Primer probe group, kit and method for detecting black sea vibrio

Abstract

The invention discloses a primer probe group, a kit and a method for detecting black sea vibrio. The primer probe group capable of specifically detecting the vibrio black sea vibrio is designed based on the recA gene of the vibrio black sea vibrio as a target, and the detection sensitivity reaches 0.01 fg / mu L. The invention not only provides a new molecular target for detecting the vibrio black sea vibrio, but also can directly and specifically distinguish the vibrio black sea vibrio from vibrio parahaemolyticus, vibrio harveyi, vibrio fluvialis, vibrio alginolyticus and vibrio cholerae through PCR reaction, and provides a simpler and more convenient detection means for disease monitoring of the vibrio black sea vibrio in the aquaculture process.

Description

Technical Field

[0001] The present invention relates to the technical field of aquatic animal disease detection, and specifically, to a primer-probe set, a kit and a method for detecting Vibrio ponticus. Background Art

[0002] Vibrio is one of the common bacterial flora in the marine environment and is also one of the most common opportunistic pathogens in mariculture. At present, the ecological environment on which aquatic animals depend for survival is deteriorating continuously, and the occurrence and prevalence of bacterial diseases are becoming more and more serious. Vibrio ponticus is a marine bacterium, which has been identified as a potential pathogen of intestinal diseases in aquatic animals such as seahorses because it has been reported to exist as a dominant bacterial flora in three seahorses suffering from intestinal diseases and other fish and oysters.

[0003] At present, there are research reports that taking the Vibrio dnaJ gene as the target gene and designing a pair of degenerate primers in the conserved regions at its head and tail can be used to detect 8 species of Vibrio bacteria including Vibrio ponticus. The lowest detection limit of this method for Vibrio is 10 2 CFU / mL. However, this method ultimately still depends on sequencing technology to construct a phylogenetic evolution tree and cannot directly distinguish Vibrio ponticus from Vibrio parahaemolyticus, Vibrio harveyi, Vibrio fluvialis, Vibrio alginolyticus and Vibrio cholerae through PCR reaction (Vibrio PCR detection method based on the dnaJ gene, publication date: July 11, 2018). In addition, there are few reports on molecular detection technologies for other targets of Vibrio ponticus, making the PCR technology relatively single in the actual application process. Therefore, it is urgent to systematically and comprehensively explore new specific detection targets to improve the accuracy of the PCR detection technology for Vibrio ponticus.

[0004] Quantitative Real-time PCR is a method that adds a fluorescent dye or a fluorescent probe on the basis of the conventional PCR technology, uses the accumulation of fluorescent signals to monitor the whole PCR process in real time, and quantitatively analyzes the template with unknown concentration through a standard curve. As an internationally recognized standard method for nucleic acid molecule quantification, Quantitative Real-time PCR is the preferred method for rapidly detecting gene expression levels. It not only realizes the leap from qualitative to quantitative PCR, but also has the characteristics of higher sensitivity, stronger specificity, better repeatability and higher automation degree compared with the conventional PCR.

[0005] At present, the detection methods for Vibrio ponticus suitable for application in the aquaculture field are still very limited. Therefore, developing a rapid and reliable method for identifying Vibrio ponticus is of great significance in aquaculture. Summary of the Invention

[0006] To overcome the above-mentioned defects and deficiencies in the prior art, the present invention provides a primer-probe set, a kit and a method for detecting Vibrio nigripulchritudo.

[0007] The first object of the present invention is to provide a primer-probe set for detecting Vibrio nigripulchritudo.

[0008] The second object of the present invention is to provide the application of the above-mentioned primer-probe set in the preparation of a product for detecting Vibrio nigripulchritudo.

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

[0010] The fourth object of the present invention is to provide the application of the above-mentioned composition in the preparation of a product for detecting Vibrio nigripulchritudo.

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

[0012] The sixth object of the present invention is to provide a method for detecting Vibrio nigripulchritudo.

[0013] The present invention claims the following:

[0014] A primer-probe set for detecting Vibrio nigripulchritudo, the primer-probe set consists 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.

[0015] As an implementable manner, 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.

[0016] The application of the above-mentioned primer-probe set in the preparation of a product for detecting Vibrio nigripulchritudo.

[0017] A composition for detecting Vibrio nigripulchritudo, the composition contains the above-mentioned primer-probe set.

