Specific molecular targets of Vibrio vulnificus and their detection methods

Abstract

The present invention discloses specific molecular targets of Vibrio vulnificus and a detection method thereof. The nucleotide sequences of the specific molecular targets are as shown in SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3 or SEQ ID NO.4. The present invention not only discloses four specific molecular targets for identifying Vibrio vulnificus, but also provides related specific primer sequences and corresponding PCR detection methods. The present invention does not require physiological and biochemical identification, and has the advantages of short detection time, low cost, strong specificity, etc.

Description

Technical Field

[0001] The present invention relates to the technology of bacterial detection, and more particularly to a specific molecular target of Vibrio vulnificus and a detection method thereof. Background Art

[0002] Vibrio vulnificus is a halophilic Gram-negative pathogenic marine bacterium with a cell size of 0.7×2 - 3.5 μm. It grows in seawater or salt lakes with a growth temperature of 15°C - 27°C and a salinity of 0.7% - 1.6%, and is often present in warm coastal areas. Summer is the peak period of Vibrio vulnificus infection, especially in areas with subtropical monsoon climate where the proliferation of Vibrioaceae is particularly likely to occur. This bacterium infects human hosts by consuming contaminated seafood, direct contact of wounds with contaminated water or seafood, resulting in septicemia, severe wound infection and gastroenteritis. Although the infection rate is low, failure to promptly identify and treat this infection can lead to high morbidity and mortality. Vibrio vulnificus often survives on plankton and shellfish, such as oysters. If a person sensitive to this bacterium ingests it in raw oysters, the mortality rate can reach about 60%. With global warming, Vibrio vulnificus grows continuously in freshwater environments, and terrestrial animals, plants and insects may come into contact with Vibrio vulnificus and may become disease vectors. Therefore, we need accurate detection means to reduce the occurrence of food poisoning incidents caused by Vibrio vulnificus.

[0003] Currently, for the detection of Vibrio vulnificus, the traditional detection method is the plate culture method. It has advantages such as low cost and strong specificity, but at the same time, the plate culture counting method also has some disadvantages, such as long culture time, cumbersome operation, and relatively strict requirements for operators and the operation process. To make up for the deficiencies of the plate detection method, molecular detection methods based on nucleic acid amplification have been applied to the detection of Vibrio vulnificus in food.

[0004] The specificity and sensitivity of molecular detection methods based on nucleic acid amplification mainly depend on the selection of corresponding detection targets. Currently, the PCR detection method is one of the most commonly used molecular detection methods, and its primers are designed according to the specific conserved sequences (detection targets) of the target bacterial genomic DNA. Therefore, the detection targets and primers are crucial for the accuracy and sensitivity of PCR detection.

[0005] In the process of using PCR technology to detect Vibrio vulnificus, many research institutions and scholars still use the genes discovered earlier as the detection targets for Vibrio vulnificus, such as 16S rDNA and vvhA genes. However, current relevant research has proven that the nucleic acid sequence of 16S rDNA has a consistency as high as 96% with Vibrio navarrensis ( Vibrio navarrensis ), and also has a nucleic acid sequence similarity of more than 90% with 33 other Vibrio strains; and vvhAAs a specific gene of Vibrio vulnificus, some researchers have also found that when vvhA gene is used as a target for PCR detection of Vibrio vulnificus, for vvhA gene mutants with the same pathogenic ability, they cannot be detected, indicating that the existing specific detection targets for Vibrio vulnificus currently cannot meet the specificity requirements of nucleic acid detection technology.

[0006] Therefore, there is an urgent need to systematically and comprehensively explore new specific detection targets to improve the accuracy of the PCR detection technology for Vibrio vulnificus. Summary of the Invention

[0007] The purpose of the present invention is to overcome the deficiencies of the prior art. By using pan-genomics technology, a comprehensive analysis of the core genes and accessory genes of Vibrio vulnificus is carried out, and then BLAST analysis is performed on the core gene part to further lock the specific gene region in the core gene, so as to provide a specific molecular target for Vibrio vulnificus and its detection method.

[0008] To achieve the above purpose, the present invention adopts the following technical solutions:

[0009] A specific molecular target for detecting Vibrio vulnificus, the nucleotide sequence of the specific molecular target is as shown in SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3 or SEQ ID NO.4.

[0010] The present invention also provides a set of primers for detecting the above specific molecular target. The nucleotide sequence of the upstream primer of the specific molecular target SEQ ID NO.1 is as shown in SEQ ID NO.5, and the nucleotide sequence of the downstream primer is as shown in SEQ ID NO.6;

[0011] The nucleotide sequence of the upstream primer of the specific molecular target SEQ ID NO.2 is as shown in SEQ ID NO.7, and the nucleotide sequence of the downstream primer is as shown in SEQ ID NO.8;

[0012] The nucleotide sequence of the upstream primer of the specific molecular target SEQ ID NO.3 is as shown in SEQ ID NO.9, and the nucleotide sequence of the downstream primer is as shown in SEQ ID NO.10;

[0013] The nucleotide sequence of the upstream primer of the specific molecular target SEQ ID NO.4 is as shown in SEQ ID NO.11, and the nucleotide sequence of the downstream primer is as shown in SEQ ID NO.12;

[0014] The present invention also provides the application of the above molecular target in the preparation of reagents for detecting Vibrio vulnificus.

