A primer-probe combination, kit and application for constant temperature rapid detection of Vibrio corallilyticus

Abstract

The present invention discloses a primer-probe combination, a kit and an application for the constant temperature rapid detection of Vibrio coralliiformis. The primers are shown in SEQ ID NO.1 and SEQ ID NO.2, and the fluorescent probe is 5'-GATGCAACGCGAAGAACCTTACC TACTCT(FAM-dT)GAC(THF)(BHQ1-dT)CCTCAGAAGAGACT-3'. The primer-probe combination of the present invention is used to perform real-time fluorescent recombinase polymerase amplification on the sample to be tested, and the test results can be obtained within 20 minutes at 39°C. The detection equipment is small and easy to carry, providing a more convenient detection means for on-site and resource-scarce places. In addition, the detection sensitivity of the kit of the present invention is 10 times that of qPCR, and the reaction speed is faster than qPCR.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to a primer-probe combination, kit, and application for isothermal rapid detection of Vibrio lysodeana. Background Technology

[0002] Vibrio coralliilyticus, a pathogen causing coral bleaching and dissolution, was first discovered by Aurora-Rizzi Velasquez-Rodriguez in 2004 and is now recognized as a global marine pathogen. Due to its highly pathogenic nature, rapid and accurate detection of Vibrio coralliilyticus in corals has become a major concern. In the absence of effective treatments, establishing a simple, sensitive, rapid, and accurate method for detecting Vibrio coralliilyticus, promptly eliminating the source of infection, and interrupting the transmission route can effectively prevent the damage caused by Vibrio coralliilyticus disease to corals.

[0003] Currently, the main methods for detecting Vibrio lysosporioides include pure culture, gene sequencing, enzyme-linked immunosorbent assay (ELISA), immunoblotting, and molecular biological detection techniques such as PCR. However, these methods have significant limitations, including complex operation, low efficiency, high cost, and cumbersome procedures. Therefore, establishing a simple, accurate, rapid, and visualized detection method is the future research direction for the detection of Vibrio lysosporioides. Currently, RPA technology has been applied to the detection of various nucleic acid viruses due to its simple operation, high specificity, and sensitivity. Summary of the Invention

[0004] The purpose of this invention is to overcome the technical problem that the existing PCR technology is limited to laboratory use, thus restricting its application in resource-scarce environments, and to provide a primer and probe combination, kit, and application for isothermal rapid detection of Vibrio lysodemi.

[0005] The primer and probe combination kit of the present invention can be used to detect environmental samples and can quickly determine whether Vibrio lysosporioides is present in the environment.

[0006] To achieve the above-mentioned objectives of the present invention, the present invention adopts the following technical solution:

[0007] A primer-probe combination for rapid isothermal detection of Vibrio lysinensis includes an RPA primer pair and a fluorescent probe; the sequence of the RPA primer pair is as follows:

[0008] Forward primer: 5'-ACAAGCGGTGGAGCATGTGGTTTAATTCGA-3' (SEQ ID NO.1);

[0009] Reverse primer: 5'-CTGTCTCTCAGTTCCCGAAGGCACAAGACTGTCTC-3' (SEQ ID NO.2);

[0010] The sequence of the fluorescent probe is as follows:

[0011] 5'-GATGCAACGCGAAGAACCTTACCTACTCT(FAM-dT)GAC(THF)(BHQ1-dT)CCTCAGAAGAGACT-3'.

[0012] In the above fluorescent probe sequence, G at position 29 is a FAM-dT modified base; C at position 32 is a BHQ1-dT modified base.

[0013] Another object of the present invention is to provide the application of the above-described primer-probe combination in the preparation of a kit for the rapid isothermal detection of Vibrio lysodeana.

[0014] Another object of the present invention is to provide a kit for the rapid isothermal detection of Vibrio lysodeana, comprising the above-described primer-probe combination.

[0015] Preferably, the kit further comprises a fluorescent basic buffer, magnesium acetate, and ultrapure water.

[0016] Another object of the present invention is to provide the application of the above-described kit in the isothermal rapid detection of Vibrio lysodeoxycholate. This application is used in the isothermal rapid detection of whether environmental samples contain Vibrio lysodeoxycholate.

[0017] Preferably, the constant temperature is 39°C.

[0018] Another object of the present invention is to provide a method for rapid detection of Vibrio lysodeana at isothermal temperature, comprising the following steps:

[0019] a. Extract DNA from the sample to be tested;

[0020] b. Using the DNA from step a as a template, perform real-time fluorescent recombinase polymerase amplification using the primer-probe combination described above or the kit described above to obtain the amplification product.

