Specific molecular targets of vibrio alginolyticus and vibrio fluvialis and methods of detecting the same

Abstract

The application discloses specific molecular targets of Vibrio alginolyticus and Vibrio fluvialis and a detection method thereof, wherein the nucleotide sequence of the specific molecular target of the Vibrio alginolyticus is as shown in SEQ ID NO. 1, and the nucleotide sequence of the specific molecular target of the Vibrio fluvialis is as shown in SEQ ID NO. 2, SEQ ID NO. 3, SEQ ID NO. 4 or SEQ ID NO. 5. The application not only discloses one specific molecular target for identifying the Vibrio alginolyticus and four specific molecular targets for identifying the Vibrio fluvialis, but also provides related specific primer sequences and corresponding PCR detection methods. The application has the advantages of short detection time, low cost, strong specificity and the like without physiological and biochemical identification.

Description

TECHNICAL FIELD

[0001] The present application relates to the technology of bacterial detection, in particular, to specific molecular targets of Vibrio alginolyticus and Vibrio fluvialis and the detection method thereof. BACKGROUND

[0002] Vibrio alginolyticus is a gram-negative bacterium, which is in the form of "comma" or spheroplast. It was isolated from Vibrio parahaemolyticus and officially named Vibrio alginolyticus in 1968. It is a halophilic bacterium, which cannot grow on CLED agar, but can grow in the presence of 10% sodium chloride. It forms large yellow (sucrose fermentation) colonies on TCBS. There are obvious bee colonies on non-selective solid medium. This organism is widely distributed in seawater and seafood, and most infections are related to contact with seawater or seafood. Vibrio alginolyticus found in seawater is mainly related to gastroenteritis, otitis media and otitis externa, but Vibrio alginolyticus usually does not have the virulence potential of Vibrio vulnificus, and the clinical features include severe skin infection and pus. Vibrio alginolyticus is an important pathogen that must be closely monitored and controlled in the aquaculture industry, because it has strong pathogenicity, rapid onset after infection and high mortality of aquatic animals, so a rapid, simple and specific method is needed for on-site detection to effectively control the epidemic and prevent economic losses.

[0003] Vibrio fluvialis is a halophilic gram-negative bacterium. It has a straight to slightly curved rod cell morphology and can move by polar flagella. Vibrio fluvialis is considered to be one of the foodborne pathogens, which is related to outbreaks of diarrhea and sporadic cases. It was first isolated from a patient with diarrhea in 1975 and was later named Vibrio fluvialis. This bacterium naturally distributes in warm, salt-containing seawater and river water, and can survive at temperatures ranging from 9°C to 31°C, and reproduce when the water temperature rises above 18°C. Infection with Vibrio fluvialis shows seasonality, and most of the diseases occur under the most suitable temperature and salinity. It can cause cholera-like diarrhea, skin infection (clinical symptoms similar to skin infection in long-term aquatic environment), and septicemia in immunocompromised individuals. In developing countries with poor sanitation, Vibrio fluvialis is the second most pathogenic Vibrio after Vibrio cholera and Vibrio parahaemolyticus, which not only endangers human health but also causes damage to freshwater and marine environments. Therefore, in order to prevent new diseases caused by Vibrio fluvialis, we must establish an effective monitoring system to provide early warning of infection.

[0004] Currently, the traditional plate culture method is commonly used for the detection of Vibrio alginolyticus and Vibrio fluvialis. While plate detection has advantages such as low cost and high specificity, it also has some drawbacks, such as long incubation time and strict requirements for operators and procedures. To overcome the shortcomings of plate detection, molecular detection methods based on nucleic acid amplification have been applied to the detection of Vibrio alginolyticus and Vibrio fluvialis in food.

