PCR detection method for vibrio parahaemolyticus serotype o10 variant and application thereof
By designing specific PCR primers targeting the O antigen gene cluster of Vibrio parahaemolyticus O10 variant, a PCR detection technology was established, which solved the problems of long detection time and insufficient sensitivity in existing technologies, and realized rapid and accurate detection of O10 variant, which is suitable for food safety and epidemiological monitoring.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- NANKAI UNIV
- Filing Date
- 2025-11-03
- Publication Date
- 2026-04-17
AI Technical Summary
Existing methods for detecting Vibrio parahaemolyticus suffer from problems such as long detection time, insufficient sensitivity, and inability to accurately distinguish O10 variants. Traditional serological testing is cumbersome, time-consuming, difficult to produce antiserum, and has serious cross-reactivity issues.
Using molecular biology-based methods, specific PCR primers were designed to target the O antigen gene cluster of Vibrio parahaemolyticus O10 variant. A PCR detection technology was established, and specific oligonucleotide sequences were used to detect the O10 variant, providing a specific PCR detection kit.
It enables accurate and rapid identification of Vibrio parahaemolyticus O10 variant, shortening the detection time to within 3 hours, with a sensitivity of 104 CFU/ml, making it suitable for rapid detection and epidemiological monitoring in the field of food safety.
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Figure CN121046558B_ABST
Abstract
Description
Technical Field
[0001] This invention belongs to the technical field of bacterial detection methods, and relates to a PCR detection method and its application for Vibrio parahaemolyticus serotype O10 variant, which has been isolated in large quantities in my country in recent years. Background Technology
[0002] Vibrio parahaemolyticus is a halophilic, Gram-negative bacillus widely distributed in coastal and estuarine areas, and is a parasite of many shellfish. Since causing a large-scale food poisoning incident in Japan in the 1950s, Vibrio parahaemolyticus has frequently caused foodborne illnesses globally. In coastal areas of my country, such as Guangdong, Shanghai, and Zhejiang, the isolation rate of this bacterium is high, and it frequently causes sporadic food poisoning incidents, posing a serious threat to food safety. Therefore, rapid and accurate detection and traceability of Vibrio parahaemolyticus are of great significance in the field of food safety.
[0003] Currently, my country's national food safety standard (GB4789.7-2013) uses bacterial culture and biochemical testing methods for the detection of Vibrio parahaemolyticus. Qualitative identification takes approximately 8-18 hours, while quantitative identification takes approximately 4-5 days. The 2019 national standard for entry-exit inspection and quarantine in my country uses real-time quantitative PCR for the detection of Vibrio parahaemolyticus in aquatic animals, and the entire process can be completed within 24 hours. However, these methods can only detect Vibrio parahaemolyticus at the species level, and their low throughput makes them unsuitable for meeting the needs of epidemiological tracing and rapid detection.
[0004] Serological testing methods are characterized by their ease of operation and intuitive result interpretation, and have been widely used since the 1930s. This method can effectively distinguish between different pathogenic strains within a single species / genus. Currently, most important pathogenic bacteria have established serological typing systems based on surface polysaccharide antigens, and these typing systems are widely used in inspection and quarantine and disease prevention and control systems. my country's national food safety standard (GB4789.7-2013) also includes serology as an optional item for Vibrio parahaemolyticus detection. However, although traditional serological identification methods are widely used, they still have many shortcomings. These mainly include: 1) Establishing a serological typing system is cumbersome and time-consuming; 2) The production and quality control of antisera are difficult; 3) Many antisera are produced and stored by only a few units internationally, and the antisera used for identification are severely incomplete; 4) The experimental process for serological identification is time-consuming (2-6 days) and cross-reactivity is serious.
[0005] Compared with traditional serological methods, molecular biology-based bacterial detection technologies have the advantages of high accuracy, speed and sensitivity, and have been widely used in bacterial detection and typing.
