CN117265150B - Vibrio alginolyticus quantitative detection method based on microfluidic chip type digital PCR technology - Google Patents

Abstract

The invention discloses a vibrio alginolyticus quantitative detection method based on a microfluidic chip type digital PCR technology, and belongs to the technical field of food safety detection. The novel specific target spot which can be used for detecting the vibrio alginolyticus and is obtained through screening has better specificity compared with the traditional detection target spot. The chip type digital PCR method established based on the self-screening target point N646_0363 has better specificity and sensitivity, the detection sensitivity of the genome DNA is 3.18 fg/mu L, and the detection sensitivity of bacterial liquid is 4.75X10 ¹ CFU/mL can effectively reduce deviation caused by food matrix, and is suitable for quantitative detection of low-concentration targets. The method is convenient to operate, simple in result judgment, free of standard curve, short in detection time to within 2 h, and important in practical application value in the fields of food safety and environmental monitoring.

Description

Vibrio alginolyticus quantitative detection method based on microfluidic chip type digital PCR technology

Technical Field

The invention belongs to the technical field of food safety detection, and relates to a vibrio alginolyticus quantitative detection method based on a microfluidic chip type digital PCR technology.

Background

Vibrio alginolyticusVibrio alginolyticus,Va) Is a halophilic gram-negative bacterium widely distributed in estuaries, oceans and animal aquatic products, and is a main pathogenic bacterium for aquaculture. The bacteria are pathogenic bacteria for human and veterinary co-diseases, can cause ulcer disease, septicemia and the like of various aquatic products such as fishes, shrimps and the like, and cause great loss to the breeding industry. Meanwhile, the strain can cause diarrhea, wound infection, ear infection (otitis media and otitis externa) and the like of human beings, and can cause serious threat to human health. Currently, only vibrio parahaemolyticus (GB 4789.7-2013) and vibrio vulnificus (GB 4789.44-2020) in four common pathogenic vibrio parahaemolyticus, vibrio alginolyticus, vibrio vulnificus and vibrio cholerae have corresponding national standard detection methods, and the whole culture and identification process generally requires 3-6 days.

The nucleic acid molecule detection technology is one of the most important development directions in the field of rapid detection of food-borne pathogenic bacteria, mainly comprises common PCR, fluorescent quantitative PCR, digital PCR and the like, and the accuracy and the specificity of the method mainly depend on the selection of detection target genes. At present, the common vibrio alginolyticus detection target is mainly concentrated on collagenase gene #colH) Stress regulating generpoX) Coding gene of transmembrane transcription regulating factortoxR) Molecular chaperone genegroEL) DNA gyrase B subunitBasic coding gene [ ]gyrB) And the like, and the specificity of part of traditional target genes is not strong in the actual detection process, so that the accuracy of a nucleic acid detection result is reduced, and the advantages of a PCR rapid detection technology cannot be fully exerted. Aiming at vibrio alginolyticus industry standard SN/T2754.12-2011, a loop-mediated isothermal amplification detection method is provided, and collagenase genes are selected as detection targets; SN/T5364.3-2021 prescribes a microdroplet digital PCR detection method, which selectsgroELThe gene is used as a detection target. Digital PCR is called a "third generation PCR technique", an absolute quantitative analysis technique for endpoint detection, with higher tolerance, sensitivity and accuracy than fluorescent quantitative PCR. The technology is divided into two types of micro-droplet type and chip type, and compared with micro-droplet type digital PCR, the chip type digital PCR has the advantages of more convenient operation, lower pollution risk, shorter detection time, lower cost and the like. At present, the research on the specific target of vibrio alginolyticus is less, so that the establishment of the microfluidic chip type digital PCR detection technology based on the novel self-screening and high-specificity recognition molecular target has important significance.

Disclosure of Invention

In view of the shortcomings of the prior art, the first object of the present invention is the use of any one of the following 1) -8) gene fragments as specific targets in the detection or identification of Vibrio alginolyticus, which is not aimed at diagnosis of disease: 1) N646_0350; 2) N646_0363; 3) N646_0367; 4) N646_0380; 5) N646_0458; 6) N646_0782; 7) N646_1442; 8) N646_4545, wherein,

the nucleotide sequence of the N646-0350 is shown as SEQ ID NO. 1;

the nucleotide sequence of the N646_0363 is shown in SEQ ID NO. 2;

the nucleotide sequence of the N646_0367 is shown in SEQ ID NO. 3;

the nucleotide sequence of the N646_0380 is shown in SEQ ID NO. 4;

the nucleotide sequence of the N646_0458 is shown in SEQ ID NO. 5;

the nucleotide sequence of the N646_0782 is shown in SEQ ID NO. 6;

the nucleotide sequence of the N646-1442 is shown in SEQ ID NO. 7;

the nucleotide sequence of the N646_4545 is shown in SEQ ID NO. 8.