[0021] c. If a fluorescent signal is present, the sample to be tested contains Vibrio lysodesiforme; if no fluorescent signal is present, the sample to be tested does not contain Vibrio lysodesiforme.

[0022] Preferably, the reaction system for real-time fluorescent recombinase polymerase amplification is as follows: 2 μL of 10 μM forward primer, 2 μL of 10 μM reverse primer, 0.8 μL of 10 μM probe, 25 μL of fluorescent basal buffer, 12.7 μL of ultrapure water, 5 μL of DNA template, and 2.5 μL of magnesium acetate solution, for a total of 50 μL.

[0023] Preferably, the reaction conditions for the real-time fluorescent recombinase polymerase amplification are: reaction at 39°C for 20 min.

[0024] Preferably, the concentration of the DNA is 10-15 ng / μL.

[0025] The present invention has the following beneficial effects:

[0026] 1. The real-time RPA (real-time fluorescent recombinase polymerase amplification) technology established by this kit can obtain detection results within 20 minutes at 39℃. The detection device is small and easy to carry, providing a more convenient detection method for field and resource-scarce areas.

[0027] 2. This kit is more sensitive than qPCR and has a faster reaction rate. Attached Figure Description

[0028] Figure 1 These are RPA amplification curves for different primer-probe combinations; A is the positive control, and B is the amplification curve for the combination of forward primer vibrioF4, reverse primer vibrioR4, and probe probe_vibrio4 (40 cycles, 30s per cycle, 20min total).

[0029] Figure 2 This is the amplification curve of RPA under 10-fold serial dilution of DNA using the combination of forward primer vibrioF4, reverse primer vibrioR4, and probe probe_vibrio4. Detailed Implementation

[0030] The following embodiments are further illustrations of the present invention, but not limitations thereof.

[0031] Example 1:

[0032] 1. Primer and probe design: Design the required primers and probes.

[0033] The RPA primer set was designed with 5 pairs of primers and probes, based on different target fragments of 16S rRNA.

[0034] The 16S rRNA sequence is as follows:

[0035] GAACGCTGGCGGCAGGCCTAACACATGCAAGTCGAGCGGAAACGAGTTGTCTTGAA

[0036] CCTTCGGGGAACGATAACGGCGTCGAGCGGCGGACGGGTGAGTAATGCCTGGGAAATT

[0037] GCCCTGATGTGGGGGATAACCATTGGAAACGATGGCTAATACCGCATAATAGCTTCGG

[0038] CTCAAAGAGGGGGACCTTCGGGCCTCTCGCGTCAGGATATGCCCAGGTGGGATTAGCT

[0039] AGTTGGTGAGGTAATGGCTCACCAAGGCGACGATCCCTAGCTGGTCTGAGAGGATGAT

[0040] CAGCCACACTGGAACTGAGACACGGTCCAGACTCCTACGGGAGGCAGCAGTGGGGAA

[0041] TATTGCACAATGGGCGCAAGCCTGATGCAGCCATGCCGCGTGTATGAAGAAGGCCTTC

[0042] GGGTTGTAAAGTACTTTCAGCAGTGAGGAAGGTGGATGTGTTAATAGCNCNTTCATTT

[0043] GACGTTAGCTGCAGAAGAAGCACCGGCTAACTCCGTGCCAGCAGCCGCGGTAATACGG

[0044] AGGGTGCGAGCGTTAATCGGAATTACTGGGCGTAAAGCGCATGCAGGTGGTTTGTTAA

[0045] GTCAGATGTGAAAGCCCGGGGCTCAACCTCGGAATTGCATTTGAAACTGGCAGACTAG

[0046] AGTACTGTAGAGGGGGGTAGAATTTCAGGTGTANCGGTGAAATGCGTAGAGATCTAAA

[0047] GGAATACCGGTGGGCGAAGGCGGCCCCCTGGACAGATACTGACACTCAAGATGCGAA

[0048] AAGCGTGGGGAGCAAACAGGATTAGATACCCCTGGTAGTCCACGCCGTAAACGATGTC

[0049] TACTTGGAGGTTGTGGCCTTGAGCCGTGGCTTTCGGAGCTAACGCGTTAAGTAGACCGC

[0050] CTGGGGAGTACGGTCGCAAGATTAAAACTCAAATGAATTGACGGGGGCCCGCACAAG

[0051] CGGTGGAGCATGTGGTTTAATTCGATGCAACGCGAAGAACCTTACCTACTCTTGACATC

[0052] CTCAGAAGAGACTGGAGACAGTCTTGTGCCTTCGGGAACTGAGAGACAGGTGCTGCAT

[0053] GGCTGTCGTCAGCTCGTGTTGTGAAATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCT

[0054] TATCCTTGTTTGCCAGCGAGTCATGTCGGGAACTCCAGGGAGACTGCCGGTGATAAAC

[0055] CGGAGGAAGGTGGGGACGACGTCAAGTCATCATGGCCCTTACGAGTAGGGCTACACAC

[0056] GTGCTACAATGGCGCATACAGAGGGCGGCCAACTTGCGAAAGTGAGCGAATCCCAAA

[0057] AAGTGCGTCGTAGTCCGGATTGGAGTCTGCAACTCGACTCCATGAAGTCGGAATCGCT

[0058] AGTAATCGTAGATCAGAATGCTACGGTGAATACGTTCCCGGGCCTTGTACACACCGCC

[0059] CGTCACACCATGGGAGTGGGCTGCAAAAGAAGTGGGTAGTTTAACCTTCGGGGGGACG

[0060] CTCACCACTTTGTGGTTCA (SEQ ID NO. 11).

[0061] The sequences of the 5 primer pairs are shown below:

[0062] The target fragment size is 101bp:

[0063] Forward primer vibrioF1: GCGGCAGGCCTAACACATGCAAGTCGAGCG (SEQ ID NO.3) Reverse primer vibrioR1: TCCCAGGCATTACTCACCCGTCCGCCGCTCGACGC (SEQ ID NO.4) Target fragment size is 102 bp:

[0064] Forward primer vibrioF2: CTCCTACGGGAGGCAGCAGTGGGGAATATTGCA (SEQ ID NO.5) Reverse primer vibrioR2: GAAAGTACTTTACAACCCGAAGGCCTTCTTCATAC (SEQ ID NO.6) Target fragment size is 100bp:

[0065] Forward primer vibrioF3: GAAACTGGCAGACTAGAGTACTGTAGAGGG (SEQ ID NO.7) Reverse primer vibrioR3: GGGCCGCCTTCGCCCACCGGTATTCCTTTA (SEQ ID NO.8) Target fragment size is 113 bp:

[0066] Forward primer vibrioF4: ACAAGCGGTGGAGCATGTGGTTTAATTCGA (SEQ ID NO.1) Reverse primer vibrioR4: CTGTCTCTCAGTTCCCGAAGGCACAAGACTGTCTC (SEQ ID NO.2) Target fragment size is 153bp:

[0067] Forward primer vibrioF5: GGAACTGAGACACGGTCCAGACTCCTACGG (SEQ ID NO.9) Reverse primer vibrioR5: GCTATTAAACACATCCACCTTCCTCACTGCTGAA (SEQ ID NO.10)

[0068] The sequences of the 5 probes are shown below:

[0069] probe_vibrio1:

[0070] ACTCACCCGTCCGCCGCTCGACGCCGTTA(FAM-dT)C(THF)(BHQ1-dT)TCCCCGAAG GTTCA-C3-spacer

[0071] probe_vibrio2:

[0072] GAAGGCCTTCTTCATACACGCGGCATGGC(FAM-dT)GC(THF)(BHQ1-dT)CAGGCTT GCGCCCA-C3-spacer

[0073] probe_vibrio3:

[0074] CCGCCTTCGCCCACCGGTATTCCTTTAGA(FAM-dT)C(THF)C(BHQ1-dT)ACGCATTTC ACC-C3-spacer

[0075] probe_vibrio4:

[0076] GATGCAACGCGAAGAACCTTACCTACTCT(FAM-dT)GAC(THF)(BHQ1-dT)CCTCAGA AGAGACT

[0077] probe_vibrio5:

[0078] GACTCCAATCCGGACTACGACGCACTTTT(FAM-dT)GGG(THF)(BHQ1-dT)TCGCTCACTTTCGCA

[0079] 2. Establish a real-time fluorescent RPA reaction system

[0080] Real-time RPA reactions were performed using nucleic acid amplification reagent (fluorescent type) (catalog number S002ZC) from Hangzhou Zhongce Biotechnology Co., Ltd., in a volume of 50 μL. The RPA reaction system for each reaction tube was as follows: 2 μL of 10 μM forward primer, 2 μL of 10 μM reverse primer, 0.8 μL of 10 μM probe, 25 μL of A Buffer (fluorescent basal buffer) provided by the kit, 12.7 μL of ultrapure water, 5 μL of preserved Vibrio lysate (DSM19607) DNA template, and 2.5 μL of B Buffer (magnesium acetate solution) provided by the kit, for a total of 50 μL.