[0006] Bacterial surface polysaccharide antigens mainly include O polysaccharide (O antigen), common antigen (CA), extracellular polysaccharide antigen, spores, and capsular polysaccharide (K antigen). The structural diversity of O polysaccharide (O antigen) and capsular polysaccharide (K antigen) forms the basis of bacterial serotyping. The diversity of O antigen and / or K antigen within the same bacterium is determined by the genetic diversity of the genes encoding the various enzymes that synthesize O antigen and / or K antigen. These genes are often clustered at fixed sites on the genome, called O antigen gene clusters and / or K antigen gene clusters. Based on the diversity of its O antigen, Vibrio parahaemolyticus can be divided into 13 O antigen serotypes; based on the diversity of its K antigen, it can be divided into 71 K antigen serotypes. The O antigen gene clusters of these serotypes are located between the two housekeeping genes dgkA and gmhD, while the K antigen gene clusters are located between the two housekeeping genes gmhD and rjg, and the gene cluster information has been deciphered. Currently, some scholars have used molecular biology methods to target specific genes in the O antigen gene cluster and K antigen synthesis gene cluster to conduct serotype detection of Vibrio parahaemolyticus.
[0007] Since 2010, the O3:K6 serotype of Vibrio parahaemolyticus isolated in my country has been the most common. In 2020, a strain with serotype O10:K4 was detected in my country for the first time. In recent years, the O10:K4 strain has rapidly gained dominance and is now surpassed by the O3:K6 strain to become the most prevalent and most frequently detected Vibrio parahaemolyticus serotype in my country. The identification of the aforementioned O10:K4 strains was all done using traditional serotyping methods (i.e., antigen-antibody agglutination reaction). However, our research found that the K antigen gene cluster of these strains is completely identical to that of the standard K4 strain, while their O antigen gene cluster is not identical to that of the standard O10 strain. Specifically, the O antigen gene cluster of the standard O10 strain consists of 15 genes. Although the O antigen gene cluster of the aforementioned isolated O10:K4 strains also consists of 15 genes, the first 7 genes and the last 3 genes are the same, while the middle 5 genes are completely different. Therefore, although the traditional serotype test result is O10:K4, this result may be due to cross-reactivity in the serological agglutination experiment. From the perspective of currently used molecular biological detection methods, due to the alteration of the O antigen gene cluster, these isolates are not true O10 serotypes, and we call them O10 variants. Summary of the Invention
[0008] This invention addresses the technical shortcomings of existing Vibrio parahaemolyticus detection methods, such as long detection time, insufficient sensitivity, and inability to distinguish between O10 variants. It provides a molecular biology-based method for the specific detection of Vibrio parahaemolyticus serotype O10 variant and its application. Based on the analysis of the O antigen serotype gene cluster of Vibrio parahaemolyticus O10 variant, a specific oligonucleotide sequence for detecting Vibrio parahaemolyticus O10 variant is provided, and a corresponding PCR detection technique is established. This method is of great significance for the accurate and rapid identification and epidemiological monitoring of this bacterium.
[0009] To achieve the above-mentioned objectives, the present invention provides the following technical solution:
[0010] A specific PCR primer for detecting Vibrio parahaemolyticus serotype O10 variant, the primer comprising an upstream primer P1 and a downstream primer P2, wherein the sequence of the upstream primer P1 is shown in SEQ ID NO: 1 and the sequence of the downstream primer P2 is shown in SEQ ID NO: 2.
[0011] Furthermore, the present invention also provides a PCR detection reagent or kit for detecting Vibrio parahaemolyticus serotype O10 variant using the specific PCR primers described above.
[0012] Furthermore, the PCR system comprises the following components and concentrations: 0.25 μL of 10 mM upstream primer, 0.25 μL of 10 mM downstream primer, 3 μL of crude genomic DNA extract, 6.5 μL of ddH2O, and 10 μL of 5 u / μL 2×Taq DNA polymerase premix.
[0013] Furthermore, the specific PCR primers used to detect Vibrio parahaemolyticus serotype O10 are derived from specific regions of the IS1 and orf9 genes in the O antigen gene cluster of Vibrio parahaemolyticus serotype O10.
[0014] This invention also provides a rapid typing method for Vibrio parahaemolyticus serotype O10 variant, using reagents or kits containing specific PCR primers for the O10 variant. This method is for non-disease diagnostic purposes.
[0015] Furthermore, the above-mentioned rapid typing method is applicable to the rapid typing of Vibrio parahaemolyticus.