In one embodiment, the use comprises preparing a reagent or kit for detecting or identifying Vibrio alginolyticus.

In one embodiment, the reagent comprises a primer, probe, antibody or aptamer.

A second object of the present invention is to provide a composition for detecting Vibrio alginolyticus, comprising an upstream primer, a downstream primer and a probe of target N646_0363; the nucleotide sequence of the upstream primer is shown as SEQ ID NO. 9, the nucleotide sequence of the downstream primer is shown as SEQ ID NO. 10, and the nucleotide sequence of the probe is shown as SEQ ID NO. 11.

In one embodiment, the 5 'end of the probe is modified with a fluorescent reporter group and the 3' end of the probe is modified with a quencher group.

In one embodiment, the fluorescent reporter group is selected from FAM, HEX, JOE, CY, CY5 and the quencher group is selected from DABCYL, TAMRA, BHQ.

In one embodiment, the 5' end of the probe is modified with a fluorescent reporter group CY 5.

A third object of the present invention is to provide a kit for detecting Vibrio alginolyticus, which comprises the detection composition.

In one embodiment, the kit further comprises a standard for vibrio alginolyticus.

A fourth object of the present invention is to provide a method for detecting Vibrio alginolyticus for non-disease diagnosis purposes, which is to detect Vibrio alginolyticus using the composition or the kit.

In one embodiment, the method of detection comprises fluorescent quantitative PCR or chip-type digital PCR.

In one embodiment, the method comprises the steps of:

(1) Extracting genomic DNA of a sample to be detected or directly diluting the sample to be detected;

(2) Fluorescent quantitative PCR or chip-type digital PCR was performed using the above composition.

In one embodiment, in the step (2), the chip-type digital PCR reaction system uses 2 μl of genomic DNA of the sample to be tested or diluted sample to be tested as a template, and the detection composition, the chip-type digital PCR reaction reagent and ultrapure water are added.

In one embodiment, the chip-type digital PCR reaction system is: absolute Q ™ DNA Digital PCR Master Mix (5X) 2. Mu.L,va0363the primers (10. Mu. Mol/L) for the upstream and downstream of the gene were each 0.5. Mu.L, the probe (10. Mu. Mol/L) was 0.25. Mu.L, the DNA template was 2. Mu.L, and finally the primers were made up to 10. Mu.L with water.

In one embodiment, the chip-type digital PCR amplification procedure is: 96. 10 min at the temperature; 96. 15℃ s; 60. 30 s at C.for 40 cycles.

The invention has the following beneficial effects:

(1) Compared with the traditional detection target spot, the novel detection target spot of vibrio alginolyticus is obtained through autonomous screening, has better accuracy and specificity, and can effectively solve the problem of inaccurate detection. The invention has good specificity, in 24 strains, only the vibrio alginolyticus generates amplification signals, and other vibrio (parahaemolyticus vibrio, vulnificus vibrio, cholera vibrio) and non-vibrio negative control bacteria (salmonella typhimurium, escherichia coli, staphylococcus aureus, listeria monocytogenes, pseudomonas aeruginosa and bacillus cereus) have no amplification phenomenon when being detected by utilizing the target provided by the invention.

(2) The invention applies the chip type digital PCR to the detection of the vibrio alginolyticus for the first time, and the detection sensitivity is 3.18 fg/mu L when the genome DNA is used as a template; when the bacterial liquid is used as a template, the detection sensitivity is 4.75X10 ¹ CFU/mL; compared with the traditional fluorescent quantitative PCR, the method is more suitable for detecting trace targets.

(3) The method has the advantages that the result judgment is simple, absolute quantification can be realized without depending on a standard curve, and the detection time is shortened to be within 2 h; compared with the microdroplet digital PCR, the method is convenient to operate, low in cost and capable of meeting the actual detection requirement.