[0015] The present invention also provides a kit for detecting Vibrio vulnificus, which comprises all of the above nucleotide sequences.

[0016] The present invention also provides a method for detecting Vibrio vulnificus for non-disease diagnosis and treatment purposes, comprising the following steps:

[0017] (1) Extract the DNA of the microorganism to be detected, and use the extracted DNA as a template to perform PCR amplification on it with the above primers.

[0018] (2) Perform agarose gel electrophoresis analysis on the amplification product. If a band of the target size (154bp, 288bp, 341bp or 427bp) appears in the electrophoresis result, it is determined that the sample contains Vibrio vulnificus. If no band of the target size (154bp, 288bp, 341bp or 427bp) appears in the electrophoresis result, it is determined that the sample does not contain Vibrio vulnificus.

[0019] Preferably, in the above method for detecting Vibrio vulnificus, the reaction system for PCR amplification is 20 μL, which includes 10 μL of 2× PCR Mix, 2 μL of template DNA, and upstream and downstream primers at 10 μM.

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

[0021] Compared with the prior art, the present invention has the following beneficial effects. The present invention discloses four specific molecular targets for identifying Vibrio vulnificus, provides relevant specific primer sequences, and corresponding PCR detection methods. The present invention does not require physiological and biochemical identification, and has the advantages of short detection time, low cost, and strong specificity. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 It is a verification diagram of the sensitivity result of the primer pair of Vibrio vulnificus VV-1 in the specific embodiment.

[0023] Note: Lane M: DL1200 DNA Marker; Lanes 1-7 increase in gradient: 1.94 × 10 1 – 1.94 ×10 7 CFU / mL; Lane N: negative control;

[0024] Figure 2 It is a verification diagram of the sensitivity result of the primer pair of Vibrio vulnificus VV-2 in the specific embodiment.

[0025] Note: Lane M: DL1200 DNA Marker; Lanes 1-7 are in increasing gradient: 1.94 × 10 1 – 1.94 ×10 7 CFU / mL; Lane N: Negative control;

[0026] Figure 3 It is the verification diagram of the sensitivity result of Vibrio vulnificus VV-3 primer pair in the specific implementation manner.

[0027] Note: Lane M: DL1200 DNA Marker; Lanes 1-7 are in increasing gradient: 1.94 × 10 1 – 1.94 ×10 7 CFU / mL; Lane N: Negative control;

[0028] Figure 4 It is the verification diagram of the sensitivity result of Vibrio vulnificus VV-4 primer pair in the specific implementation manner.

[0029] Note: Lane M: DL1200 DNA Marker; Lanes 1-7 are in increasing gradient: 1.94 × 10 1 – 1.94 ×10 7 CFU / mL; Lane N: Negative control;

[0030] Figure 5 It is the verification diagram of the specificity result of Vibrio vulnificus VV-1 primer pair in the specific implementation manner.

[0031] 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.

[0032] Figure 6 It is the verification diagram of the specificity result of Vibrio vulnificus VV-2 primer pair in the specific implementation manner.

[0033] 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.

[0034] Figure 7 It is the verification diagram of the specificity result of the *Vibrio vulnificus* VV-3 primer pair in the specific implementation manner.

[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 It is the verification diagram of the specificity result of the *Vibrio vulnificus* VV-4 primer pair in the specific implementation manner.

[0037] 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. Specific implementation manner

[0038] (1)Analysis of Vibrio vulnificus specific genes and primers

[0039] A total of 873 genomic sequences of Vibrio and non-Vibrio were downloaded from the NCBI (https: / / www.ncbi.nlm.nih.gov / genome) database. After annotation of all genomic sequences by Prokka, Roary was used for analysis. Genes that were present in more than 95% of the genomic sequences of all Vibrio vulnificus strains and absent (or present in less than 5%) in the genomic sequences of other non-target strains were selected as candidate target genes. After obtaining these target genes, BLAST alignment was performed through the NCBI database for further screening, and finally, specific molecular targets of Vibrio vulnificus were obtained, such as SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3, or SEQ ID NO.4. Specific primer sequences for Vibrio vulnificus were obtained according to different molecular target genes (Table 1).

