A method for specific detection of Vibrio tectorum and its application

Abstract

The application discloses a method for specifically detecting Vibrio vulnificus and application. The application takes the recA gene of Vibrio vulnificus as a target, designs a primer probe group capable of specifically detecting Vibrio vulnificus, and the detection sensitivity can reach 0.01 fg / ul. In addition, the application can distinguish Vibrio vulnificus from its similar species, Vibrio tamarinicus and Vibrio anguillarum, in the recA gene, has good specificity and repeatability. The application can provide an effective technical means for disease detection and monitoring of Vibrio vulnificus in the process of aquatic animal breeding, and has a wide application prospect in aquaculture.

Description

Technical Field

[0001] This invention relates to the technical field of aquatic animal disease detection, specifically to a method and application for the specific detection of Vibrio tectorum. Background Technology

[0002] Vibrio neptunius is a marine bacterium that has been isolated from seawater samples and marine organisms. It is a pathogenic bacterium in mollusks such as oysters and clams, and is also considered one of the main pathogens of bacterial enteritis in seahorses. This bacterium can cause disease outbreaks, seriously affecting the healthy development of aquaculture. Currently, there is very little research on detection methods for Vibrio neptunius.

[0003] Molecular biology techniques based on DNA sequencing have been applied to the detection and identification of Vibrio. Bacterial 16S rRNA is highly conserved and universal, making it a relatively ideal gene target sequence for microbial identification. However, many different microbial species share 16S rRNA similarities exceeding 96%. For Vibrio species, studies have shown that the 16S rRNA sequence similarity of many Vibrio species reaches over 99%, such as *Vibrio alginolyticus* and *Vibrio tectorum*, which have a 100% similarity. Therefore, there is a need to find new molecular targets for Vibrio identification.

[0004] Quantitative real-time PCR (qPCR) is a method that adds fluorescent dyes or probes to conventional PCR techniques. It utilizes the accumulation of fluorescence signals to monitor the entire PCR process in real time and uses a standard curve to quantify templates of unknown concentrations. As an internationally recognized standard method for nucleic acid quantification, qPCR is the preferred method for rapid detection of gene expression levels. It not only represents a leap from qualitative to quantitative PCR but also offers higher sensitivity, stronger specificity, better repeatability, and a higher degree of automation compared to conventional PCR.

[0005] Currently, there is no suitable method for detecting Vibrio tectorum in aquaculture sites. Therefore, it is of great significance to develop a rapid and reliable method for identifying Vibrio tectorum. Summary of the Invention

[0006] To overcome the aforementioned defects and deficiencies in the existing technology, the present invention provides a method and application for the specific detection of Vibrio tectorum.

[0007] The first objective of this invention is to provide a primer and probe set for detecting Vibrio tectorum.

[0008] A second objective of this invention is to provide the application of the above-described primer-probe set in the preparation of products for detecting Vibrio tectorum.

[0009] A third object of the present invention is to provide a composition for detecting Vibrio tectorum.

[0010] A fourth object of the present invention is to provide the use of the above-described composition in the preparation of products for detecting Vibrio tectorum.

[0011] The fifth object of the present invention is to provide a kit for detecting Vibrio tectorum.

[0012] The sixth objective of this invention is to provide a method for detecting Vibrio tectorum.

[0013] This invention claims protection for the following:

[0014] A primer-probe set for detecting Vibrio tectorum, the primer-probe set comprising a forward primer F1, a reverse primer R1, and a fluorescent probe P1, wherein the nucleotide sequence of the forward primer F1 is shown in SEQ ID NO: 1, the nucleotide sequence of the reverse primer R1 is shown in SEQ ID NO: 2, and the nucleotide sequence of the fluorescent probe P1 is shown in SEQ ID NO: 3.

[0015] Existing technology CN109266724A discloses that the recA gene is a powerful molecular marker for identifying Vibrio species, but it cannot distinguish between *Vibrio neptunius*, *Vibrio corallilyticus*, and *Vibrio anguillarum* using the recA gene. Furthermore, existing technology (10.1099 / ijs.0.02963-0) also discloses that *V. corallilyticus* and *Vibrio neptunius* share 96% similarity in the recA gene. However, the results of this invention demonstrate that the primer-probe set designed for the recA gene in this invention can distinguish *Vibrio neptunius* from *V. corallilyticus* and *Vibrio anguillarum*, exhibiting good specificity.