Drawings

FIG. 1 is a diagram of self-screening targets and conventional target specificity verification electrophoresis; a: the N646_0350 gene; b: the N646_0363 gene; c: the N646_0367 gene; d: the N646_0380 gene; e: the N646_0458 gene; f: the N646_0782 gene; g: the N646_1442 gene; h: the N646_4545 gene; i:colHa gene; j:rpoXa gene; k:toxRa gene; l:groELa gene; m:gyrBand (3) a gene. Lane M: DL1000 DNA marker; lane N: blank control; lane 1: vibrio alginolyticus ATCC 17749; lane 2: vibrio alginolyticus ATCC 33787; lanes 3-12: vibrio alginolyticus isolate Va 1-Va 10; lane 13: vibrio parahaemolyticus ATCC 17802; lane 14: vibrio parahaemolyticus ATCC 33847; lane 15: vibrio vulnificus ATCC 27562; lane 16: vibrio vulnificus CICC 25009; lane 17: vibrio cholerae CICC 23794; lane 18: vibrio cholerae CICC 23795; lane 19: salmonella typhimurium ATCC 14028; lane 20: escherichia coli ATCC 25922; lane 21: staphylococcus aureus CMCC (B) 260031; lane 22: listeria monocytogenes ATCC 19115; lane 23: pseudomonas aeruginosa ATCC 9027; lane 24: bacillus cereus CMCC (B) 63303.

FIG. 2 is a one-dimensional scatter plot of different primer probe ratios; wherein R1, R2, R3 and R4 respectively represent the ratio of the primer probes to be 1:0.5, 1:1, 1:1.5 and 1:2.

FIG. 3 shows the results of chip-type digital PCR specific assays; wherein S1: vibrio alginolyticus ATCC 17749; s2: vibrio alginolyticus ATCC 33787; S3-S10: vibrio alginolyticus isolates Va 1-Va 8; s11: vibrio parahaemolyticus ATCC 17802; s12: vibrio parahaemolyticus ATCC 33847; s13: vibrio vulnificus ATCC 27562; s14: vibrio vulnificus CICC 25009; s15: vibrio cholerae CICC 23794; s16: vibrio cholerae CICC 23795; S17-S18: vibrio alginolyticus isolate Va 9-Va 10; s19: salmonella typhimurium ATCC 14028; s20: escherichia coli ATCC 25922; s21: staphylococcus aureus CMCC (B) 260031; s22: listeria monocytogenes ATCC 19115; s23: pseudomonas aeruginosa ATCC 9027; s24: bacillus cereus CMCC (B) 63303.

FIG. 4 shows genomic sensitivity results of chip-type digital PCR and fluorescent quantitative PCR assays; a: chip type digital PCR one-dimensional scatter diagram; b: chip type digitalPCR microwell array map; c: genome sensitivity is detected by fluorescent quantitative PCR, wherein D0-D7, 3.18 fg/. Mu.L-3.18×10 ⁷ fg/μL；NTC, no template control。

FIG. 5 shows the bacterial liquid sensitivity results of chip-type digital PCR and fluorescent quantitative PCR detection; a: chip type digital PCR one-dimensional scatter diagram; b: chip type digital PCR micropore array diagram; c: and detecting the sensitivity of the bacterial liquid by fluorescent quantitative PCR, wherein B0-B7, 4.75 CFU/mL-4.75X10 ⁷ CFU/mL；NTC, no template control。

Detailed Description

The invention will be further illustrated in the following drawings and specific examples, which are included to better illustrate and not to limit the invention.

Reagents and media used in the examples: absolute Q ™ DNA Digital PCR Master Mix (5×), quantum studio ™ Absolute Q ™ Isolation Buffer are all available from Applied Biosystems company, USA; 2 XSanTaq PCR Mix (blue dye-containing) (Shanghai Biotechnology Co., ltd.); premix Ex Taq (Probe qPCR) (Dalianbao bioengineering Co., ltd.); 3% sodium chloride Alkaline Peptone Water (APW), LB broth were purchased from Beijing land bridge technologies Co., ltd.

The apparatus and device used in the examples: KB240 type cryoincubator (Binder, germany); TS-200B type constant temperature cradle (Shanghai is laboratory instruments Co., ltd.); 1-14 bench centrifuge (Sigma Germany); CFX96 real-time fluorescent quantitative PCR instrument (Bio-Rad company, usa); quantum studio ™ Absolute Q ™ digital PCR System, quantum studio ™ Absolute Q ™ MAP16 Digital PCR Plate were purchased from America Applied Biosystems company.