[0005] The specificity and sensitivity of molecular detection methods based on nucleic acid amplification mainly depend on the selection of the corresponding detection target. Currently, PCR is one of the most commonly used molecular detection methods. Its primers are designed based on specific conserved sequences (detection targets) of the target bacterial genomic DNA. Therefore, the detection target and primers are crucial to the accuracy and sensitivity of PCR detection. For *Vibrio alginolyticus*, this bacterium, along with *Vibrio parahaemolyticus* and others, constitutes the core group of the *Vibrio* genus—*Vibrio harveyi*. These vibrios are very similar in phenotype and genotype, and cross-identification can easily occur during detection. Furthermore, *Vibrio fluvialis* often... toxR While genes are target genes for PCR, they are often interfered with by Vibrio parahaemolyticus during detection, leading to false positives. Therefore, in order to isolate and identify these Vibrio species, there is an urgent need for a systematic and comprehensive exploration of new specific detection targets to improve the accuracy of nucleic acid amplification-based detection technologies for Vibrio alginolyticus and Vibrio fluvialis. Summary of the Invention

[0006] The purpose of this invention is to overcome the shortcomings of the prior art by using pan-genomics technology to comprehensively analyze the core genes and accessory genes of Vibrio alginolyticus and Vibrio fluvialis. Furthermore, BLAST analysis is performed on the core gene portion to further identify specific gene regions within the core gene, thereby providing a specific molecular target for Vibrio alginolyticus and Vibrio fluvialis and its detection method.

[0007] To achieve the above objectives, the present invention adopts the following technical solution:

[0008] A specific molecular target for detecting Vibrio alginolyticus and Vibrio fluvialis, wherein the nucleotide sequence of the specific molecular target for Vibrio alginolyticus is as shown in SEQ ID NO.1; and the nucleotide sequence of the specific molecular target for Vibrio fluvialis is as shown in SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4 or SEQ ID NO.5.

[0009] The present invention also discloses a set of primers for detecting the above-mentioned specific molecular targets, characterized in that the nucleotide sequence of the upstream primer of the specific molecular target SEQ ID NO.1 is as shown in SEQ ID NO.6, and the nucleotide sequence of the downstream primer is as shown in SEQ ID NO.7;

[0010] The nucleotide sequence of the upstream primer for detecting the specific molecular target SEQ ID NO. 2 is shown in SEQ ID NO. 8, and the nucleotide sequence of the downstream primer is shown in SEQ ID NO. 9;

[0011] The nucleotide sequence of the upstream primer for detecting the specific molecular target SEQ ID NO. 3 is shown in SEQ ID NO. 10, and the nucleotide sequence of the downstream primer is shown in SEQ ID NO. 11;

[0012] The nucleotide sequence of the upstream primer for detecting the specific molecular target SEQ ID NO. 4 is shown in SEQ ID NO. 12, and the nucleotide sequence of the downstream primer is shown in SEQ ID NO. 13;

[0013] The nucleotide sequence of the upstream primer for detecting the specific molecular target SEQ ID NO. 5 is shown in SEQ ID NO. 14, and the nucleotide sequence of the downstream primer is shown in SEQ ID NO. 15.

[0014] The application also discloses application of the molecular target in preparation of a reagent for detecting Vibrio vulnificus.

[0015] The application also discloses a kit for detecting Vibrio alginolyticus and Vibrio fluvialis, which comprises all the nucleotide sequences.

[0016] The application also discloses a method for detecting Vibrio alginolyticus and Vibrio fluvialis for non-disease diagnosis and treatment purposes, which comprises the following steps:

[0017] (1) extracting DNA of the microorganism to be detected, taking the extracted DNA as a template, and performing PCR amplification on the template by using the primers;

[0018] (2) performing agarose gel electrophoresis analysis on the amplification product, and determining that the sample contains Vibrio alginolyticus if a band of a target size (155 bp) appears in the electrophoresis result, and determining that the sample does not contain Vibrio alginolyticus if a band of the target size (155 bp) does not appear in the electrophoresis result; and determining that the sample contains Vibrio fluvialis if a band of a target size (145 bp, 130 bp, 286 bp or 282 bp) appears in the electrophoresis result, and determining that the sample does not contain Vibrio fluvialis if a band of the target size (145 bp, 130 bp, 286 bp or 282 bp) does not appear in the electrophoresis result.

[0019] Preferably, in the method for detecting Vibrio alginolyticus and Vibrio fluvialis, the reaction system of the PCR amplification is 20 microliters, which comprises 10 microliters of 2x PCR Mix, 2 microliters of template DNA, and 10 micromolar of the upstream and downstream primers.

