A primer-probe set for detecting Photobacterium damselae and its application

Abstract

The present invention provides a primer-probe set, a system, a primer composition and their applications for detecting Photobacterium damselae based on the LFD-RPA technology. The primer-probe set includes primer RAA-yidC-F, primer RAA-yidC-R and probe RAA-yidC-P. The primer-probe set of the present invention has a rapid reaction and a wide temperature range, enabling rapid detection of the target gene. Moreover, the primer-probe set has good sensitivity and specificity for clinical simulated samples, can effectively detect Photobacterium damselae, and provides a rapid detection method available for in vitro diagnosis of Photobacterium damselae. The primer-probe set of the present invention has strong specificity; high detection sensitivity; high accuracy and reliability; simple and rapid operation, suitable for on-site detection, and has a wide range of application scenarios.

Description

Technical Field

[0001] The present invention relates to the technical field of molecular biology, and particularly relates to a primer-probe set for detecting Photobacterium damselae and its application. Background Art

[0002] Photobacterium damselae is one of the most common pathogenic bacteria in marine cultured fish, and is divided into two subspecies: Photobacterium damselae subsp. damselae (PDD) and Photobacterium damselae subsp. piscicida (PDP). Among them, Photobacterium damselae subsp. damselae is a zoonotic pathogen that infects fish such as Pseudosciaena crocea, Cynoglossus semilaevis, Pampus argenteus, Sebastes schlegeli, etc., crustaceans such as Litopenaeus vannamei, Scylla paramamosain, etc., as well as Tursiops truncatus and humans. Photobacterium damselae subsp. piscicida is called Pasteurella, and its infected hosts are all fish, and it is considered to be one of the most threatening bacterial diseases in worldwide marine aquaculture.

[0003] Recombinase-aid Amplification (RAA) technology is a method that can rapidly amplify nucleic acids at a constant temperature. Different from RPA, the RAA amplification method uses recombinase obtained from bacteria or fungi. At a constant temperature of 37°C, this recombinase can tightly bind to primer DNA to form a polymer of the enzyme and the primer. When the primer searches for a sequence on the template DNA that is completely complementary to it, with the help of single-stranded DNA binding protein (SSB), the template DNA is denatured, and under the action of DNA polymerase, a new DNA complementary strand is formed, and the reaction product also grows exponentially. Usually, an amplification fragment that can be detected by agarose gel electrophoresis can be obtained within 1 hour. By adding a fluorescent group to the RAA reaction system and using the accumulation of fluorescent signals to monitor the entire RAA amplification process in real time, quantitative and qualitative analysis of the starting template can be achieved within 20 minutes. The whole reaction is simple and rapid, and because it does not require high-temperature cycling, it is particularly suitable for use in non-laboratory testing sites with a large number of samples.

[0004] The inventor of the present invention considered providing a primer-probe set, a system, a primer composition for detecting Photobacterium damselae based on LFD-RAA technology and its application. Summary of the Invention

[0005] The technical problem solved by the present invention is to provide a primer-probe set, a system, a primer composition and their applications for detecting Photobacterium damselae, which are used for rapid on-site detection of Photobacterium damselae in gene detection based on the LFD-RAA technology, and have the characteristics of good specificity, high sensitivity and rapid reaction.

[0006] In view of this, the present invention provides a primer-probe set for detecting Photobacterium damselae, including: primer RAA-yidC-F, primer RAA-yidC-R and probe RAA-yidC-P; the nucleotide sequence of the primer RAA-yidC-F is shown in SEQ ID No.1, the nucleotide sequence of the primer RAA-yidC-R is shown in SEQ ID No.2, and the nucleotide sequence of the probe RAA-yidC-P is shown in SEQ ID No.3.

[0007] Preferably, the 5' end of the probe RAA-yidC-P is labeled with the fluorescent group FAM, the 31st base is modified with tetrahydrofuran, and the 3' end is labeled with the blocking group C3-spacer.