[0016] The positive effects of the PCR detection method for detecting Vibrio parahaemolyticus O10 variant disclosed in this invention and its application include, but are not limited to:
[0017] (1) For the first time, a technique for detecting Vibrio parahaemolyticus O10 variant, the most common foodborne pathogen in my country in recent years, using molecular biology methods has been disclosed, which makes up for the deficiencies of traditional serotype identification methods and can be used for accurate identification and epidemiological monitoring of this bacterium;
[0018] (2) For pure bacterial samples, the PCR detection method can accurately detect Vibrio parahaemolyticus O10 variant strain within 3 hours; for simulated samples, the PCR detection method can accurately detect Vibrio parahaemolyticus O10 variant strain within 15 hours; compared with the traditional serological detection method (which usually takes about 3 days), the detection time is greatly shortened, and it has the advantage of fast detection speed.
[0019] (3) For pure bacterial samples, the detection sensitivity of this PCR detection method is 10. 4 CFU / ml; for simulated samples, the detection sensitivity of this PCR method is 10. 1 CFU / ml; similar to other molecular biology detection methods, it has the advantage of high detection sensitivity. Attached Figure Description
[0020] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the accompanying drawings used in the description of the embodiments or the prior art are briefly introduced below.
[0021] Figure 1 Agarose gel electrophoresis showed the detection results of Vibrio parahaemolyticus O10 variant PCR detection kit for the corresponding serotypes and serotypes O1-O13: where M is DL2000 DNA molecular standard, 1 is O10 variant DB20082912; 2 is O10 variant DB 2008060; 3 is O10 variant 2023FR012; 4 is O10 variant 2023FR072; 5 is O10 variant 2024FR050; 6 is O10 variant 2024FR209; 7 is type O1; 8 is type O2; 9 is type O3; 10 is type O4; 11 is type O5; 12 is type O6; 13 is type O7; 14 is type O8; 15 is type O9; 16 is type O10; 17 is type O11; 18 is type O12; and 19 is type O13.
[0022] Figure 2 Agarose gel electrophoresis showed the sensitivity detection results of the PCR detection kit for Vibrio parahaemolyticus O10 variant at different pure bacterial concentrations.
[0023] Figure 3 Agarose gel electrophoresis showed the sensitivity detection results of the Vibrio parahaemolyticus O10 variant PCR detection kit after overnight incubation of simulated samples prepared by adding different initial concentrations of strains to 2.5g of oysters. Detailed Implementation
[0024] The present invention will now be described through specific embodiments. Unless otherwise specified, all technical means used in this invention are methods well known to those skilled in the art. Furthermore, the embodiments should be understood as illustrative, not limiting the scope of the invention; the essence and scope of the invention are defined only by the claims. For those skilled in the art, various changes or modifications to the material composition and dosage in these embodiments without departing from the essence and scope of the invention are also within the scope of protection of this invention. All raw materials and reagents used in this invention are commercially available.
[0025] The sources of the Vibrio parahaemolyticus strains are shown in Table 1.
[0026] Table 1. Vibrio parahaemolyticus strains and their sources used in this invention.
[0027] serotype source serial number O1 Department of Microbiology, Soochow University, Taiwan 1247 O2 Taiwan Center for Biological Resources Preservation and Research 12969 O3 China Center for Type Culture Collection GB 2008103 O4 China Center for Type Culture Collection GB 2008178 O5 China Center for Type Culture Collection GB 2008184 O6 China Center for Type Culture Collection GB 2008081 O7 Taiwan Center for Biological Resources Preservation and Research 12964 O8 Taiwan Center for Biological Resources Preservation and Research 1406 O9 Shanghai Municipal Center for Disease Control and Prevention 20579 O10 Taiwan Center for Biological Resources Preservation and Research 12968 O11 Taiwan Center for Biological Resources Preservation and Research 12963 O12 Shanghai Municipal Center for Disease Control and Prevention 20582 O13 Shanghai Municipal Center for Disease Control and Prevention 49–91 O10 variant China Center for Type Culture Collection DB 20082912 O10 variant China Center for Type Culture Collection DB 2008060 O10 variant Shandong Provincial Center for Disease Control and Prevention 2023FR012 O10 variant Shandong Provincial Center for Disease Control and Prevention 2023FR072 O10 variant Shandong Provincial Center for Disease Control and Prevention 2024FR050 O10 variant Shandong Provincial Center for Disease Control and Prevention 2024FR209
[0028] Example 1: Design and preparation of serotype-specific primers for Vibrio parahaemolyticus O10 variant O antigen.