[0016] As one possible implementation, the fluorescent probe P1 is labeled with the fluorescent group FAM at its 5' end and the quenching group MGB at its 3' end.

[0017] The above-mentioned primer-probe set is used in the preparation of products for detecting Vibrio tectorum.

[0018] A composition for detecting Vibrio tectorum, the composition comprising the primer and probe set described above.

[0019] The above composition is used in the preparation of products for detecting Vibrio tectorum.

[0020] A kit for detecting Vibrio tectorum, the kit containing the aforementioned primer and probe set.

[0021] Preferably, the kit further comprises a positive control reference and / or a negative control reference.

[0022] Preferably, the positive control reference is plasmid DNA containing the target gene for Vibrio tectorum detection, and the negative control reference is ddH2O;

[0023] The target gene for detection is the recA gene sequence of Vibrio tectorum.

[0024] Preferably, the kit further includes a real-time PCR detection reagent.

[0025] A method for detecting Vibrio tectorum, using the above-mentioned kit to detect a sample, wherein the detection is for non-disease treatment diagnosis purposes.

[0026] Compared with the prior art, the present invention has the following beneficial effects:

[0027] This invention discloses a method for specifically detecting Vibrio tectorum and its applications. Based on the recA gene of Vibrio tectorum as a target, this invention designs a primer-probe set capable of specifically detecting Vibrio tectorum, with a detection sensitivity reaching 0.01 fg / μL. Furthermore, this invention can also distinguish Vibrio tectorum from Vibrio corallobe and Vibrio anguillarum, which have high similarity in the recA gene, demonstrating good specificity and reproducibility. This invention can provide an effective technical means for the detection and monitoring of Vibrio tectorum diseases in aquatic animal farming, and has broad application prospects in aquaculture. Attached Figure Description

[0028] Figure 1 This shows the amplification results of different Vibrio tectorum primer and probe sets in Example 1.

[0029] Figure 2 For the specific detection of Vibrio tectorum primer and probe set, the negative controls are: Vibrio alginolyticus, Vibrio parahaemolyticus, Vibrio harveyi, Vibrio fluvibrio, Vibrio cholerae, Vibrio Sinaloa, Vibrio anguillarum, Vibrio lysozyme, Vibrio virulentii, Vibrio nigra, Flavobacterium columnare, Pseudomonas fluorescens, and Aeromonas guinea pig; the blank control is ddH2O.

[0030] Figure 3 The results show the sensitivity of the Vibrio tectorum primer and probe set.

[0031] Figure 4 This is the result of the negative stability test of the Vibrio tectorum primer and probe set.

[0032] Figure 5 The results show the detection of Vibrio tectorum in 24 actual samples. Detailed Implementation

[0033] The present invention will be further illustrated below with reference to specific embodiments, but the embodiments do not limit the present invention in any way. Unless otherwise specified, the reagents, methods, and equipment used in the present invention are conventional reagents, methods, and equipment in this technical field.

[0034] Unless otherwise specified, all reagents and materials used in the following examples are commercially available.

[0035] Example 1: Effect of primer set on the detection of Vibrio tectorum

[0036] I. Experimental Methods

[0037] 1. Plasmid DNA Synthesis

[0038] The gene sequence used for primer design of Vibrio tectorum was ligated into the pUC57 vector to construct a plasmid.

[0039] 2. Design primers

[0040] The whole genome sequence of Vibrio tectorum was searched in Genbank and compared by BLAST analysis. The recA gene was used as the target gene. Based on the principles of real-time fluorescent PCR primer and probe design, specific primers and probes were designed using Primer Express primer design software.

[0041] Vibrio tectorum primer and probe set 1 (Y1):

[0042] Forward primer VN-1-F: 5'-AAGGCGAGATGGGCGATAG-3' (SEQ ID NO: 1);

[0043] Reverse primer VN-1-R: 5'-ACGCATTGCTTGCGATAGC-3' (SEQ ID NO: 2);

[0044] Fluorescent probe VN-1-P: 5'-ACATGGGTCTTCAAGCG-3' (SEQ ID NO: 3);

[0045] The fluorescent probe VN-1-P is labeled with the fluorescent group FAM at its 5' end and the quencher group MGB at its 3' end.