[0008] Preferably, the 5' end of the primer RAA-yidC-R is labeled with Biotin.

[0009] The present invention also provides a system for detecting Photobacterium damselae, including the primer-probe set described in the above technical solution.

[0010] Preferably, it further includes an LFD nucleic acid test strip.

[0011] Correspondingly, the present invention also provides a primer composition for amplifying Photobacterium damselae DNA, including primer RAA-yidC-F and primer RAA-yidC-R; the nucleotide sequence of the primer RAA-yidC-F is shown in SEQ ID No.1, and the nucleotide sequence of the primer RAA-yidC-R is shown in SEQ ID No.2.

[0012] The present invention also provides an application of the primer-probe set described in the above technical solution in detecting or assisting in the detection of Photobacterium damselae.

[0013] The present invention also provides an application of the primer-probe set described in the above technical solution in the preparation of products for detecting or assisting in the detection of Photobacterium damselae.

[0014] The present invention also provides an application of the system described in the above technical solution in the preparation of products for detecting or assisting in the detection of Photobacterium damselae.

[0015] The present invention also provides an application of the primer composition described in the above technical solution in the preparation of a product for detecting or assisting in the detection of Photobacterium damselae.

[0016] The present invention provides a primer-probe set, a system, a primer composition and an application thereof for detecting Photobacterium damselae based on the LFD-RPA technology. Compared with the prior art, the primer RAA-yidC-F and the primer RAA-yidC-R can be used for rapid amplification of the yidC gene sequence of Photobacterium damselae by RAA to obtain the RAA amplification product of yidC; the probe RAA-yidC-P matches the target DNA sequence of the RAA amplification product of yidC for LFD-RAA detection. The primer-probe set provided by the present invention has a rapid reaction and a wide temperature range, and can achieve rapid detection of the target gene. Moreover, the primer-probe set has good sensitivity and specificity for clinical simulated samples, can effectively detect Photobacterium damselae, and provides a rapid detection method available for in vitro diagnosis of Photobacterium damselae. The primer-probe set of the present invention has strong specificity; high detection sensitivity; high accuracy, reliability; simple and rapid operation, is suitable for on-site detection, and has a wide range of application scenarios. Brief Description of the Drawings

[0017] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for use in the description of the embodiments or the prior art. Obviously, the drawings in the following description are only the embodiments of the present invention, and those of ordinary skill in the art can also obtain other drawings according to the provided drawings without creative efforts.

[0018] Figure 1 It is the PCR amplification result of the yidC gene of Photobacterium damselae in Example 1 of the present invention;

[0019] Figure 2 It is the PCR amplification result of the tehA gene of Photobacterium damselae in Example 1 of the present invention;

[0020] Figure 3 It is the detection result diagram of the LFD-RAA detection method for Photobacterium damselae in Example 2 of the present invention;

[0021] Figure 4 It is the specificity verification result diagram of the LFD-RAA detection method for Photobacterium damselae in Example 3 of the present invention. Detailed Embodiments

[0022] In order to further understand the present invention, the following describes the preferred implementation embodiments of the present invention in combination with examples. However, it should be understood that these descriptions are only for further illustrating the features and advantages of the present invention, rather than limiting the claims of the present invention.

[0023] An embodiment of the present invention discloses a primer-probe set for detecting Photobacterium damselae, including: primer RAA-yidC-F, primer RAA-yidC-R, and probe RAA-yidC-P; the nucleotide sequence of the primer RAA-yidC-F is as shown in SEQ ID No.1, the nucleotide sequence of the primer RAA-yidC-R is as shown in SEQ ID No.2, and the nucleotide sequence of the probe RAA-yidC-P is as shown in SEQ ID No.3.