Example 1: specific target spot excavation of vibrio alginolyticus

The 8 novel targets N646_0350, N646_0363, N646_0367, N646_0380, N646_0458, N646_0782, N646_1442 and N646_4545 of the vibrio alginolyticus are obtained through a bioinformatics method. The PCR primers were designed using Primer Premier 5.0 software, all of which required preliminary specificity verification by Primer-BLAST on-line tool (https:// www.ncbi.nlm.nih.gov/tools/Primer-BLAST/index. Cgi), followed by delivery of the satisfactory primers to Nanjing gold Biotechnology Co. The self-screening targets and the conventional detection targets in the existing literature were specifically evaluated by using common PCR according to 24 strains existing in the laboratory (Table 1), and the primer information is shown in Table 2 and Table 3. The vibrio strain is inoculated into 10 mL of 3% NaCl Alkaline Peptone Water (APW), the other strains are inoculated into 10 mL of LB culture medium, shake culture is carried out for 16-18 h at 36+/-1 ℃, and cultures are collected. Extracting bacterial genome DNA by adopting a boiling method: taking 1. 1 mL fresh culture, centrifuging at 6,000Xg for 5 min, discarding supernatant, adding 50 μl of double distilled water to resuspend thallus, boiling at 100deg.C for 10 min, immediately ice-bathing for 10 min, centrifuging at 14,000Xg for 5 min, and collecting supernatant for PCR detection or preserving at-20deg.C for use. Ordinary PCR reaction system (25. Mu.L): 2 XSanTaq PCR Mix (blue dye containing) 12.5. Mu.L, 1. Mu.L each of the upstream and downstream primers (10. Mu.M), 2. Mu.L of DNA template, and 25. Mu.L of double distilled water. Amplification conditions: pre-denaturation at 94℃for 5 min; denaturation at 94℃for 30 s, annealing at 58℃for 30 s, elongation at 72℃for 20 s,30 cycles; finally, the reaction is carried out at 72 ℃ for 7 min. Detection of amplification products: and directly carrying out 2% agarose gel (adding nucleic acid dye) constant pressure (100V) electrophoresis on the PCR product, observing the result under an ultraviolet gel imager after 40 min, and selecting a target spot with better specificity for the establishment of a subsequent detection method.

Table 1 target specificity verification Strain and detection results

Note that: ATCC (American Type Culture Collection), american type culture Collection; CICC (China Center of Industrial Culture Collection), china center for type culture Collection; CMCC (China Medical Culture Collection), china medical Collection management center; va1 to Va10 represent 10 Vibrio alginolyticus isolates.

The specificity verification result shows that (figure 1) from the screening targets N646-0350, N646-0363, N646-0367, N646-0380, N646-0458, N646-0782,N646_1442 and N646_4545 can be detected in the positive strain of the vibrio alginolyticus, and the amplification is not found in the non-vibrio alginolyticus, which indicates that the self-screening target point has high specificity. Likewise, traditional targetscolH、rpoXAndgyrBcan be detected in positive strains; whiletoxRFailure to detect in Vibrio alginolyticus;groELalthough amplified in positive strains, weak bands were generated in non-target bacteria (Vibrio parahaemolyticus), indicating conventional targetstoxRAndgroELthe specificity is not high.

TABLE 2 screening results of Vibrio alginolyticus targets

TABLE 3 Vibrio alginolyticus traditional target information

Example 2: primer probe design and chip type digital PCR detection

Selected from screening targets N646_0363 (accession number: CP006718.1, designated asva0363) The conserved sequence is designed into a high-specificity primer and a probe, and the specific primer and probe sequences are as follows:

va0363-F：5’-CGCCTTTACTGGTGAGCCT-3’，SEQ ID NO.9；

va0363-R：5’-GAGCACGAGCAACGATTTCT-3’，SEQ ID NO.10；

va0363-P：5’-CY5-TTCACTTCCAATTCAGAGGCGATAGA-BHQ2-3’，SEQ ID NO.11。

chip type digital PCR reaction system (10. Mu.L): absolute Q ™ DNA Digital PCR Master Mix (5X) 2. Mu.L,va0363primers for upstream and downstream of geneva0363F/R, 10. Mu. Mol/L) each 0.5. Mu.L of probe [. Sup.va0363P, 10. Mu. Mol/L) 0.25. Mu.L, 2. Mu.L of DNA template, and finally made up to 10. Mu.L with water.

Amplification conditions: 96. 10 min at the temperature; 96. 15℃ s; 60. 30 s at C.for 40 cycles.

