Method for detecting five pilus adhesion subtypes of enterotoxigenic escherichia coli in pig farm based on 5-plex mPCR
By constructing a 5-plex mPCR detection method and designing specific primer pairs to simultaneously detect five fimbrial adhesins subtypes of enterotoxin-producing Escherichia coli from pig farms, this method solves the problem of the difficulty in simultaneously detecting multiple fimbrial genotypes in existing technologies, and achieves efficient pathogen detection and vaccine selection.
Patent Information
- Application Number
- CN202511155277.6
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-08-18
- Publication Date
- 2025-11-21
AI Technical Summary
Current technology lacks a method to simultaneously detect five fimbriated adhesins subtypes of enterotoxin-producing Escherichia coli from pig farms, making disease prevention and control in piglets difficult.
A 5-plex mPCR detection method was constructed, and specific primer pairs were designed for the simultaneous detection of F5, F18, F6, F4, F41 and K99 fimbrial genes. The simultaneous detection of the five fimbrial genotypes was achieved through multiplex PCR reaction.
It enables simultaneous detection of five fimbrial genotypes in one go, supports the selection of multivalent vaccines, and improves the accuracy and efficiency of detection.
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Figure CN120989270A_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the field of biological detection technology, specifically to a method for detecting five fimbrial adhesins subtypes of enterotoxin-producing Escherichia coli from pig farms based on 5-plex mPCR. Background Technology
[0002] Enterotoxigenic Escherichia coli (ETEC) is an important pathogen that causes diarrhea in young animals (newborn piglets, calves, lambs, and weaned piglets). Newborn animals infected with ETEC often die from severe watery diarrhea and rapid dehydration, with both morbidity and mortality rates being very high.
[0003] ETEC produces virulence factors including adhesin, enterotoxin (Ent), edema disease principle (EDP), endotoxin, and hemolysin. Enterotoxin and adhesin play crucial roles from both pathogenesis and immunology perspectives. Enterotoxins are generally classified into heat-stable enterotoxin (ST) and heat-labile enterotoxin (LT). Both types can be produced individually or simultaneously, and genetically, their production is controlled by plasmids.
[0004] Adhesion is fundamental to microbial colonization and a prerequisite for bacterial pathogenicity. All bacterial structural components with adhesive properties are collectively called adhesins or colonization factors. These are typically macromolecular structural components on the bacterial surface, primarily pili of Gram-negative bacteria, and secondarily non-pilial adhesins, such as certain outer membrane proteins and lipoteichoic acid of Gram-positive bacteria. The proteinaceous pili of ETECs (Epidermal-Activated Tetracycline Adhesins) are fibrous and encapsulate the surface of bacterial antigens, protecting bacteria from complement killing and macrophage phagocytosis. These ETEC-specific pili differ from type I pili commonly found in *Escherichia coli*; their agglutination reaction on erythrocytes is not inhibited by D-mannose, exhibiting a mannose-resistant hemagglutination reaction. The main adhesin antigens include K88 (F4, K88ab, K88ac, K88ad), K99 (F5), 987p (F6), F41, and F42.
[0005] Post-weaning diarrhea in piglets is primarily caused by the pathogenic ETEC strain expressing F4 (K88) or F18 adhesin-containing fimbriae. Common fimbriae also include K99 (F5), 987p (F6), and F41. Rapid detection of these fimbriae types is helpful for disease prevention and control in piglets. While multiplex PCR methods exist for detecting fimbriae genes, methods that can simultaneously detect all five of these fimbriae genes are still lacking. Summary of the Invention
[0006] In view of this, the present invention provides a method for detecting five fimbrial adhesins subtypes of enterotoxigenic Escherichia coli in pig farms based on 5-plex mPCR, and constructs a stable mPCR system that can simultaneously detect five serological subtypes (F4, F18, F6, F41, K99) in one go, so as to realize the simultaneous detection of the above fimbrial genotypes.
