Double PCR (Polymerase Chain Reaction) detection primer for flavobacterium psychrophilum and aeromonas salmonicida and application

Abstract

The invention discloses a duplex PCR (Polymerase Chain Reaction) detection primer for flavobacterium psychrophilum and aeromonas salmonicida, and relates to a detection primer for flavobacterium psychrophilum and aeromonas salmonicida and application of the detection primer. The duplex PCR detection primer comprises an rpoC upstream primer / downstream primer and an fstA upstream primer / downstream primer, and the application of the duplex PCR detection primer of flavobacterium psychrophilum and aeromonas salmonicida in fish mixed pathogen detection is provided. When the rpoC and fstA primer combination is used for detecting flavobacterium psychrophilum and aeromonas salmonicida, the sensitivity of the rpoC and fstA primer combination can reach 1 * 10 <-4 > ng / mu L, and the sensitivity is good. The rainbow trout multiple bacterial diseases caused by flavobacterium psychrophilum and aeromonas salmonicida and the mixed infection of the rainbow trout multiple bacterial diseases have good application value in rapid identification of rainbow trout multiple bacterial diseases caused by flavobacterium psychrophilum and aeromonas salmonicida.

Description

Technical Field

[0001] The invention relates to a detection primer for psychrophilic Flavobacterium and Aeromonas salmonicida and application thereof. Background Art

[0002] Flavobacterium psychrophilum and Aeromonas salmonicida are major pathogens infecting rainbow trout and other salmonids. Both can cause highly lethal diseases (such as bacterial coldwater sickness and furunculosis). In aquaculture environments, they often coexist due to fluctuating water temperatures and host immunosuppression, significantly increasing the risk of mixed infections. Mixed infections can cause additive tissue damage (such as skin ulcers and internal bleeding) and immune disruption, making clinical diagnosis and treatment far more complex than with a single infection. Therefore, developing detection methods for these two pathogens is crucial to ensure the sustainable development of the aquaculture industry through scientific detection methods.

[0003] The pathological symptoms of rainbow trout infected with Psychrophilic Flavobacterium and Aeromonas salmonicida are similar. In clinical testing, separation and purification methods are usually used to confirm whether it is a mixed infection. Therefore, PCR detection of the pathogens of rainbow trout infected with Psychrophilic Flavobacterium and Aeromonas salmonicida should be performed separately. Among them, the isolation and culture time of Psychrophilic Flavobacterium is usually 72 h - 96 h, which is very time-consuming. Therefore, the construction of an efficient and accurate mixed pathogen detection technology has important diagnostic and application significance.

[0004] In order to solve the problem that the current method for detecting Flavobacterium psychrophilum and Aeromonas salmonicida pathogens cannot reflect the situation of mixed infection, the present invention provides dual PCR detection primers for Flavobacterium psychrophilum and Aeromonas salmonicida and applications thereof.

[0005] The dual PCR detection primers for psychrophilic Flavobacterium and salmonicidal Aeromonas include: a primer pair rpoC and a primer pair fstA, wherein the upstream primer of the primer pair rpoC is 5'-ATAGAACGCATAAGCCAGAA-3', and the downstream primer of the primer pair rpoC is 5'-CTACACCACAACGGTCACAA-3'; the upstream primer of the primer pair fstA is 5'-CCGTTTCCCGCTTTTCCT-3', and the downstream primer of the primer pair fstA is 5'-CCTTGTAGTGTCCTTCTCCCG-3'.

[0006] Application of the above-mentioned dual PCR detection primers for psychrophilic bacterium and salmonicidal Aeromonas in the detection of mixed pathogens in fish.

[0007] Furthermore, the application method is as follows: extract the genomic DNA of the fish to be tested, perform a dual PCR reaction using the above-mentioned dual PCR detection primers, and perform agarose gel electrophoresis after amplification; what kind of test results do they correspond to; the volume ratio of the primer pair rpoC to the primer pair fstA is 1:1, and the concentrations of both are 10 μmol / L.