ChipThe copy number (copies/mu L) of the target gene in each reaction can be directly obtained by digital PCR, and the conversion formula of the copy number and the concentration of the corresponding bacterial liquid is as follows: c=c ₀ ×V ₀ /(V ₁ ×V ₂ ) Wherein, C is the concentration of bacterial liquid (CFU/mL) obtained by converting the copy number of digital PCR, C ₀ Copy number concentration (copies/. Mu.L) in 10. Mu.L reaction system, V ₀ -final volumetric volume (μl), V, of DNA extraction ₁ DNA template dosage ([ mu ] L), V in digital PCR reaction system ₂ Bacterial liquid volume (mL) for DNA extraction.

Primer probe proportion optimization: on the basis of guaranteeing the distinguishing effect of the positive microdroplet and the negative microdroplet, different primer probe concentration ratios (1:0.5, 1:1, 1:1.5 and 1:2) are set, and the primer probe ratios are determined. As can be seen from FIG. 2, with increasing probe concentration, the fluorescence signal of positive droplets is increased, indicating that primer probe concentration ratios of 1:0.5-1:2 are available for detection. When the ratio of the primer probes is 1:0.5, the positive micro-droplets and the negative micro-droplets can be well distinguished, and the 1:0.5 can be selected as the ratio of the primer probes by comprehensively considering the resolution and the detection cost.

Example 3: chip type digital PCR specificity verification

Using Vibrio alginolyticus and Vibrio non-alginolyticus genomic DNA as templates (strains see Table 1), detection was performed using the chip-type digital PCR system optimized in example 2. Chip type digital PCR reaction system (10. Mu.L): absolute Q ™ DNA Digital PCR Master Mix (5X) 2. Mu.L,va0363primers for upstream and downstream of geneva0363F/R, 10. Mu. Mol/L) each 0.5. Mu.L of probe [. Sup.va0363P, 10. Mu. Mol/L) 0.25. Mu.L, 2. Mu.L of DNA template, and finally made up to 10. Mu.L with water.

As shown in FIG. 3, the chip-type digital PCR detection method established by the screening target point N646_0363 has good specificity, only the vibrio alginolyticus has positive microdrops, and none of the vibrio alginolyticus has amplification.

Example 4: detection sensitivity and linear range verification

(1) Genome sensitivity: the genomic DNA of Vibrio alginolyticus ATCC 17749 is used withoutBacterial water is subjected to 10-time gradient dilution, chip type digital PCR and fluorescent quantitative PCR amplification are carried out by using the dilution as a template, and the experiment is set in three ways. Wherein, the fluorescent quantitative PCR reaction system (25. Mu.L) is as follows: premix Ex Taq (Probe qPCR) 12.5. Mu.L,va0363primers for upstream and downstream of geneva0363F/R, 10. Mu. Mol/L) 1.0. Mu.L each, probeva0363P, 10. Mu. Mol/L) 0.5. Mu.L, 5. Mu.L of DNA template, and finally made up to 25. Mu.L with water. Amplification conditions: 95. 30 ℃ s; 95. DEG C5 s,60 ℃ 30 s (CY 5 fluorescence collection), 40 cycles.

As can be seen from FIG. 4, the sensitivity of the chip-type digital PCR detection genome is 3.18 fg/. Mu.L, and the sensitivity of the fluorescent quantitative PCR detection genome is 31.8 fg/. Mu.L, and the result shows that the sensitivity of the chip-type digital PCR detection genome is higher than that of the fluorescent quantitative PCR method.

(2) Bacterial liquid sensitivity and linear range: taking the bacterial liquid cultured to the logarithmic phase, and carrying out 10-time gradient dilution to 10 by using sterile physiological saline ^-8 The DNA was extracted from the dilutions of each gradient using the boiling method, and chip-type digital PCR and fluorescent quantitative PCR amplification were performed using this as a template. Simultaneous selection of 10 ^-5 、10 ^-6 And 10 ^-7 Plate counts were performed at three dilutions, and experiments were performed in triplicate, with results showing that the bacterial fluid concentration was 4.75X10 ⁸ CFU/mL。