[0029] Select Vibrio parahaemolyticus O10 variant IS1 and orf9 The target sequence was imported into the primer design software PrimerPrimier 5.0, and the parameters were set as follows: positive and negative strand output modes were selected; sequence amplification length was 300-800 bp; Haripin: none; Dimer: none; False Priming: none; Cross Dimer: none. The program was run to obtain one specific primer sequence each for the positive and negative strands. The primer specificity was verified by comparing their sequences with all sequences in GenBank using a BLAST search. The primer sequences were: upstream primer P1, as shown in SEQ ID NO: 1, 5'-AGCACAAAATCCGAGAGAGT-3'; downstream primer P2, as shown in SEQ ID NO: 2, 5'-ATTATCTTTTGTCGCCTGG-3'. The designed primer sequences were sent to Thermo Fisher Scientific (China) Co., Ltd. for DNA synthesis and purification using PAGE, and then stored for later use.
[0030] Example 2 Preparation of crude genomic DNA extract from the strain to be tested
[0031] 1. The obtained pure bacterial cultures were processed using the following methods:
[0032] (1) Pick a single bacterial colony and put it into 10 µL of deionized water, or 10 µL of bacterial culture that has been cultured overnight, and treat it in a boiling water bath for 15 minutes.
[0033] (2) Place on ice for 1 minute, then centrifuge at 8000 rpm for 1 minute;
[0034] (3) Take 3µL of supernatant as the template for the next PCR reaction.
[0035] 2. The bacteria in the simulated samples were treated using the following methods:
[0036] (1) After washing the oysters purchased from the market with sterile water, grind them into small pieces using a rotary blade homogenizer, divide them into 2.5g portions, and dissolve them in 24ml of brine (sodium chloride content 3%).
[0037] (2) The Vibrio parahaemolyticus O10 strain cultured overnight at 37 degrees Celsius was diluted 10-fold in a series of 10-fold increments, with each strain and each dilution containing 10 ml of the diluted strain. 4 10 3 10 2 10 1 and 10 0 Each bacterium was added to the sample prepared in (1), and after thorough mixing, 3 ml was added to 27 ml of high-salt LB medium (sodium chloride content 3%), and cultured at 37 degrees with shaking for 12 hours.
[0038] (3) Take 100 µL of the overnight culture sample from (2), incubate in a boiling water bath for 15 minutes, then place on ice for 1 minute, and centrifuge at 8000 rpm for 1 minute to obtain the crude DNA extract of each sample;
[0039] (4) Take 3 µL of supernatant as a template for the next PCR reaction.
[0040] Example 3: PCR amplification and agarose gel electrophoresis identification
[0041] The crude genomic DNA extract obtained in Example 2 was used as a template for PCR amplification. The PCR reaction system was prepared according to the various reagent components and concentrations shown in Table 2.
[0042] Table 2. PCR reaction system for detecting Vibrio parahaemolyticus O10 variant.
[0043] Element concentration Sample volume (µL) <![CDATA[ddH2O]]> - 6.5 upstream primer P1 10 mM 0.25 Downstream primer P2 10 mM 0.25 2×Taq DNA polymerase premix 5 u / μL 10 crude genomic DNA extract - 3 Total volume - 20
[0044] In the PCR instrument (Biometra), set the cycling parameters and run the test. The cycling parameters are shown in Table 3:
[0045] Table 3 PCR reaction process
[0046]
[0047] After the reaction, electrophoresis was performed using a 1.5% agarose gel (130V, 30 minutes). The results showed that this PCR reaction system specifically detected the O10 variant strain (with a single, clearly visible 545 bp band), while no amplification bands were produced for the O1-O13 serotype standard strains. Details are as follows: Figure 1 As shown in Table 4, the specific detection details of this PCR reaction system for pure cultures of Vibrio parahaemolyticus O10 variant strains at different concentrations are shown in Table 5. The specific detection details of this PCR reaction system for simulated samples prepared from different initial concentrations of Vibrio parahaemolyticus O10 variant strains after overnight incubation are also shown in Table 5.