[0020] Preferably, in the above-mentioned method for detecting Vibrio alginolyticus and Vibrio fluvialis, the PCR amplification reaction conditions are: 95 °C pre-denaturation for 3 min; 95 °C denaturation for 15 s, 60 °C annealing for 15 s, 72 °C extension for 30 s, for a total of 35 cycles, 72 °C extension for 5 min, and 4 °C hold.

[0021] Compared with existing technologies, the present invention has the following advantages. The present invention not only discloses a specific molecular target for identifying *Vibrio alginolyticus* and four specific molecular targets for identifying *Vibrio fluvialis*, but also provides related specific primer sequences and corresponding PCR detection methods. The present invention eliminates the need for physiological and biochemical identification, and has advantages such as short detection time, low cost, and high specificity. Attached Figure Description

[0022] Figure 1 The results show the sensitivity of the Vibrio alginolyticus VA1 primer pair in the specific implementation method.

[0023] Note: Lane M: DL1200 DNA Marker; Lanes 1-8 increase in sequence: 4 × 10⁸ 1 – 4 × 10 8 CFU / mL; Lane N: negative control.

[0024] Figure 2 The specific results of the Vibrio alginolyticus VA1 primer pair are shown in the specific implementation method;

[0025] Note: Lane M: DL2000 DNA Marker; Lane 1: Vibrio parahaemolyticus; Lane 2: Vibrio alginolyticus; Lane 3: Vibrio vulnificus; Lane 4: Vibrio fluvialis; Lane 5: Bacillus cereus; Lane 6: Escherichia coli CICC 10411; Lane 7: Escherichia coli CICC 10412; Lane 8: Escherichia coli CICC 10662; Lane 9: Escherichia coli CICC 10667; Lane 10: Escherichia coli CICC 24189; Lane 11: Pseudomonas aeruginosa ATCC 15442; Lane 12: Pseudomonas aeruginosa ATCC 27853; Lane 13: Cronobacter sakazakii; Lane 14: Salmonella enteritidis; Lane 15: Salmonella typhimurium; Lane N: Negative control.

[0026] Figure 3 The results show the sensitivity of the Vibrio fluvibacterium VF1 primer pair in the specific implementation method;

[0027] Note: Lane M: DL1200 DNA Marker; Lanes 1-7 increase in value by a gradient of 1.32 × 10⁻⁶. 1 CFU / mL – 1.32 × 10 7 CFU / mL; Lane N: negative control.

[0028] Figure 4 VF2 primer pair sensitivity results for the specific embodiment of Vibrio fluvialis;

[0029] Note: Lane M: DL2000 DNA Marker; Lanes 1-7 in ascending order: 1.32 x 10 1 CFU / mL - 1.32 x 10 7 CFU / mL; Lane N: Negative control.

[0030] Figure 5 VF3 primer pair sensitivity results for the specific embodiment of Vibrio fluvialis;

[0031] Note: Lane M: DL2000 DNA Marker; Lanes 1-7 in ascending order: 1.32 x 10 1 CFU / mL - 1.32 x 10 7 CFU / mL; Lane N: Negative control.

[0032] Figure 6 VF4 primer pair sensitivity results for the specific embodiment of Vibrio fluvialis;

[0033] Note: Lane M: DL2000 DNA Marker; Lanes 1-7 in ascending order: 1.32 x 10 1 CFU / mL - 1.32 x 10 7 CFU / mL; Lane N: Negative control.