[0018] The application of the above-mentioned composition in the preparation of a product for detecting Vibrio nigripulchritudo.

[0019] A kit for detecting Vibrio nigripulchritudo, the kit contains the above-mentioned primer-probe set.

[0020] Preferably, the kit further comprises a positive control reference and / or a negative control reference.

[0021] Preferably, the positive control reference is plasmid DNA containing the detection target gene of Vibrio nigripulchritudo, and the negative control reference is ddH 2 O;

[0022] The detection target gene is the recA gene sequence of Vibrio nigripulchritudo.

[0023] Preferably, the kit further comprises a fluorescence quantitative PCR detection reagent.

[0024] A method for detecting Vibrio nigripulchritudo, which uses the above kit to detect a sample to be tested, and the detection is for non-disease treatment diagnosis purposes.

[0025] Compared with the prior art, the present invention has the following beneficial effects:

[0026] The present invention discloses a primer-probe set, a kit and a method for detecting Vibrio nigripulchritudo. Based on the recA gene of Vibrio nigripulchritudo as a target, the present invention designs a primer-probe set capable of specifically detecting Vibrio nigripulchritudo, and the detection sensitivity reaches 0.01 fg / μL. The present invention not only provides a new molecular target for detecting Vibrio nigripulchritudo, but also can directly distinguish Vibrio nigripulchritudo from Vibrio parahaemolyticus, Vibrio harveyi, Vibrio fluvialis, Vibrio alginolyticus and Vibrio cholerae through PCR reaction, providing a more convenient detection means for disease monitoring of Vibrio nigripulchritudo in the aquaculture process. Description of the Drawings

[0027] Figure 1 It is the amplification situation of different primer-probe sets of Vibrio nigripulchritudo in Example 1.

[0028] Figure 2 It is the specific detection of the primer-probe set of Vibrio nigripulchritudo. The negative control is: Vibrio alginolyticus, Vibrio parahaemolyticus, Vibrio harveyi, Vibrio fluvialis, Vibrio cholerae, Vibrio cincinnatiensis, Vibrio anguillarum, Vibrio coralliilyticus, Vibrio fortis, Vibrio nereis, Flavobacterium columnare, Pseudomonas fluorescens, Aeromonas caviae; the blank control is ddH 2 O.

[0029] Figure 3 It is the sensitivity detection result of the primer-probe set of Vibrio nigripulchritudo.

[0030] Figure 4 It is the negative stability detection result of the primer-probe set of Vibrio nigripulchritudo.

[0031] Figure 5 It is the detection result of Vibrio nigripulchritudo in 24 actual samples. Detailed Embodiments

[0032] The present invention will be further described below in conjunction with specific embodiments, but the embodiments do not limit the present invention in any form. Unless otherwise specified, the reagents, methods, and equipment used in the present invention are conventional reagents, methods, and equipment in the technical field.

[0033] Unless otherwise specified, the reagents and materials used in the following examples are all commercially available.

[0034] Example 1 Influence of primer sets on the detection of Vibrio nigripulchritudo

[0035] I. Experimental method

[0036] 1. Plasmid DNA synthesis

[0037] The gene sequence designed for the primers of Vibrio nigripulchritudo was ligated to the pUC57 vector to construct a plasmid.

[0038] 2. Primer design

[0039] The complete genome sequence of Vibrio nigripulchritudo was searched on Genbank, and through BLAST analysis and comparison, with the recA gene as the target gene, specific primers and probes were designed using the Primer Express primer design software according to the principles of real-time fluorescence PCR primer and probe design.

[0040] Vibrio nigripulchritudo primer-probe set 1 (Y1):

[0041] Forward primer VPO-1-F: 5'-CGACGTTATGGTTGTCGATTCA-3' (SEQ ID NO: 1); Reverse primer VPO-1-R: 5'-CCATTTCACCTTCAATTTCTGCTT-3' (SEQ ID NO: 2);

[0042] Fluorescent probe VPO-1-P: 5'-TAGCCGCACTGACACC-3' (SEQ ID NO: 3);

[0043] The 5' end of the fluorescent probe VPO-1-P is labeled with the fluorescent group FAM, and the 3' end is labeled with the quenching group MGB.