[0081] Add the reactants to each reaction tube in the following order: 12.7 μL ultrapure water, 25 μL A Buffer, 5 μL DNA template, 2 μL 10 μM forward primer, 2 μL 10 μM reverse primer, 0.8 μL 10 μM probe, and 2.5 μL B Buffer. Then place the reaction tubes in a fluorescence analyzer equipped with a miniature optical detector and react at 39 °C for 20 min.

[0082] 3. Specificity and sensitivity testing

[0083] The stability of RPA with different primer-probe combinations was tested. The results showed that only the combination of forward primer vibrioF4, reverse primer vibrioR4, and probe probe_vibrio4 exhibited a stable specific amplification curve. Figure 1 (A is the positive control, B is the amplification curve of Vibrio lysate) Other combinations did not have stable amplification curves. Among them, the positive control was tested after adding reagents according to the kit instructions: 1 tube of reaction powder, 25 μL of A Buffer, 4 μL of C Buffer, 13.5 μL of pure water, 5 μL of positive control, 2.5 μL of B Buffer, and a total volume of 50 μL.

[0084] For sensitivity testing, the cultured Vibrio lysate DNA (sample template concentration 126.3 ng / μL) was serially diluted 10 times. 1 10 2 5*10 2 10 3 5*10 3 10 4 5*10 4 Then, using the forward primer vibrioF4, the reverse primer vibrioR4, and the probe probe_vibrio4, RPA testing was performed, and stable RPA amplification fluorescence signals were detected in all cases. Figure 2 The detection limit can reach 10. -4 ng / μL.

[0085] Figure 2 This is the amplification curve of RPA under 10-fold serial dilution of DNA using the combination of forward primer vibrioF4, reverse primer vibrioR4, and probe probe_vibrio4. To collect fluorescence more concentratedly, 300 cycles were set, with each cycle lasting 15 seconds, for a total of 75 minutes. The experimental results show that a clear curve appeared after 80 cycles and 20 minutes (the thickened line is shown).

[0086] The above are merely preferred embodiments of the present invention. It should be noted that the above preferred embodiments should not be construed as limiting the present invention, and the scope of protection of the present invention should be determined by the scope defined in the claims. Persons skilled in the art will appreciate that improvements and modifications may be made without departing from the spirit and scope of the present invention, and such improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A primer-probe combination for rapid isothermal detection of Vibrio lysinensis, characterized in that, It includes an RPA primer pair and a fluorescent probe; the sequence of the RPA primer pair is as follows: Forward primer: 5'-ACAAGCGGTGGAGCATGTGGTTTAATTCGA-3'; Reverse primer: 5'-CTGTCTCTCAGTTCCCGAAGGCACAAGACTGTCTC-3'; The sequence of the fluorescent probe is as follows: 5'-GATGCAACGCGAAGAACCTTACCTACTCT(FAM-dT)GAC(THF)(BHQ1-dT)CCTCA GAAGAGACT-3'.

2. The application of the primer-probe combination according to claim 1 in the preparation of a kit for the rapid isothermal detection of Vibrio lysodeana.

3. A kit for rapid detection of Vibrio lysate at isothermal temperature, characterized in that, It includes the primer-probe combination as described in claim 1.

4. The reagent kit according to claim 3, characterized in that, The kit also includes a fluorescent basic buffer, magnesium acetate, and ultrapure water.

5. The application of the primer-probe combination of claim 1 or the kit of claim 3 or 4 in the isothermal rapid detection of Vibrio lysodeana.

6. The application according to claim 5, characterized in that, The constant temperature is 39℃.

7. A method for rapid detection of Vibrio lysinensis at isothermal temperature, characterized in that, Includes the following steps: a. Extract DNA from the sample to be tested; b. Using the DNA from step a as a template, perform real-time fluorescent recombinase polymerase amplification using the primer-probe combination described in claim 1 or the kit described in claim 3 or 4 to obtain the amplification product; c. If a fluorescent signal is present, the sample to be tested contains Vibrio lysodesiforme; if no fluorescent signal is present, the sample to be tested does not contain Vibrio lysodesiforme.

8. The method according to claim 7, characterized in that, The reaction system for real-time fluorescent recombinase polymerase amplification is as follows: 2 μL of 10 μM forward primer, 2 μL of 10 μM reverse primer, 0.8 μL of 10 μM probe, 25 μL of fluorescent basal buffer, 12.7 μL of ultrapure water, 5 μL of DNA template, and 2.5 μL of magnesium acetate solution, totaling 50 μL.

9. The method according to claim 7 or 8, characterized in that, The reaction conditions for the real-time fluorescent recombinase polymerase amplification were: 39℃ for 20 min.

10. The method according to claim 7 or 8, characterized in that, The concentration of the DNA is 10-15 ng / μL.

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