[0048] Table 4. Detection results of pure cultures of Vibrio parahaemolyticus variant O10 at different concentrations
[0049] Pure bacterial sample concentration (CFU / ml) Number of repeated tests Average detection time (h) Detection rate <![CDATA[10 7 ]]> 3 3 100% <![CDATA[10 6 ]]> 3 3 100% <![CDATA[10 5 ]]> 3 3 100% <![CDATA[10 4 ]]> 3 3 100% <![CDATA[10 3 ]]> 3 3 0 <![CDATA[10 2 ]]> 3 3 0 <![CDATA[10 1 ]]> 3 3 0
[0050] Table 5. Detection results of simulated samples of Vibrio parahaemolyticus O10 variant strain at different concentrations.
[0051] Simulated initial sample concentration (CFU / ml) Number of repeated tests Detection time (h) Detection rate <![CDATA[10 4 ]]> 3 15 100% <![CDATA[10 3 ]]> 3 15 100% <![CDATA[10 2 ]]> 3 15 100% <![CDATA[10 1 ]]> 3 15 100% <![CDATA[10 0 ]]> 3 15 0
[0052] The above experimental results show that this PCR detection kit can accurately detect Vibrio parahaemolyticus variant O10 and does not cross-react with other serotypes (see [link to kit]). Figure 1 For pure bacterial samples, this PCR detection kit can accurately detect Vibrio parahaemolyticus O10 variant strain within 3 hours, with a detection sensitivity of 10. 4 CFU / ml; For simulated samples, after overnight incubation, this PCR detection kit can accurately detect Vibrio parahaemolyticus O10 variant strain within 15 hours, with a detection sensitivity of 10. 1 CFU / ml.
[0053] Example 4: Application example of the PCR reaction system of the present invention
[0054] To verify the practicality of the PCR reaction system of this invention, we collected 220 Vibrio parahaemolyticus strains isolated from food between 2023 and 2024. After conventional serotyping, 198 strains were identified as O10:K4, 26 as O3:K6, and 6 as O2:K3. Furthermore, genomic analysis showed that the results for the strains identified as O3:K6 and O2:K3 were consistent with the results of conventional serotyping. The K antigen gene cluster of the 198 strains identified as O10:K4 was identical to that of K4, while the O antigen gene cluster belonged to the O10 variant.
[0055] Next, using the steps in Examples 2 and 3, the above 220 Vibrio parahaemolyticus strains were identified by PCR. The results showed that 198 strains could amplify the specific band of the O10 variant, which was completely consistent with the conclusions drawn from the genomic analysis; the remaining strains could not amplify. This result verifies the practicality of the present invention.
[0056] For those skilled in the art, this invention is not limited to the details of the exemplary embodiments described above, and can be implemented in other specific forms without departing from the spirit or essential characteristics of this invention. Therefore, the embodiments should be considered exemplary and non-limiting in all respects, and the scope of this invention is defined by the appended claims rather than the foregoing description. Thus, it is intended that all variations falling within the meaning and scope of equivalents of the claims be included within this invention. No reference numerals in the claims should be construed as limiting the scope of the claims.
[0057] The above description is merely a preferred embodiment of the present invention and is not intended to limit the scope of the invention. Any minor modifications, equivalent substitutions, and improvements made to the above embodiments based on the technical essence of the present invention should be included within the protection scope of the present invention.
Claims
1. A rapid detection method of the bacterial species of Vibrio parahaemolyticus serotype O10:K4 variant, characterized in that, The detection is performed using specific PCR primers for the O10:K4 variant or reagents or kits containing such specific PCR primers. The method is for non-disease diagnostic purposes. The primers include an upstream primer P1 and a downstream primer P2. The sequence of the upstream primer P1 is shown in SEQ ID NO: 1, and the sequence of the downstream primer P2 is shown in SEQ ID NO:
2. The primer amplification length in the detection method is 545 bp.
2. The rapid detection method for Vibrio parahaemolyticus serotype O10:K4 variant according to claim 1, characterized in that, The PCR reaction system used in the detection method comprises the following components and concentrations: 0.25 μL of 10 mM upstream primer, 0.25 μL of 10 mM downstream primer, 3 µL of crude genomic DNA extract, 6.5 μL of ddH2O, and 10 μL of 5 u / μL 2×Taq DNA polymerase premix.
3. The rapid detection method for Vibrio parahaemolyticus serotype O10:K4 variant according to claim 1, characterized in that, Suitable for rapid typing of Vibrio parahaemolyticus.