[0044] Vibrio nigripulchritudo primer-probe set 2 (Y2):

[0045] Forward primer VPO-2-F: 5'-GTAGCCGCACTGACACCAAA-3' (SEQ ID NO: 4);

[0046] Reverse primer VPO-2-R: 5'-TTGAAGGCCCATGTGGCTAT-3' (SEQ ID NO: 5);

[0047] Fluorescent probe VPO-2-P: 5'-TTGAAGGTGAAATGGG-3' (SEQ ID NO: 6);

[0048] The 5'-end of the fluorescent probe VPO-2-P is labeled with the fluorescent group FAM, and the 3'-end is labeled with the quenching group MGB.

[0049] 3. Perform the PCR reaction

[0050] The reaction system is shown in Table 1.

[0051] Table 1 Real-time fluorescence PCR reaction system

[0052] Reagent Name Working Solution Concentration Sample Loading Volume Final Concentration Reaction Premix 2× 12.5 μL 1× Forward Primer F 10 μM 1 μL 0.4 μM Reverse Primer R 10 μM 1 μL 0.4 μM Probe P 10 μM 0.5 μL 0.2 μM DNA Template 10 - 100 ng / μL 5 μL - <![CDATA[ddH 2 O]]> - 5 μL -

[0053] The reaction program is as follows: The decontamination program is 50°C for 5 min, 1 cycle; the Holding Stage program is 95°C for 5 min, 1 cycle; the Cycling Stage is 95°C for 15 s, 60°C for 30 s, 45 cycles; collect the fluorescence signal at 60°C for 30 s, and observe the reaction results using a fluorescence PCR instrument.

[0054] II. Experimental results

[0055] The results are as Figure 1 shown. Both primer-probe groups can perform amplification, but the Ct value of primer-probe group 1 is smaller and the repeatability is better. There is no non-specific amplification in the negative control. Therefore, primer-probe group 1 is determined as the optimal primer-probe group.

[0056] Example 2 A kit for detecting Vibrio nigripulchritudo in aquatic animals

[0057] I. Composition

[0058] 1. Primer-probe group for detecting Vibrio nigripulchritudo

[0059] Forward primer VPO-1-F: 5'-CGACGTTATGGTTGTCGATTCA-3' (SEQ ID NO: 1); Reverse primer VPO-1-R: 5'-CCATTTCACCTTCAATTTCTGCTT-3' (SEQ ID NO: 2);

[0060] Fluorescent probe VPO-1-P: 5'-TAGCCGCACTGACACC-3' (SEQ ID NO: 3);

[0061] The 5'-end of the fluorescent probe VPO-1-P is labeled with the fluorescent group FAM, and the 3'-end is labeled with the quenching group MGB.

[0062] 2. PCR reaction reagents

[0063] PCR reaction premix, positive control reference, negative control reference;

[0064] The positive control reference is a plasmid containing the recA gene sequence of Vibrio alginolyticus, and the negative control reference is ddH 2 O.

[0065] II. Usage method

[0066] Use the primer-probe set of Vibrio alginolyticus to perform PCR reaction on the DNA of the sample to be tested, and the reaction system is shown in Table 2.

[0067] Table 2 Real-time fluorescence PCR reaction system

[0068] Reagent Name Working Solution Concentration Sample Loading Volume Final Concentration Reaction Premix 2× 12.5 μL 1× Forward Primer F 10 μM 1 μL 0.4 μM Reverse Primer R 10 μM 1 μL 0.4 μM Probe P 10 μM 0.5 μL 0.2 μM DNA Template 10 - 100 ng / μL 5 μL - <![CDATA[ddH 2 O]]> - 5 μL -

[0069] The reaction procedure is as follows: the decontamination procedure is 50°C for 5 min, 1 cycle; the Holding Stage procedure is 95°C for 5 min, 1 cycle; the Cycling Stage is 95°C for 15 s, 60°C for 30 s, 45 cycles; collect fluorescence signals at 60°C for 30 s, and observe the reaction results using a fluorescence PCR instrument.

[0070] III. Interpretation method

[0071] If the Ct value of the test sample is ≥45 or there is no Ct value, the curve is a straight line or a slight slope, and there is no "S"-shaped amplification curve, it can be determined that the sample does not contain Vibrio alginolyticus or the content is lower than the detection limit;

[0072] 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 alginolyticus.

[0073] Example 3 Specificity experiment

[0074] 1. Experimental method

[0075] Extract DNA from each sample according to a commercially available DNA extraction kit, and use a full-wavelength micro-spectrophotometer to measure the purity and concentration of the mentioned DNA, and store it at -20°C for later use.