[0008] Furthermore, the total volume of the duplex PCR reaction system was 25 μL, and the configuration method was as follows: 12.5 μL of 2 × Rapid Taq MasterMix, 1 μL of genomic DNA of the fish to be tested, 1 μL each of rpoC-upstream primer and downstream primer, 1 μL each of fstA-upstream primer and downstream primer, and water was added to 25 μL; the PCR program was pre-denaturation at 95°C for 3 min; denaturation at 95°C for 15 s, annealing at 52°C-62°C for 15 s, and extension at 72°C for 15 s, for a total of 30 cycles; final extension at 72°C for 5 min.

[0009] The present study compared the full genomes of Flavobacterium psychrophilum and Aeromonas salmonicida to identify specific sequences, eliminating cross-reactions with other pathogens (such as Vibrio anguillarum and Edwardsiella edwardsiella). The gene sequences were then aligned and analyzed using DNAman software. Finally, species-specific primers were designed using the rpoC gene of Flavobacterium psychrophilum and the fstA gene of Aeromonas salmonicida as templates.

[0010] The dual PCR detection primers described in the present invention can simultaneously detect the presence of two pathogenic bacteria, Flavobacterium psychrophilum and Aeromonas salmonicida, through a single PCR reaction. This allows immediate confirmation of whether the diseased fish are co-infected, thus avoiding missed detection due to similar symptoms. The rpoC and fstA primer combination described in the present invention can detect Flavobacterium psychrophilum and Aeromonas salmonicida with a sensitivity of up to 1×10 -4 ng / μL, with good sensitivity, and the invented dual PCR detection system also has good specificity. The invention has good application value for the rapid identification of multiple bacterial diseases of rainbow trout caused by psychrophilic Flavobacterium and Aeromonas salmonicida and their mixed infections.

[0011] The dual PCR technology of the present invention is of great significance in the rapid diagnosis of mixed infections of fish pathogens due to its high efficiency and cost advantages. However, its development and application face multiple technical challenges, requiring breakthroughs in multiple dimensions such as primer design, reaction system optimization, detection sensitivity balance, and sample complexity. The present invention uses specific genes (rpoC gene and fstA gene) of psychrophilic Flavobacterium and salmonicidal Aeromonas to ensure that the two pairs of primers only specifically bind to their respective target genes, avoiding nonspecific amplification, and gradient PCR is used to screen for the optimal annealing temperature. To avoid confusion between amplified fragments of similar sizes during gel electrophoresis, the sizes of the PCR amplification products are 135bp (rpoC) and 348bp (fstA), respectively.

[0012] This invention addresses the problem that current methods for detecting the pathogens Flavobacterium psychrophilum and Aeromonas salmonicida cannot detect mixed infections. Using the dual PCR method, the specific genes of both pathogens can be simultaneously amplified in a single reaction, significantly shortening detection time and reducing sample consumption. In cases of overlapping symptoms (such as surface ulcers and visceral lesions), dual PCR can clearly distinguish the pathogen type and the degree of infection, providing a basis for targeted and precise medication. Therefore, the development of efficient and accurate mixed pathogen detection technology has important diagnostic and application significance. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Figure 1 The results of agarose gel electrophoresis in the conventional PCR reaction in Example 1 are shown;

[0014] Figure 2 The agarose gel electrophoresis results of the duplex PCR reaction in Example 1;

[0015] Figure 3 The electrophoresis results of different annealing temperatures in Example 1 are shown;

[0016] Figure 4 The primer ratio optimization results in Example 1 are as follows;

[0017] Figure 5 The primer specificity verification results in Example 1 are as follows;

[0018] Figure 6 The sensitivity test results of the dual PCR reaction system in Example 1 are as follows;

[0019] Figure 7 This is the test result in Example 2. DETAILED DESCRIPTION

[0020] The following will be combined with the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the embodiments described are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making any creative efforts shall fall within the scope of protection of the present invention.

[0021] It should be noted that, in the absence of conflict, the embodiments of the present invention and the features in the embodiments may be combined with each other.