[0007] To achieve the above objectives, the present invention provides a method for detecting five fimbrial adhesins subtypes of enterotoxin-producing Escherichia coli based on 5-plex mPCR, comprising a multiplex PCR primer set, wherein the multiplex PCR primer set includes primer pairs for detecting the F5 fimbrial gene of Escherichia coli, primer pairs for detecting the F18 fimbrial gene of Escherichia coli, primer pairs for detecting the F6 fimbrial gene of Escherichia coli, primer pairs for detecting the F4 fimbrial gene of Escherichia coli, and primer pairs for detecting the F41 fimbrial gene of Escherichia coli; The primer pair used to detect the F5 fimbrial gene in Escherichia coli is: F5-F: 5'-ACTCTGGTTCTTCTTGGCTGT-3' (shown in SEQ ID NO. 1) F5-R: 5'-TACCTTCCCAGGTCGGAGTT-3' (shown in SEQ ID NO. 2); The primer pair used to detect the F18 fimbrial gene in Escherichia coli is: F18-F: 5'-ACCAAGCGGAACTGGTAGTG-3' (shown in SEQ ID NO.3) F18-R: 5'-TCACGGTTTTCAGAGCGACA-3' (shown in SEQ ID NO.4); The primer pair used to detect the F6 fimbrial gene in Escherichia coli is: F6-F: 5'-CCCGCTGAAAACAACACCAG-3' (shown in SEQ ID NO. 5) F6-R: 5'-GCCCCTTGCCAGTAGCTTTA-3' (shown in SEQ ID NO. 6); The primer pair used to detect the F4 fimbrial gene in Escherichia coli is: F4-F: 5'-CCGAGGGGTTCTGAATCGT-3' (shown in SEQ ID NO.7) F4-R: 5'-CGTTTGCAATACCCAGTGCAT-3' (shown in SEQ ID NO.8); The primer pair used to detect the F41 fimbrial gene in Escherichia coli is: F41-F: 5'-CTGCTGATTGGACGGAAGGT-3' (shown in SEQ ID NO.9) F41-R: 5'-CTGCTGATTGGACGGAAGGT-3' (shown in SEQ ID NO.10).
[0008] Optionally, the following steps are included: PCR amplification template preparation, PCR reaction, and electrophoresis detection of results; the PCR reaction system includes 10 μL DNA annealing buffer, 200 μM dNTP mixture, 10 μM each of five forward primers, 10 μM each of five reverse primers, 1 μL template, and 1.25 U Taq polymerase.
[0009] Optionally, the parameters for the PCR reaction are: 94℃ pre-denaturation for 30s; 94℃ denaturation for 30s, 50℃ annealing for 90s, 72℃ extension for 60s, 30 cycles; 72℃ final extension for 5min; and product storage at 4℃.
[0010] Optionally, the PCR amplification template preparation involves picking a single colony, inoculating it into 50 μL of deionized water, heating it at 95°C for 10 min, gently centrifuging it, covering it with water, and then using it as a template stock solution for subsequent PCR reactions.
[0011] The above-described technical solution of the present invention has at least the following beneficial effects: The technical solution provided by this invention can output a complete five-serum subtype profile in a single test, accurately supporting the selection of multivalent vaccines. Attached Figure Description
[0012] Figure 1 This is an electrophoresis diagram of the PCR detection results of five fimbrial genes in Example 1 of the present invention; Figure 2 This is an electrophoresis diagram of the PCR detection results of five fimbrial genes in Example 3 of the present invention. Detailed Implementation
[0013] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the following will be described in conjunction with the accompanying drawings of the embodiments of the present invention. Figure 1 The technical solutions of the embodiments of the present invention will be clearly and completely described herein. Obviously, the described embodiments are only a part of the embodiments of the present invention, and not all of them. All other embodiments obtained by those skilled in the art based on the described embodiments of the present invention are within the scope of protection of the present invention.
[0014] Example 1 Primer design Design specific primer pairs for simultaneous detection of the F5, F18, F6, F4, and F41 fimbrial genes. The nucleotide sequences are shown below: The primer pair used to detect the F5 fimbrial gene in Escherichia coli is: F5-F: 5'-ACTCTGGTTCTTCTTGGCTGT-3' (shown in SEQ ID NO. 1) F5-R: 5'-TACCTTCCCAGGTCGGAGTT-3' (shown in SEQ ID NO. 2); The primer pair used to detect the F18 fimbrial gene in Escherichia coli is: F18-F: 5'-ACCAAGCGGAACTGGTAGTG-3' (shown in SEQ ID NO.3) F18-R: 5'-TCACGGTTTTCAGAGCGACA-3' (shown in SEQ ID NO.4); The primer pair used to detect the F6 fimbrial gene in Escherichia coli is: F6-F: 5'-CCCGCTGAAAACAACACCAG-3' (shown in SEQ ID NO. 5) F6-R: 5'-GCCCCTTGCCAGTAGCTTTA-3' (shown in SEQ ID NO. 6); The primer pair used to detect the F4 fimbrial gene in Escherichia coli is: F4-F: 5'-CCGAGGGGTTCTGAATCGT-3' (shown in SEQ ID NO.7) F4-R: 5'-CGTTTGCAATACCCAGTGCAT-3' (shown in SEQ ID NO.8); The primer pair used to detect the F41 fimbrial gene in Escherichia coli is: F41-F: 5'-CTGCTGATTGGACGGAAGGT-3' (shown in SEQ ID NO.9) F41-R: 5'-CTGCTGATTGGACGGAAGGT-3' (shown in SEQ ID NO.10).