[0040]

[0041] (2)Strain culture and DNA extraction

[0042] Using the standard strain of Vibrio vulnificus ATCC 27562 as the target bacterium for detection, it was inoculated into 2216E liquid medium and subcultured at 37 °C, serially diluted with sterile normal saline, and spread onto 2216E solid medium for plate counting. Strains such as Vibrio parahaemolyticus, Vibrio alginolyticus, Vibrio vulnificus, Vibrio fluvialis, Escherichia coli, Cronobacter sakazakii, and Salmonella were used for primer specificity verification. Vibrio was inoculated into 2216E liquid medium, and non-Vibrio was inoculated into LB liquid medium, and cultured at 37 °C until the logarithmic growth phase. A DNA extraction kit was used to extract the bacterial DNA, which was used as the template for subsequent PCR amplification and stored at -20 °C for long-term storage.

[0043] (3)Amplification system and amplification conditions

[0044] The PCR system was 20 μL, including 10 µL 2× PCR Mix, 2 µL template DNA, and 10 µM upstream and downstream primers. The PCR amplification program was pre-denaturation at 95 °C for 3 min; denaturation at 95 °C for 15 s, annealing at 60 °C for 15 s, extension at 72 °C for 30 s, for a total of 35 cycles, extension at 72 °C for 5 min, and hold at 4 °C.

[0045] (4)Verification of primer sensitivity

[0046] The bacterial liquid of Vibrio vulnificus cultured to the logarithmic growth phase was serially diluted with sterile normal saline to obtain bacterial liquids with different concentrations (1.94×101 CFU / mL – 1.94×10 7 CFU / mL), DNA extraction was carried out. DNA at different concentrations was used as a template for PCR amplification to investigate the amplification efficiency of different primers.

[0047] Such as Figures 1-4 , among the four pairs of Vibrio vulnificus primer sequences screened, it can be seen from the PCR gel electrophoresis result diagram that the screened primer sequences can successfully amplify the genomic DNA of Vibrio vulnificus ATCC 27562 (1.94 × 10 1 CFU / mL – 1.94 × 10 7 CFU / mL). The detection limit of primer VV1 is 1.94 × 10 3 CFU / mL, the detection limit of primer VV2 is 1.94 × 10 3 CFU / mL, the detection limit of primer VV3 is 1.94×10 2 CFU / mL, and the detection limit of primer VV4 is 1.94×10 2 CFU / mL.

[0048] (5) Specificity verification

[0049] Strains such as Vibrio vulnificus, Vibrio alginolyticus, Vibrio parahaemolyticus, Vibrio fluvialis, Escherichia coli, Cronobacter sakazakii, Salmonella, etc. were used for primer specificity verification (as shown in Table 2). Vibrio strains were inoculated into 2216E liquid medium, and non-Vibrio strains were inoculated into LB liquid medium and cultured at 37°C until the logarithmic growth phase. The bacterial liquid was diluted with sterile normal saline, and DNA of different bacterial liquids was extracted using a kit as a template for subsequent PCR verification. The results are as Figures 5-8 shown. The specificity of the four groups of primers for Vibrio vulnificus was verified. When primers VV1 and VV2 amplified non-target bacteria, some large fragments were amplified (band size > 1000 bp), but specific-sized fragments only appeared in the target bacteria (the target fragment size in VV1 was 154 bp, and the target fragment size in VV2 was 288 bp), and they did not appear in other non-target bacteria. Therefore, primers VV1 and VV2 have good specificity. In the specificity detection of primers VV3 and VV4, specific fragments only appeared in the target bacteria (the target fragment size in VV3 was 341 bp, and the target fragment size in VV4 was 427 bp), and no amplification bands appeared in non-target bacteria, proving that the involved primers VV3 and VV4 both have high specificity and can be used for the detection of Vibrio vulnificus.

[0050] Table 2 Strains used for the specificity evaluation of Vibrio vulnificus primers of the present invention

[0051] .

Claims

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

12.

2. A method for detecting Vibrio vulnificus for non-diagnostic and therapeutic purposes, characterized in that, It includes the following steps: (1) Extract the DNA of the microorganism to be detected. Using the extracted DNA as a template, perform PCR amplification on it with the primer pair described in claim 1; (2) Perform agarose gel electrophoresis analysis on the amplification product. If a band with a target size of 154bp, 288bp, 341bp or 427bp appears in the electrophoresis result, it is determined that Vibrio vulnificus is contained in the sample. If no band with a target size of 154bp, 288bp, 341bp or 427bp appears in the electrophoresis result, it is determined that Vibrio vulnificus is not contained in the sample.

3. The method for detecting Vibrio vulnificus according to claim 2, wherein The reaction system of the PCR amplification is 20 μL, which includes 10 µL 2× PCR Mix, 2µL template DNA, and upstream and downstream primers at 10µM.

4. The method for detecting Vibrio vulnificus according to claim 2, wherein The reaction conditions of the PCR amplification are pre-denaturation at 95°C for 3 min; denaturation at 95°C for 15 s, annealing at 60°C for 15 s, extension at 72°C for 30 s, for a total of 35 cycles, extension at 72°C for 5 min, and hold at 4°C.

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