[0024] The present invention provides an RAA isothermal fluorescence nucleic acid detection primer-probe set for Photobacterium damselae. Preferably, according to the above primer-probe set, the 5' end of the probe RAA-yidC-P is labeled with the fluorescent group FAM, the 31st base is modified with tetrahydrofuran, and the 3' end is labeled with the blocking group C3-spacer. The 5' end of the primer RAA-yidC-R is labeled with Biotin.

[0025] The fluorescent group of the specific fluorescent probe is preferably selected from one of FAM, VIC, JOE, TET, CY3, CY5, ROX, TexasRed, and LC RED460.

[0026] The above primer-probe set further includes at least one of A Buffer, B Buffer, RAA dry powder reagent, Photobacterium damselae standard, and DEPC-treated water. The A Buffer is 20% PEG; the B Buffer is 280 mM MgAc. The components of the RAA dry powder reagent are preferably as follows: 1 mmol / L dNTP, 90 ng / μL SSB protein, 120 ng / μL recA recombinase protein (SC-recA / BS-recA) or 30 ng / μL Rad51, 30 ng / μL Bsu DNA polymerase, 30 ng / mL RTE reverse transcriptase, 100 mmol / L Tricine, 20% PEG, 5 mmol / L dithiothreitol, 100 ng / μL creatine kinase, Exo exonuclease.

[0027] For the above primer-probe set, the Photobacterium damselae standard is a positive plasmid containing a partial sequence of the yidC gene of Photobacterium damselae.

[0028] The present invention also provides a system for Photobacterium damselae, including the above primer-probe set.

[0029] According to the above system, the system further contains an LFD nucleic acid detection test strip.

[0030] The present invention also provides a primer composition for amplifying the DNA of Photobacterium damselae, comprising the above-mentioned primer RAA-yidC-F and primer RAA-yidC-R; the nucleotide sequence of the primer RAA-yidC-F is shown in SEQ ID No.1, and the nucleotide sequence of the primer RAA-yidC-R is shown in SEQ ID No.2.

[0031] The present invention also provides a method for detecting Staphylococcus aureus, which includes using the DNA of a sample to be tested as a template, and performing LFD-RAA detection with the above-mentioned primer-probe set or the above-mentioned system to obtain a specific band; determining whether the sample to be tested contains Photobacterium damselae according to the specific band.

[0032] The application of the above-mentioned primer-probe set also belongs to the protection scope of the present invention. The specific application is any one of the following:

[0033] (1) Detecting or assisting in detecting Photobacterium damselae;

[0034] (2) Preparing a product for detecting or assisting in detecting Photobacterium damselae.

[0035] The application of the above-mentioned system also belongs to the protection scope of the present invention. The specific application is any one of the following:

[0036] (1) Detecting or assisting in detecting Photobacterium damselae;

[0037] (2) Preparing a product for detecting or assisting in detecting Photobacterium damselae.

[0038] The application of the above-mentioned primer composition also belongs to the protection scope of the present invention. The specific application is any one of the following:

[0039] (1) Detecting or assisting in detecting Photobacterium damselae;

[0040] (2) Preparing a product for detecting or assisting in detecting Photobacterium damselae;

[0041] (3) Amplifying the DNA of Photobacterium damselae;

[0042] (4) Preparing a product for amplifying the DNA of Photobacterium damselae.

[0043] The primer composition provided by the present invention and its paired probe RAA-yidC-P can be used for rapid on-site detection of Photobacterium damselae in gene detection based on the LFD-RAA technology, and have the advantages of good specificity, high sensitivity, and very fast reaction.

[0044] In the primer-probe set provided by the present invention, primer RAA-yidC-F and primer RAA-yidC-R can be used for rapid amplification of the yidC gene sequence of Photobacterium damselae by RAA to obtain the RAA amplification product of yidC; probe RAA-yidC-P matches the target DNA sequence of the RAA amplification product of yidC for LFD-RAA detection. The primer-probe set of the present invention has a rapid reaction and a wide temperature range (37-42 °C), and can achieve rapid detection of the target gene. This primer-probe set has good sensitivity and specificity for clinical simulation samples, can effectively detect Photobacterium damselae, and provides a rapid detection method available for in vitro diagnosis of Photobacterium damselae.