[0046] Vibrio tectorum primer and probe set 2 (Y2):

[0047] Forward primer VN-2-F: 5'-GGTCTTCAAGCGCGTATGC-3' (SEQ ID NO: 4); Reverse primer VN-2-R: 5'-AGACTGCTTGAGGTTACCCGTTA-3' (SEQ ID NO: 5);

[0048] Fluorescent probe VN-2-P: 5'-TCGCAAGCAATGCGTA-3' (SEQ ID NO: 6);

[0049] The fluorescent probe VN-2-P is labeled with the fluorescent group FAM at its 5' end and the quencher group MGB at its 3' end.

[0050] 3. Perform PCR reaction

[0051] The reaction system is shown in Table 1.

[0052] Table 1 Real-time fluorescence PCR reaction system

[0053] Reagent Name working fluid concentration Sample volume Final concentration Reaction premix 2× 12.5μL 1× upstream primer F 10μM 1μL 0.4μM Downstream primer R 10μM 1μL 0.4μM Fluorescent probe P 10μM 0.5μL 0.2μM DNA template 10–100 ng / μL 5μL - <![CDATA[ddH2O]]> - 5μL -

[0054] The reaction program was as follows: the decontamination program was 50℃ for 5 min, 1 cycle; the honing stage program was 95℃ for 5 min, 1 cycle; the cycling stage was 95℃ for 15 s, 60℃ for 30 s, 45 cycles; the fluorescence signal was collected at 60℃ for 30 s, and the reaction results were observed using a fluorescence PCR instrument.

[0055] II. Experimental Results

[0056] The results are as follows Figure 1 As shown, both primer and probe sets can perform amplification, but primer and probe set 1 has a smaller Ct value and better repeatability. The negative control showed no non-specific amplification. Therefore, primer and probe set 1 was determined to be the optimal primer and probe set.

[0057] Example 2: A kit for detecting Vibrio tectorum in aquatic animals

[0058] I. Composition

[0059] 1. Primer and probe set for detecting Vibrio tectorum

[0060] Forward primer VN-1-F: 5'-AAGGCGAGATGGGCGATAG-3' (SEQ ID NO: 1);

[0061] Reverse primer VN-1-R: 5'-ACGCATTGCTTGCGATAGC-3' (SEQ ID NO: 2);

[0062] Fluorescent probe VN-1-P: 5'-ACATGGGTCTTCAAGCG-3' (SEQ ID NO: 3);

[0063] The fluorescent probe VN-1-P is labeled with the fluorescent group FAM at its 5' end and the quencher group MGB at its 3' end.

[0064] 2. PCR reaction reagents

[0065] PCR reaction premix, positive control reference, negative control reference;

[0066] The positive control reference material was a plasmid containing the recA gene sequence of Vibrio tectorum, and the negative control reference material was ddH2O.

[0067] II. Instructions for Use

[0068] The Vibrio tectorum primer and probe set was used to perform PCR reaction on the DNA of the test sample. The reaction system is shown in Table 2.

[0069] Table 2 Real-time fluorescence PCR reaction system

[0070] Reagent Name working fluid concentration Sample volume Final concentration Reaction premix 2× 12.5μL 1× upstream primer F 10μM 1μL 0.4μM Downstream primer R 10μM 1μL 0.4μM Fluorescent probe P 10μM 0.5μL 0.2μM DNA template 10–100 ng / μL 5μL - <![CDATA[ddH2O]]> - 5μL -

[0071] The reaction program was as follows: the decontamination program was 50℃ for 5 min, 1 cycle; the honing stage program was 95℃ for 5 min, 1 cycle; the cycling stage was 95℃ for 15 s, 60℃ for 30 s, 45 cycles; the fluorescence signal was collected at 60℃ for 30 s, and the reaction results were observed using a fluorescence PCR instrument.

[0072] III. Interpretation Methods

[0073] If the Ct value of the test sample is ≥45 or there is no Ct value, the curve is a straight line or a slightly sloping line, and there is no "S"-shaped amplification curve, it can be determined that the sample does not contain Vibrio tectorum or the content is lower than the detection limit.

[0074] If the Ct value of the test sample is <45 and the curve shows an "S"-shaped amplification curve, it can be determined that the sample contains Vibrio tectorum.

[0075] Example 3 Specificity Experiment

[0076] I. Experimental Methods

[0077] DNA was extracted from each sample using a commercially available DNA extraction kit, and the purity and concentration of the extracted DNA were determined using a full-wavelength micro spectrophotometer. The DNA was then stored at -20°C for later use.

