extension at 72 °C for 5 min.

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