[0034] Figure 7 VF1 primer pair specificity results for the specific embodiment of Vibrio fluvialis;

[0035] Note: Lane M: DL2000 DNA Marker; Lane 1 is Vibrio parahaemolyticus; Lane 2 is Vibrio alginolyticus; Lane 3 is Vibrio vulnificus; Lane 4 is Vibrio fluvialis; Lane 5 is Bacillus cereus; Lane 6 is Escherichia coli CICC 10411; Lane 7 is Escherichia coli CICC 10412; Lane 8 is Escherichia coli CICC 10662; Lane 9 is Escherichia coli CICC 10667; Lane 10 is Escherichia coli CICC 24189; Lane 11 is Pseudomonas aeruginosa ATCC 15442; Lane 12 is Pseudomonas aeruginosa ATCC 27853; Lane 13 is Cronobacter sakazakii; Lane 14 is Salmonella enteritidis; Lane 15 is Salmonella typhimurium; Lane N: Negative control;

[0036] Figure 8 VF2 primer pair specificity results for the specific embodiment of Vibrio fluvialis;

[0037] Note: Lane M: DL2000 DNA Marker; Lane 1: Vibrio parahaemolyticus; Lane 2: Vibrio alginolyticus; Lane 3: Vibrio vulnificus; Lane 4: Vibrio fluvialis; Lane 5: Bacillus cereus; Lane 6: Escherichia coli CICC 10411; Lane 7: Escherichia coli CICC 10412; Lane 8: Escherichia coli CICC 10662; Lane 9: Escherichia coli CICC 10667; Lane 10: Escherichia coli CICC 24189; Lane 11: Pseudomonas aeruginosa ATCC 15442; Lane 12: Pseudomonas aeruginosa ATCC 27853; Lane 13: Cronobacter sakazakii; Lane 14: Salmonella enteritidis; Lane 15: Salmonella typhimurium; Lane N: Negative control.

[0038] Figure 9 VF3 primer pair specificity results in the specific embodiment of Vibrio fluvialis;

[0039] Note: Lane M: DL2000 DNA Marker; Lane 1: Vibrio parahaemolyticus; Lane 2: Vibrio alginolyticus; Lane 3: Vibrio vulnificus; Lane 4: Vibrio fluvialis; Lane 5: Bacillus cereus; Lane 6: Escherichia coli CICC 10411; Lane 7: Escherichia coli CICC 10412; Lane 8: Escherichia coli CICC 10662; Lane 9: Escherichia coli CICC 10667; Lane 10: Escherichia coli CICC 24189; Lane 11: Pseudomonas aeruginosa ATCC 15442; Lane 12: Pseudomonas aeruginosa ATCC 27853; Lane 13: Cronobacter sakazakii; Lane 14: Salmonella enteritidis; Lane 15: Salmonella typhimurium; Lane N: Negative control.

[0040] Figure 10 VF4 primer pair specificity results in the specific embodiment of Vibrio fluvialis;

[0041] Note: Lane M: DL2000 DNA Marker; Lane 1: Vibrio parahaemolyticus; Lane 2: Vibrio alginolyticus; Lane 3: Vibrio vulnificus; Lane 4: Vibrio fluvialis; Lane 5: Bacillus cereus; Lane 6: Escherichia coli CICC 10411; Lane 7: Escherichia coli CICC 10412; Lane 8: Escherichia coli CICC 10662; Lane 9: Escherichia coli CICC 10667; Lane 10: Escherichia coli CICC 24189; Lane 11: Pseudomonas aeruginosa ATCC 15442; Lane 12: Pseudomonas aeruginosa ATCC 27853; Lane 13: Cronobacter sakazakii; Lane 14: Salmonella enteritidis; Lane 15: Salmonella typhimurium; Lane N: Negative control. DETAILED DESCRIPTION

[0042] (1) Vibrio alginolyticus and Vibrio fluvialis specific genes and primer analysis

[0043] A total of 873 Vibrio and non-Vibrio genome sequences were downloaded from the NCBI (https: / / www.ncbi.nlm.nih.gov / genome) database. The qualified genomes were annotated by the annotation software Prokka, and then analyzed by Roary. The genes that exist in more than 95% of all Vibrio alginolyticus strain genome sequences and do not exist (or exist in less than 5%) in other non-target strain genome sequences were selected as Vibrio alginolyticus candidate target genes; similarly, the genes that exist in more than 95% of all Vibrio fluvialis strain genome sequences and do not exist (or exist in less than 5%) in other non-target strain genome sequences were selected as Vibrio fluvialis candidate target genes. After obtaining these target genes, BLAST comparison was performed through the NCBI database for further screening, and finally the specific target gene sequences of Vibrio alginolyticus and Vibrio fluvialis were obtained.