[0022] Specific embodiment one: The dual PCR detection primers for psychrophilic bacterium and salmonicidal Aeromonas in this embodiment are: primer pair rpoC and primer pair fstA, wherein the upstream primer of primer pair rpoC is 5'-ATAGAACGCATAAGCCAGAA-3', and the downstream primer of primer pair rpoC is 5'-CTACACCACAACGGTCACAA-3'; the upstream primer of primer pair fstA is 5'-CCGTTTCCCGCTTTTCCT-3', and the downstream primer of primer pair fstA is 5'-CCTTGTAGTGTCCTTCTCCCG-3'.

[0023] Specific embodiment 2: Application of the dual PCR detection primers of psychrophilic Flavobacterium and Aeromonas salmonicida described in specific embodiment 1 in the detection of mixed pathogens in fish.

[0024] Specific embodiment 3: This embodiment differs from specific embodiment 2 in the following: The application method is to extract genomic DNA from the fish to be tested, perform a dual PCR reaction using the dual PCR detection primers described in specific embodiment 1, and perform amplification by agarose gel electrophoresis; the corresponding test results are as follows: the volume ratio of the rpoC primer pair to the fstA primer pair is 1:1, and the concentration of each is 10 μmol / L. Other steps and parameters are the same as those in specific embodiment 2.

[0025] According to the brightness screening results of the gel electrophoresis bands, when the volume ratio of the rpoC primer pair to the fstA primer pair in the dual PCR detection described in this embodiment is 1:1, the two bands of the gel electrophoresis are the clearest and the effect is the best.

[0026] Specific embodiment 4: This embodiment differs from specific embodiment 3 in that the total volume of the duplex PCR reaction system is 25 μL and is prepared as follows: 12.5 μL of 2 × Rapid Taq Master Mix, 1 μL of genomic DNA from the fish to be tested, 1 μL each of the rpoC upstream primer and downstream primer, and 1 μL each of the fstA upstream primer and downstream primer, with water added to 25 μL. The PCR program is: initial denaturation at 95°C for 3 minutes; denaturation at 95°C for 15 seconds, annealing at 52°C-62°C for 15 seconds, and extension at 72°C for 15 seconds, for a total of 30 cycles; and a final extension at 72°C for 5 minutes. All other steps and parameters are the same as those in specific embodiment 3.

[0027] Example 1 Establishment of Duplex PCR Method

[0028] The psychrophilic Flavobacterium used in this example was the CH06 strain from a previous laboratory study (Chai Jingru, Wang Di, Lu Tongyan, et al. Isolation, Identification, and Pathogenicity of Psychrophilic Flavobacterium from Rainbow Trout [J]. Journal of Fisheries of China, 2021, 45(06):929-939). Aeromonas salmonicida was a standard strain (ATCC 33658) from the ATCC Culture Collection. Both bacterial strains were cultured and genomic DNA was extracted using a bacterial genomic DNA extraction kit or other recognized, equally effective extraction method and stored at -20°C until ready for use.

[0029] The total volume of the duplex PCR reaction system was 25 μL and was prepared as follows: 12.5 μL of 2 × Rapid Taq Master Mix, 1 μL of the test sample DNA, 1 μL each of rpoC-F / R, and 1 μL each of fstA-F / R, and the mixture was made up to 25 μL with water. The PCR program was an initial denaturation at 95°C for 3 min; 30 cycles of denaturation at 95°C for 15 s, annealing at 52°C–62°C for 15 s, and extension at 72°C for 15 s; and a final extension at 72°C for 5 min.

[0030] 1. Screening of primer pairs

[0031] Three pairs of primers (As1, As2, As3) were designed based on the fstA gene (AM712656.1) of Aeromonas salmonicida, and three pairs of primers (Fp1, Fp2, Fp3) were designed based on the rpoC gene (66553079) of Flavobacterium psychrophilum. Conventional PCR reactions were performed to verify the specificity of the primers.