[0078] The samples included: Vibrio alginolyticus, Vibrio parahaemolyticus, Vibrio harveyi, Vibrio fluvialis, Vibrio cholerae, Vibrio Sinaloa, Vibrio anguillarum, Vibrio lysozyme, Vibrio viridissus, Vibrio nigricans, Flavobacterium columnare, Pseudomonas fluorescens, and Aeromonas guinea pig.

[0079] PCR amplification was performed using the kit from Example 2.

[0080] 2. Experimental Results

[0081] The specificity results of the primer and probe set for Vibrio alginolyticus showed that no amplified fluorescent signal was detected in the DNA samples of Vibrio alginolyticus and other aquatic animal diseases. Figure 2 This indicates that the established real-time fluorescence PCR detection method has good specificity.

[0082] Example 4: Sensitivity and Repeatability Experiment

[0083] I. Experimental Methods

[0084] The synthesized plasmid DNA was quantified using a spectrophotometer, yielding a plasmid concentration of 100 ng / μL. The plasmid was then serially diluted, with seven concentrations of DNA (10 pg / μL, 1 pg / μL, 100 fg / μL, 10 fg / μL, 1 fg / μL, 0.1 fg / μL, and 0.01 fg / μL) used as templates for detection using the kit described in Example 2.

[0085] 2. Experimental Results

[0086] The results showed a typical amplification curve, and the detection sensitivity of this method for plasmids containing the recA gene of Vibrio tectorum reached 0.01 fg / μL. Figure 3 The results of three-parallel experiments for each of the seven dilution gradients showed that the coefficients of variation ranged from 0.11% to 2.79%, indicating that the established real-time fluorescence PCR detection method has good reproducibility (Table 3).

[0087] Table 3. Repeatability experiments of primer and probe sets for Vibrio tectoris

[0088]

[0089] Example 5 Negative Stability Test

[0090] I. Experimental Methods

[0091] Using ddH2O as a negative control, the experiment was repeated 20 times, with 3 additional positive control references, to conduct a negative stability test.

[0092] II. Experimental Results

[0093] The results showed that the established real-time fluorescence PCR detection method had good negative stability and no non-specific amplification occurred. Figure 4 ).

[0094] Example 6: Detection of Vibrio tectorum in actual samples

[0095] I. Experimental Methods

[0096] DNA was extracted from 24 actual samples, including 6 oysters, 6 sea cucumbers, 6 seahorses, and 6 sea urchins, and the kit from Example 2 was used to detect whether they were infected with Vibrio tectorum.

[0097] II. Experimental Results

[0098] The DNA from 24 actual samples was tested using the kit from Example 2, and the results are as follows: Figure 5 As shown in the figure, two actual samples tested positive for Vibrio tectorum in DNA, while 22 actual samples tested negative for Vibrio tectorum in DNA.

[0099] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. Use of a primer probe set in the manufacture of a product for detecting Vibrio coralliilyticus in Crassostrea gigas, characterized in that, The primer probe set is composed of a forward primer F1, a reverse primer R1 and a fluorescent probe P1, the nucleotide sequence of the forward primer F1 is shown as SEQ ID NO: 1, the nucleotide sequence of the reverse primer R1 is shown as SEQ ID NO: 2, and the nucleotide sequence of the fluorescent probe P1 is shown as SEQ ID NO: 3, and the application is for non-diagnostic purposes.

2. Use according to claim 1, characterized in that, The 5' end of the fluorescent probe P1 is labeled with a fluorescent group FAM, and the 3' end is labeled with a quenching group MGB.

3. Use of a kit in the manufacture of a product for detecting Vibrio mimicus in Crassostrea gigas, characterized in that, The kit comprises the primer probe set of claim 1 or 2, and the application is for non-diagnostic purposes.

4. Use according to claim 3, characterized in that, The kit further comprises a positive control reference and a negative control reference.

5. Use according to claim 4, characterized in that, The positive control reference is plasmid DNA containing the detection target gene of Vibrio vulnificus, and the negative control reference is ddH2O.

6. Use according to claim 3, characterized in that, The kit further comprises a fluorescent quantitative PCR detection reagent.

7. A method of detecting Vibrio mimicus in Crassostrea gigas, characterized in that, The kit of any one of claims 3-6 is used to detect a sample to be tested, and the method is for non-diagnostic purposes.

Images