[0045] The present invention also provides an RAA isothermal fluorescence detection method for Photobacterium damselae. DNA of a sample to be tested is extracted, and using the DNA of the sample to be tested as a template, an RAA reaction is carried out in the presence of an upstream primer, a downstream primer, a specific fluorescent probe, RAA dry powder reagent, A Buffer, B Buffer and DEPC-treated water of Photobacterium damselae, and the sample to be tested is analyzed according to the RAA reaction.

[0046] To further understand the present invention, the technical solutions provided by the present invention will be described in detail below in conjunction with embodiments. The protection scope of the present invention is not limited by the following embodiments.

[0047] The raw materials used in the embodiments of the present invention are all commercially available.

[0048] Example 1

[0049] Construction of a detection method for Photobacterium damselae

[0050] 1. Experimental materials:

[0051] Photobacterium damselae, other strains (Vibrio harveyi, Vibrio splendidus, Vibrio alginolyticus and Vibrio parahaemolyticus) were all provided by the Key Laboratory of Marine Organism Breeding and High-value Utilization of Fujian Province, Fisheries Research Institute of Fujian Province.

[0052] RAA primers and paired fluorescent probes were synthesized by Shanghai Jierui Biotechnology Co., Ltd.

[0053] The Photobacterium damselae strain was sourced from the Key Laboratory of Marine Organism Breeding and High-value Utilization of Fujian Province, Fisheries Research Institute of Fujian Province.

[0054] 2. Experimental instruments

[0055] Centrifuge, vortex mixer, constant temperature water bath, PCR instrument.

[0056] Example 1:

[0057] 1. Design of RAA amplification primer pairs and RAA probes.

[0058] Through the whole-genome alignment and analysis of Photobacterium damselae, the DNAMAN 6.0 software was used to align multiple sequences to find the conserved regions. Two sets of primers and probes were designed in the conserved regions and BLAST alignment was performed in the NCBI database. The primer and probe sequences are shown in Table 1.

[0059] Table 1 Primer and Probe Sequences

[0060]

[0061]

[0062] 2. Primer Screening Results

[0063] PCR amplification of the two pairs of primers and 1.5% agarose gel electrophoresis were carried out, and the results are as Figure 1 and Figure 2 shown. The primer pair RAA-yidC-F / RAA-yidC-R showed good specificity in the amplification of DNA from Photobacterium damselae, Escherichia coli, Staphylococcus aureus, Vibrio harveyi, Vibrio splendidus, Vibrio alginolyticus, and Vibrio parahaemolyticus. Only Photobacterium damselae amplified a band, and no bands were observed in other strains. The primer pair RAA-tehA-F / RAA-tehA-R amplified a band for Photobacterium damselae, but also had bands for Escherichia coli and Staphylococcus aureus. The primer pair RAA-yidC-F / RAA-yidC-R was determined for the next experiment.

[0064] Example 2:

[0065] 1. Construction of the LFD-RAA detection method for Photobacterium damselae.

[0066] The reaction system used was 50 μL: 2.0 μL of 2 μmol / L upstream and downstream primers, 0.6 μL of 2 μmol / L probe, 25 μL of A Buffer, 12.9 μL of water. One tube of reaction dry powder was added, and after thorough mixing, 5 μL of the DNA sample to be tested was added. 2.5 μL of B Buffer was added to the tube cap, and the mixture was gently shaken up and down 5 - 6 times and centrifuged at low speed for 10 sec, and then reacted in a 39 °C constant temperature water bath for 30 min. After the reaction, the 50 μL reaction solution system was diluted with sterile water or PBS (50 μL reaction system solution: 300 μL diluent). 8 - 10 μL of the nucleic acid amplification product was aspirated and dropped onto the sample pad of the nucleic acid detection test strip. The sample pad of the test strip was inserted downward into a microplate containing 100 μL of buffer, and the detection result in the reading area was read within 15 - 30 min ( Figure 3 shown).