[0032] The total volume of the PCR reaction system was 25 μL and was prepared as follows: 12.5 μL of 2 × Rapid Taq Master Mix, 1 μL of the test sample DNA, 2 μL of F / R, and water to 25 μL. The PCR program was 95°C for 3 minutes of initial denaturation; 30 cycles of denaturation at 95°C for 15 seconds, annealing at 52°C–62°C for 15 seconds, and extension at 72°C for 15 seconds; and a final extension at 72°C for 5 minutes. The PCR products were analyzed by 2% agarose gel electrophoresis. Figure 1 ( Figure 1 (Agarose gel electrophoresis results in a common PCR reaction) are shown, where (M) DL500Marker, (1) As1; (2) As2; (3) As3; (4) Fp1; (5) Fp2; (6) Fp3. Figure 1 It can be seen that in the ordinary PCR reaction, all six pairs of primers can bind to DNA and amplify the target band.

[0033] Three primer pairs (As1, As2, As3) for Aeromonas salmonicida were combined with three primer pairs (Fp1, Fp2, Fp3) for Flavobacterium psychrophilum. The total volume of the duplex PCR reaction system was 25 μL. The following configuration was used: 12.5 μL of 2 × Rapid Taq Master Mix, 1 μL of the test sample DNA, 1 μL each of As-F / R, and 1 μL each of Fp-F / R, and the mixture was made up to 25 μL with water. The PCR program was a preliminary denaturation at 95°C for 3 minutes; 30 cycles of denaturation at 95°C for 15 seconds, annealing at 52°C–62°C for 15 seconds, and extension at 72°C for 15 seconds; and a final extension at 72°C for 5 minutes. The duplex PCR products were analyzed by 2% agarose gel electrophoresis. The results are shown in Figure 2. Figure 2 ( Figure 2 The results of agarose gel electrophoresis in the duplex PCR reaction are shown in Figure 2, where (M) DL500 Marker, (1) As1+Fp1; (2) As2+Fp1; (3) As3+Fp1; (4) As1+Fp2; (5) As2+Fp2; (6) As3+Fp2; (7) As1+Fp3; (8) As2+Fp3; (9) As3+Fp3.

[0034] Depend on Figure 2 It can be seen that in the duplex PCR reaction, (1) the primer pair As1+Fp1 can ensure the amplification of the target band, and the target bands are farther apart, which can better distinguish between Aeromonas salmonicida and Flavobacterium psychrophilum. Therefore, As1 (fstA) and Fp1 (rpoC) were finally selected as primers.

[0035] 2. Different annealing temperatures

[0036] The duplex PCR products were analyzed by 2% agarose gel electrophoresis using different annealing temperatures, namely (1) 52°C; (2) 54°C; (3) 56°C; (4) 58°C; (5) 60°C; and (6) 62°C. The results are shown in the figure. Figure 3 ( Figure 3 (shown as the electrophoresis results at different annealing temperatures in Example 1), where (M) is DL500 Marker. Figure 3 As can be seen, the amplification effect is good within the annealing temperature range of 56-62°C, the band specificity is good, and the band size is consistent with the expected. The corresponding band was excised and sent back for sequencing. The resulting sequence is consistent with the target fragment sequence, indicating that the amplified fragment in this example is indeed the target gene fragment.

[0037] 3. Ratio of two primer pairs

[0038] Under the condition that the total volume of the PCR reaction system remains unchanged at 25 μL, three treatments were set: treatment 1: the volume ratio of the rpoC primer pair to the fstA primer pair was 1:1, treatment 2: the volume ratio of the rpoC primer pair to the fstA primer pair was 2:1, and treatment 3: the volume ratio of the rpoC primer pair to the fstA primer pair was 1:2. The PCR procedure was as described above, and the annealing temperature was set at 60°C for amplification. After amplification, agarose gel electrophoresis was performed, and the most suitable volume ratio was screened according to the brightness of the gel electrophoresis bands. Figure 4 The results of primer ratio optimization are shown in the figure. (M) DL500 Marker, (1) Treatment 1, (2) Treatment 2, and (3) Treatment 3. Figure 4 From the results, it can be seen that the two bands of treatment 1 (the volume ratio of rpoC primer pair to fstA primer pair is 1:1) gel electrophoresis have the best effect.