[0015] Specific primer pairs for the F5, F18, F6, F4, and F41 fimbriae genes amplified 593bp, 179bp, 148bp, 211bp, and 449bp, respectively.
[0016] Example 2 PCR amplification PCR amplification template preparation: Pick a single colony and inoculate it into 50 μL of deionized water, and heat at 95℃ for 10 min; PCR reaction system: 5x buffer 10 μL dNTP mixture 200 μM each 1 μL (10 μM) of forward primer (5 forward primers in total) 1 μL (10 μM) of reverse primers (out of 5) Template 1 μL Taq polymerase 1.25 U / 50 μl 50 μL in total The product was stored at 4°C after being obtained.
[0017] Interpretation of PCR test results The amplification product was spotted into the wells of a 4% agarose gel and electrophoresed at 160V for 60 minutes. Figure 1 .
[0018] Depend on Figure 1 It is understood that the method for detecting five fimbrial adhesins subtypes of enterotoxin-producing Escherichia coli based on 5-plex mPCR provided by the present invention constructs a stable mPCR system that can simultaneously detect five serum subtypes (F4, F18, F6, F41, K99) at one time, and can realize the simultaneous detection of the above five fimbrial genes.
[0019] Example 3 Following the methods described in Examples 1 and 2, 5-plex mPCR was performed on random samples 45-65 from the pig farm. After the PCR reaction, the amplification products were collected and loaded into the wells of a 4% agarose gel. Electrophoresis was performed at 160V for 60 minutes to obtain the desired product. Figure 2 .
[0020] Depend on Figure 2It is known that the 5-plex mPCR method for detecting five virulence factors of enterotoxin-producing Escherichia coli provided by the present invention can accurately identify different Escherichia coli fimbriatin subtypes in one step, and the designed primers have high specificity.
[0021] The above are preferred embodiments of the present invention. It should be noted that, for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.
Claims
1. A method for detecting five fimbriatin subtypes of enterotoxigenic Escherichia coli from swine farms based on 5-plex mPCR, characterized in that, The multiplex PCR primer set includes primer pairs for detecting the Escherichia coli F5 fimbrial gene, primer pairs for detecting the Escherichia coli F18 fimbrial gene, primer pairs for detecting the Escherichia coli F6 fimbrial gene, primer pairs for detecting the Escherichia coli F4 fimbrial gene, and primer pairs for detecting the Escherichia coli F41 fimbrial gene. The primer pair used to detect the F5 fimbrial gene in Escherichia coli is: F5-F:5'-ACTCTGGTTCTTCTTGGCTGT-3' F5-R: 5'-TACCTTCCCAGGTCGGAGTT-3'; The primer pair used to detect the F18 fimbrial gene in Escherichia coli is: F18-F:5'-ACCAAGCGGAACTGGTAGTG-3' F18-R: 5'-TCACGGTTTTCAGAGCGACA-3'; The primer pair used to detect the F6 fimbrial gene in Escherichia coli is: F6-F:5'-CCCGCTGAAAACAACACCAG-3' F6-R: 5'-GCCCCTTGCCAGTAGCTTTA-3'; The primer pair used to detect the F4 fimbrial gene in Escherichia coli is: F4-F: 5'-CCGAGGGGTTCTGAATCGT-3' F4-R: 5'-CGTTTGCAATACCCAGTGCAT-3'; The primer pair used to detect the F41 fimbrial gene in Escherichia coli is: F41-F:5'-CTGCTGATTGGACGGAAGGT-3' F41-R: 5'-CTGCTGATTGGACGGAAGGT-3'.
2. The method for detecting five fimbriated adhesin subtypes of enterotoxigenic Escherichia coli based on 5-plex mPCR according to claim 1, characterized in that, Includes the following steps: PCR amplification template preparation, PCR reaction, and electrophoresis detection results; the PCR reaction system includes 10 μL DNA annealing buffer, 200 μM dNTP mixture, 10 μM each of five forward primers, 10 μM each of five reverse primers, 1 μL template, and 1.25 U Taq polymerase.
3. The method for detecting five fimbriated adhesins subtypes of enterotoxigenic Escherichia coli from swine farms based on 5-plex mPCR according to claim 2, characterized in that, The PCR reaction parameters were: 94℃ pre-denaturation for 30 s; 94℃ denaturation for 30 s, 50℃ annealing for 90 s, 72℃ extension for 60 s, 30 cycles; 72℃ final extension for 5 min; and product storage at 4℃.
4. The method for detecting five fimbriated adhesin subtypes of enterotoxigenic Escherichia coli from swine farms based on 5-plex mPCR according to claim 1, characterized in that, The PCR amplification template was prepared by picking a single colony and inoculating it into 50 μL of deionized water, heating it at 95°C for 10 min, gently centrifuging it, covering it with water, and then using it as the template stock solution for subsequent PCR reactions.