[0044] (2) According to the specific target genes of Vibrio alginolyticus and Vibrio fluvialis screened in the early stage, primers were designed, and the designed primers were subjected to BLAST comparison on the NCBI website to ensure primer specificity (Tables 1 and 2).

[0045] (3) Bacterial culture and DNA extraction

[0046] Vibrio alginolyticus and Vibrio fluvialis were used as detection target bacteria, inoculated in 2216E liquid medium and cultured at 37°C, gradient diluted with sterile saline, and plated on 2216E solid medium for plate counting. Vibrio parahaemolyticus, Vibrio alginolyticus, Vibrio vulnificus, Vibrio fluvialis, Escherichia coli, Cronobacter sakazakii, Salmonella strains and other strains were used for primer specificity verification. The Vibrio was inoculated in 2216E liquid medium, and the non-Vibrio was inoculated in LB liquid medium, and cultured at 37°C to the logarithmic growth phase. The Nuaidan DNA extraction kit was used for bacterial liquid DNA extraction, which was used as the subsequent PCR amplification template, and stored at -20°C for a long time.

[0047] (4) Amplification system and amplification conditions

[0048] The PCR system was 25 microliters, including 12.5 µL 2× PCR Mix, 2 µL template DNA, and 10 µM upper and lower primers. The PCR amplification program was 95 ℃ pre-denaturation for 3 min; 95 ℃ denaturation for 15 s, 60 ℃ annealing for 15 s, 72 ℃ extension for 30 s, a total of 35 cycles, 72 ℃ extension for 5 min, and 4 ℃ holding.

[0049] (5) Primer sensitivity verification

[0050] Vibrio alginolyticus and Vibrio fluvialis cultured to the logarithmic growth phase were serially diluted with sterile physiological saline to obtain different concentrations of bacterial solutions for DNA extraction. The DNA at different concentrations was used as templates for PCR amplification, and the amplification efficiency of different primers was investigated. Figure 1 , Figures 3-6 ).like Figure 1 The image shows the results of primer sensitivity verification for Vibrio alginolyticus. The designed primer sequences were successfully applied to the genomic DNA of Vibrio alginolyticus ATCC 33787 (4 × 10⁻⁶). 1 CFU / mL – 4 × 10 8 Amplification was performed using CFU / mL at a bacterial concentration of 4 × 10⁻⁶. 3 CFU / mL – 4 × 10 8 An amplification band appeared when the CFU / mL concentration was lower than 4 × 10⁻⁶. 3 No target band appeared at CFU / mL; therefore, the detection limit for primer VA1 is 4 × 10⁻⁶. 3 CFU / mL.

[0051] like Figures 3-6 These are four groups of specific primer sequences for Vibrio fluvialis selected. As can be seen from the PCR gel run results, the selected primer sequences can successfully target the genomic DNA of Vibrio fluvialis ATCC 33809 (1.32 × 10⁻⁶). 1 – 1.32×10 7 Amplification was performed using CFU / mL, and the detection limit of primer VF1 was 1.32 × 10⁻⁶. 3 CFU / mL, the detection limit of primer VF2 is 1.32 × 10⁻⁶. 3 CFU / mL, the detection limit of primer VF3 is 1.32 × 10⁻⁶. 3 CFU / mL, the detection limit of primer VF4 is 1.32 × 10⁻⁶. 3 CFU / mL.

[0052] (6) Primer specificity verification

[0053] Vibrio parahaemolyticus, Vibrio alginolyticus, Vibrio vulnificus, Vibrio fluvialis, Escherichia coli, Cronobacter sakazakii, and Salmonella were cultured at 37°C to the logarithmic growth phase. The cultures were diluted with sterile physiological saline, and DNA from different bacterial cultures was extracted and used as templates for PCR amplification. The specificity of the primers was investigated. Figure 2 , Figures 7-10 ).

[0054]

[0055]

[0056]

[0057] like Figure 2 As shown, this is the result of the specificity verification of the Vibrio alginolyticus VA1 primers. Except for the target strain, no bands were found in the other non-target strains, which proves that the designed Vibrio alginolyticus VA1 primers not only have good sensitivity, but also good specificity.