[0039] 4. Duplex PCR reaction system specificity test

[0040] To verify the specificity of the duplex PCR detection system constructed in this invention, genomic DNA from Aeromonas vernii, Aeromonas sobria, Aeromonas hydrophila, Pseudomonas filamentosus, Staphylococcus saprophyticus, Aeromonas salmonicida, and Flavobacterium psychrophilum was extracted and used as templates for duplex PCR reactions. The strain numbers, Latin names, and sources of each strain are shown in Table 1.

[0041] Duplex PCR amplification was performed with a primer pair volume ratio of 1:1 and an annealing temperature of 60°C. Figure 5 The results of primer specificity verification are shown in the figure: (M) DL500 Marker, (1) Aeromonas veseri, (2) Aeromonas sobria, (3) Aeromonas hydrophila, (4) Pseudomonas fluorescens, (5) Yersinia ruckeri, (6) Aeromonas salmonicida, (7) Flavobacterium psychrophilum, and (8) Aeromonas salmonicida + Flavobacterium psychrophilum. Figure 5 The results showed that the dual PCR detection system of the present invention has good specificity.

[0042] Table 1 Strains and sources

[0043] Table 1 Strains and sources

[0044]

[0045] 5. Duplex PCR reaction system sensitivity test

[0046] In order to verify the sensitivity of the dual PCR detection system constructed in the present invention, the bacterial genomic DNA was diluted 10 times in a gradient to make the DNA concentration gradient 10 0 , 10-1 , 10 -2 , 10 -3 , 10 -4 , 10 -5 , 10 -6 ng / μL and duplex PCR amplification was performed according to the kit and method established as described above, wherein the annealing temperature was set to 60°C.

[0047] The sensitivity test results of the dual PCR reaction system are as follows Figure 6 As shown, Figure 6 Medium (M) DL500 Marker; (1) 10 0 ; (2) 10 -1 ; (3) 10 -2 ; (4) 10 -3 ; (5) 10 -4 ; (6) 10 -5 ; (7) 10 -6 . Figure 6 The results showed that the sensitivity of the detection kit and method of the present invention to psychrophilic Flavobacterium and salmonicidal Aeromonas can reach 1×10 -4 ng / μL. The detection method of psychrophilic Flavobacterium has been published in the patent (Jiang Hucheng. Multiplex PCR primer pair combination for detection of psychrophilic Flavobacterium and its application: CN119040495 A[P]. 2024-11-29), with a sensitivity of 10 -3 ng / μL, the detection method of Aeromonas salmonicida in the published patent (Li Jie. A PCR detection kit for Aeromonas salmonicida subsp. salmonicida and its use method: CN 115651989 A[P]. 2023-01-31) has a sensitivity of 10 -3 Compared with the above-mentioned detection method, the detection sensitivity of the present invention is good.

[0048] The present invention obtains two pairs of primers for Flavobacterium psychrophilum and Aeromonas salmonicida with good specificity and combined detection activity, and optimizes the amplification system and primer concentration. The rpoC and fstA primer combination of the present invention, when added at a concentration of 1:1, can efficiently complete the rapid amplification of the target gene, and the detection sensitivity can reach 1×10 -4 The present invention has good application value for the rapid identification of multiple bacterial diseases of rainbow trout caused by psychrophilic Flavobacterium and Aeromonas salmonicida and their mixed infections.

[0049] Example 2 Specific application method in fish detection

[0050] 1. Experimental fish

[0051] One hundred healthy rainbow trout (15±2 g) were placed in an indoor aquarium for one week. The water temperature during the experiment was (14±1)°C and the stocking density was 30 trout / m 3 90 healthy fish of uniform size were randomly selected for the experiment. Before artificial infection, the experimental fish were bacteriologically tested to ensure that they did not carry specific pathogens.