As can be seen from FIG. 5, the sensitivity of the chip-type digital PCR detection bacterial liquid is 4.75X10 ¹ CFU/mL, copy number and bacterial liquid concentration of 10 ¹ ~10 ⁵ Exhibits good linear relation in CFU/mL range, and the standard curve equation is log ₁₀ copies/mL=1.0032 log ₁₀ CFU/mL + 0.1265（R ² = 0.9992). The sensitivity of the fluorescence quantitative PCR detection genome is 4.75X10 ¹ CFU/mL, cq value and genomic DNA concentration at 10 ¹ ~10 ⁷ Exhibits good linear relation in CFU/mL range, and standard curve equation is Cq= -3.2654 log ₁₀ CFU/mL + 42.488（R ² =0.9997). The results show that the sensitivity of the bacterial liquid of the chip type digital PCR and the fluorescence quantitative PCR method is the same and can reach 4.75X10 ¹ CFU/mL; however, it can be seen that at this concentration, the relative standard deviation of the fluorescent quantitative PCR detection resultsThe difference is significantly higher, indicating that the accuracy of the fluorescent quantitative PCR assay is reduced when the copy number of the sample is small. In contrast, chip-type digital PCR has the advantage of absolute quantification, and is more suitable for detection of trace targets.

Example 5: detection of artificially contaminated samples

Preparing salmon as an aquatic product with negative detection result of target bacteria, weighing 25 g sample into a filtering homogenizing bag, and adding 10 ³ ~10 ⁷ CFU/mL Vibrio alginolyticus (ATCC 17749) bacterial suspension 2.5. 2.5 mL, an artificially contaminated sample was prepared. Sterile PBS solution 222.5. 222.5 mL is added into the artificially contaminated sample, and the sample homogenate of 1:10 is prepared by homogenization (the bacterial liquid dilution ratio is 1:100). Meanwhile, a sterile PBS solution was added to the sample as a negative control. The 1. 1 mL homogenized liquid is sucked into a 1.5 mL centrifuge tube, washed twice by sterile water, and subjected to fluorescent quantitative PCR and chip type digital PCR detection after shaking and mixing uniformly.

As can be seen from Table 4, the chip-type digital PCR detection of artificially contaminated salmon sample was found to be at least 6.88X10 ¹ CFU/mL of Vibrio alginolyticus, the Relative Standard Deviation (RSD) values were all less than 25%, within acceptable limits. The lowest detectable fluorescence quantitative PCR is 6.88×10 in artificially contaminated salmon ¹ CFU/mL vibrio alginolyticus, when the concentration of bacterial liquid is less than or equal to 10 ³ And when CFU/mL is carried out, the detection value of the CFU/mL has larger deviation from the theoretical addition amount. Therefore, the accuracy and the stability of the chip type digital PCR are obviously superior to those of the fluorescent quantitative PCR method when the chip type digital PCR is used for detecting a low-concentration bacteria-containing sample.

TABLE 4 detection of Vibrio alginolyticus in artificially contaminated samples

Note that: ND, undetected; N/A, inapplicable.

While the invention has been described with reference to the preferred embodiments, it is not limited thereto, and various changes and modifications can be made therein by those skilled in the art without departing from the spirit and scope of the invention as defined in the appended claims.

Claims (8)

1. A composition for detecting vibrio alginolyticus, characterized in that the composition comprises an upstream primer, a downstream primer and a probe of target N646_0363; the nucleotide sequence of the upstream primer is shown as SEQ ID NO. 9, the nucleotide sequence of the downstream primer is shown as SEQ ID NO. 10, and the nucleotide sequence of the probe is shown as SEQ ID NO. 11; the base sequence of the N646_0363 is shown in SEQ ID NO. 2.

2. The composition of claim 1, wherein the 5 'end of the probe is modified with a fluorescent reporter group and the 3' end of the probe is modified with a quencher group.

3. The composition of claim 2, wherein the fluorescent reporter group is selected from FAM, HEX, JOE, CY, CY5 and the quencher group is selected from DABCYL, TAMRA, BHQ.

4. A kit for detecting vibrio alginolyticus, characterized in that the kit comprises the composition according to any one of claims 1-3.

5. The kit according to claim 4, wherein the kit further comprises a standard of Vibrio alginolyticus.

6. A method for detecting vibrio alginolyticus for the purpose of non-disease diagnosis is characterized in that the method is to detect the vibrio alginolyticus by using the composition according to any one of claims 1-3 or the kit according to claim 4 or 5.

7. The method of claim 6, wherein the method of detection comprises fluorescent quantitative PCR or chip-type digital PCR.

8. The method according to claim 6 or 7, comprising the steps of:

(1) Extracting genomic DNA of a sample to be detected or directly diluting the sample to be detected;

(2) Fluorescent quantitative PCR or chip-type digital PCR using the composition of any one of claims 1 to 3.