[0058] like Figures 7-10 The specificity of four primer sets for *Vibrio fluvialis* was validated. Primer VF1 amplified some large fragments (fragment size greater than 1000 bp) when amplifying non-target bacteria, but only a specific fragment (145 bp) appeared in the target bacteria, while no amplification bands were observed in other non-target bacteria. Therefore, primer VF1 has good specificity. In the specificity detection of primers VF2, VF3, and VF4, only the specific fragment appeared in the target bacteria (the target fragment size was 130 bp in VF2, 286 bp in VF3, and 282 bp in VF4), while no amplification bands appeared in non-target bacteria. This proves that the designed primers VF2, VF3, and VF4 all have strong specificity and can be used for the detection of *Vibrio fluvialis*.

Claims

1. A set of primers for detecting a specific molecular target, characterized in that, The specific molecular target is a specific molecular target for detecting Vibrio alginolyticus and Vibrio fluvialis, the nucleotide sequence of the specific molecular target of Vibrio alginolyticus is as shown in SEQ ID NO. 1, and the nucleotide sequence of the specific molecular target of Vibrio fluvialis is as shown in SEQ ID NO. 2, SEQ ID NO. 3, SEQ ID NO. 4 and SEQ ID NO. 5; The nucleotide sequence of the upstream primer of the specific molecular target SEQ ID NO. 1 is as shown in SEQ ID NO. 6, and the nucleotide sequence of the downstream primer is as shown in SEQ ID NO. 7; The nucleotide sequence of the upstream primer of the specific molecular target SEQ ID NO. 2 is as shown in SEQ ID NO. 8, and the nucleotide sequence of the downstream primer is as shown in SEQ ID NO. 9; The nucleotide sequence of the upstream primer of the specific molecular target SEQ ID NO. 3 is as shown in SEQ ID NO. 10, and the nucleotide sequence of the downstream primer is as shown in SEQ ID NO. 11; The nucleotide sequence of the upstream primer of the specific molecular target SEQ ID NO. 4 is as shown in SEQ ID NO. 12, and the nucleotide sequence of the downstream primer is as shown in SEQ ID NO. 13; The nucleotide sequence of the upstream primer of the specific molecular target SEQ ID NO. 5 is as shown in SEQ ID NO. 14, and the nucleotide sequence of the downstream primer is as shown in SEQ ID NO.

15.

2. A kit for detecting Vibrio alginolyticus and Vibrio fluvialis, characterized by, The primer as claimed in claim 1 is included.

3. A method for detecting Vibrio alginolyticus and Vibrio fluvialis for non-diagnostic therapeutic purposes, characterized by, The steps include: (1) extracting the DNA of the microorganism to be detected, using the extracted DNA as a template, and using the primer as claimed in claim 1 to perform PCR amplification; (2) performing agarose gel electrophoresis analysis on the amplification product, and if a band of target size 155 bp appears in the electrophoresis result, it is determined that the sample contains Vibrio alginolyticus, and if a band of target size 155 bp does not appear in the electrophoresis result, it is determined that the sample does not contain Vibrio alginolyticus; if a band of target size 145 bp, 130 bp, 286 bp or 282 bp appears in the electrophoresis result, it is determined that the sample contains Vibrio fluvialis, and if a band of target size 145 bp, 130 bp, 286 bp or 282 bp does not appear in the electrophoresis result, it is determined that the sample does not contain Vibrio fluvialis.

4. The method for detecting Vibrio alginolyticus and Vibrio fluvialis according to claim 3, wherein The reaction system of the PCR amplification is 20 microliters, including 10 μL 2× PCR Mix, 2 μL template DNA, 10 μM upstream and downstream primers.

5. The method for detecting Vibrio alginolyticus and Vibrio fluvialis according to claim 3, wherein The reaction conditions of the PCR amplification are 95 ℃ pre-denaturation for 3 min; 95 ℃ denaturation for 15 s, 60 ℃ annealing for 15 s, 72 ℃ extension for 30 s, a total of 35 cycles, 72 ℃ extension for 5 min, and 4 ℃ keeping.

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