[0052] 2. Artificial infection experiment and sample collection

[0053] Ten fish were tested in each experimental group (Aeromonas salmonicida; Flavobacterium psychrophilum; Flavobacterium psychrophilum:Aeromonas salmonicida 1:1 ratio; Aeromonas veseri; Aeromonas sobria; Aeromonas hydrophila; Pseudomonas fluorescens; and Yersinia ruckeri) for 14 days. Muscle tissue samples were collected from six fish in each group on day 14 and stored at -80°C for subsequent analysis.

[0054] 3. Bacterial infection detection

[0055] DNA was extracted from muscle tissue using a tissue DNA extraction kit or other recognized, equally effective extraction method. The total volume of the duplex PCR reaction system was 25 μL, using the following configuration: 12.5 μL of 2 × Rapid Taq Master Mix, 1 μL of the test sample DNA, 1 μL each of rpoC-F / R, and 1 μL each of fstA-F / R, and the mixture was diluted to 25 μL. The PCR program was a 95°C initial denaturation for 3 min, followed by 30 cycles of denaturation at 95°C for 15 s, annealing at 52°C–62°C for 15 s, and extension at 72°C for 15 s, followed by a final extension at 72°C for 5 min. Results are shown in the table. Figure 7 ( Figure 7 is the detection result in Example 2).

[0056] Figure 7 Medium (M) DL500 Marker; (1) Aeromonas salmonicida; (2) Psychrophilic Flavobacterium; (3) Psychrophilic Flavobacterium: Aeromonas salmonicida 1:1; (4) Aeromonas veseri; (5) Aeromonas sobria; (6) Aeromonas hydrophila; (7) Pseudomonas fluorescens; (8) Yersinia ruckeri. Figure 7 It can be seen that the DNA of the positive sample detected a positive band, while no positive band was detected in the other groups, indicating that the detection kit and detection method of the present invention can accurately detect Aeromonas salmonicida and Flavobacterium psychrophilum in aquatic products.

Claims

1. Dual PCR detection primers for Flavobacterium psychrophilum and Aeromonas salmonicida, characterized in that The dual PCR detection primers for Flavobacterium psychrophilum and Aeromonas salmonicida are: primer pair rpoC and primer pair fstA, among which the upstream primer of primer pair rpoC is 5'-ATAGAACGCATAAGCCAGAA-3', and the downstream primer of primer pair rpoC is 5'-CTACACCACAACGGTCACAA-3'; the upstream primer of primer pair fstA is 5'-CCGTTTCCCGCTTTTCCT-3', and the downstream primer of primer pair fstA is 5'-CCTTGTAGTGTCCTTCTCCCG-3'.

2. Use of the dual PCR detection primers for Flavobacterium psychrophilum and Aeromonas salmonicida according to claim 1 in the detection of mixed pathogens in fish.

3. The use of the dual PCR detection primers for Flavobacterium psychrophilum and Aeromonas salmonicida in the detection of mixed pathogens in fish according to claim 2, characterized in that: Extract genomic DNA from the fish to be tested, perform a dual PCR reaction using the dual PCR detection primers of claim 1, and perform agarose gel electrophoresis after amplification; what kind of test results do they correspond to; wherein the volume ratio of the primer pair rpoC to the primer pair fstA is 1:1, and the concentration of each is 10 μmol / L.

4. Use of the dual PCR detection primers for Flavobacterium psychrophilum and Aeromonas salmonicida in the detection of mixed pathogens in fish according to claim 3, characterized in that: The total volume of the duplex PCR reaction system was 25 μL, and the configuration method was as follows: 12.5 μL of 2 × RapidTaq Master Mix, 1 μL of genomic DNA of the fish to be tested, 1 μL each of rpoC upstream primer and downstream primer, 1 μL each of fstA upstream primer and downstream primer, and water was added to 25 μL; the PCR program was: pre-denaturation at 95°C for 3 min; denaturation at 95°C for 15 s, annealing at 52°C–62°C for 15 s, and extension at 72°C for 15 s, for a total of 30 cycles; and final extension at 72°C for 5 min.

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