[0067] Example 3:

[0068] 1. Specificity verification of the LFD-RAA detection method for Photobacterium damselae subsp. piscicida

[0069] Select the DNA of Photobacterium damselae subsp. piscicida as the positive detection object, the DNA of non-Photobacterium damselae subsp. piscicida such as Vibrio splendidus, Vibrio harveyi, and Staphylococcus aureus as negative controls, and water as the blank control group for LFD-RAA detection. Select a reaction temperature of 39 °C for 30 min, and analyze the results in combination with a nucleic acid test strip.

[0070] In the specific detection of Photobacterium damselae subsp. piscicida by LFD-RAA, RAA-yidC-F / RAA-yidC-R / RAA-yidC-P showed specific detection bands, and the detection results of non-Photobacterium damselae subsp. piscicida were all negative, and only the control group showed color on the test strip ( Figure 4 as shown).

[0071] Example 4:

[0072] 1. Application of the method of the present invention in the nucleic acid detection of Photobacterium damselae subsp. piscicida in simulated clinical samples

[0073] In order to verify the practicability of the LFD-RAA detection method for Photobacterium damselae subsp. piscicida, 20 samples of Apostichopus japonicus suspected of being infected with Photobacterium damselae subsp. piscicida were detected. The experimental results showed that among the 20 samples of the present invention, 15 were positive and 5 were negative by PCR amplification; the results detected by the LFD-RAA method were 15 positive and 5 negative. In the simulated samples, the results detected by the LFD-RAA method were consistent with the positive detection results of PCR, indicating that the detection reagent of the present invention has high accuracy.

[0074] The above description of the embodiments is only used to help understand the method of the present invention and its core idea. It should be noted that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and modifications can be made to the present invention, and these improvements and modifications also fall within the protection scope of the claims of the present invention.

[0075] The above description of the disclosed embodiments enables those skilled in the art to implement or use the present invention. Various modifications to these embodiments will be obvious to those skilled in the art, and the general principles defined herein can be implemented in other embodiments without departing from the spirit or scope of the present invention. Therefore, the present invention will not be limited to the embodiments shown herein, but rather to the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A primer-probe set for detecting Photobacterium damselae, characterized in that, Comprising: Primer RAA-yidC-F, primer RAA-yidC-R and probe RAA-yidC-P; the nucleotide sequence of the primer RAA-yidC-F is as shown in SEQ ID No.1, the nucleotide sequence of the primer RAA-yidC-R is as shown in SEQ ID No.2, the nucleotide sequence of the probe RAA-yidC-P is as shown in SEQ ID No.3, the 5' end of the probe RAA-yidC-P is labeled with the fluorescent group FAM, the 31st base is modified with tetrahydrofuran, and the 3' end is labeled with the blocking group C3-spacer, and the 5' end of the primer RAA-yidC-R is labeled with Biotin.

2. A system for detecting Photobacterium damselae, characterized in that, Comprising the primer-probe group described in claim 1.

3. The system according to claim 2, characterized in that, Also comprising an LFD nucleic acid test strip.

4. A primer composition for amplifying Photobacterium damselae DNA, characterized in that, Comprising primer RAA-yidC-F and primer RAA-yidC-R; the nucleotide sequence of the primer RAA-yidC-F is as shown in SEQ ID No.1, and the nucleotide sequence of the primer RAA-yidC-R is as shown in SEQ ID No.

2.

5. Use of the primer-probe group described in claim 1 in the preparation of a product for detecting or assisting in the detection of Photobacterium damselae.

6. Use of the system according to any one of claims 2-3 in the preparation of a product for detecting or assisting in the detection of Photobacterium damselae.

7. Use of the primer composition described in claim 4 in the preparation of a product for detecting or assisting in the detection of Photobacterium damselae.

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