[0076] The samples include: Vibrio alginolyticus, Vibrio parahaemolyticus, Vibrio harveyi, Vibrio fluvialis, Vibrio cholerae, Vibrio cincinnatiensis, Vibrio anguillarum, Vibrio coralliilyticus, Vibrio fortis, Vibrio nereis, Flavobacterium columnare, Pseudomonas fluorescens, Aeromonas caviae.

[0077] Perform PCR amplification using the kit in Example 2.

[0078] 2. Experimental results

[0079] The specificity experiment results of the primer-probe group for Vibrio nigripulchritudo showed that no amplified fluorescence signal was detected in the sample DNA of other aquatic animal diseases such as Vibrio alginolyticus( Figure 2 ), indicating that the established real-time fluorescence PCR detection method has good specificity.

[0080] Example 4 Sensitivity and Repeatability Experiments

[0081] I. Experimental Method

[0082] The synthesized plasmid DNA was quantified on a spectrophotometer, and the plasmid concentration was found to be 100 ng / μL. The plasmid was serially diluted, and DNA at seven concentrations of 10 pg / μL, 1 pg / μL, 100 fg / μL, 10 fg / μL, 1 fg / μL, 0.1 fg / μL, and 0.01 fg / μL was used as templates for detection using the kit in Example 2.

[0083] 2. Experimental Results

[0084] The results showed that typical amplification curves appeared, and the detection sensitivity of the method for the plasmid containing the recA gene of Vibrio nigripulchritudo reached 0.01 fg / μL( Figure 3 ). Among the seven dilution gradients, the results of three parallel experiments for each dilution gradient showed that the coefficient of variation was between 0.16% and 3.52%, indicating that the established real-time fluorescence PCR detection method had good repeatability (Table 3).

[0085] Table 3 Repeatability Experiment of the Primer-Probe Group for Vibrio nigripulchritudo

[0086]

[0087] Example 5 Negative Stability Test

[0088] I. Experimental Method

[0089] ddH 2 O was used as a negative control and repeated 20 times. Another 3 positive control references were added for the negative stability experiment.

[0090] II. Experimental Results

[0091] The results showed that the established real-time fluorescence PCR detection method had good negative stability, and no non-specific amplification occurred( Figure 4 ).

[0092] Example 6 Detection of Vibrio nigripulchritudo in Actual Samples

[0093] I. Experimental Method

[0094] DNA extraction was performed on 24 actual samples, including 6 hippocampi, 6 oysters, 6 sea cucumbers, and 6 sea urchins, and the kit of Example 2 was used to detect whether Vibrio nigripulchritudo was infected.

[0095] II. Experimental Results

[0096] The DNA of the 24 actual samples extracted was detected using the kit of Example 2, and the results are as Figure 5 shown. Among them, the DNA of 4 actual samples was detected as positive for Vibrio nigripulchritudo, and the DNA of 20 actual samples was detected as negative for Vibrio nigripulchritudo.

[0097] 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 other changes, modifications, substitutions, combinations, and simplifications made without departing from the spirit and principle of the present invention shall be equivalent replacement methods and are all included in the protection scope of the present invention.

Claims

1. A primer probe set for detecting Vibrio nigromaculata, characterized in that: The primer probe set consists of a forward primer F1, a reverse primer R1, and a fluorescent probe P1. 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.

2. The primer probe set 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 the primer probe set according to claim 1 or 2 in the preparation of a product for detecting Vibrio nigromaculata.

4. A composition for detecting Vibrio nigromaculata, characterized in that: The composition comprises the primer probe set according to claim 1 or 2.

5. Use of the composition according to claim 4 in preparing a product for detecting Vibrio maritima.

6. A kit for detecting Vibrio nigromaculata, characterized in that: The kit contains the primer probe set according to claim 1 or 2.

7. The kit according to claim 6, characterized in that The kit also includes a positive control reference substance and / or a negative control reference substance.

8. The kit according to claim 7, characterized in that The positive control reference substance is a plasmid DNA containing the target gene for detecting Vibrio nigromatrix, and the negative control reference substance is ddH2O.

9. The kit according to claim 6, characterized in that The kit also contains a fluorescent quantitative PCR detection reagent.

10. A method for detecting Vibrio nigromaculata, characterized in that: The test sample is tested using the kit according to any one of claims 6 to 9, wherein the test is for the purpose of non-disease treatment diagnosis.

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