A primer set, kit, method, and application for detecting Aeromonas dacca.

Abstract

This invention belongs to the field of molecular biology, specifically disclosing a primer set, kit, method, and application for detecting Aeromonas dacca. The primer set includes an upstream primer DK1F and a downstream primer DK1R. The nucleotide sequence of the upstream primer DK1F is shown in SEQ ID NO.1, and the nucleotide sequence of the downstream primer DK1R is shown in SEQ ID NO.2. This invention provides a novel genetic marker, DK1, as a new target for Aeromonas dacca, and designs a corresponding primer set. The detection method based on this primer set exhibits high specificity and sensitivity, achieving a sensitivity of 1.38 cfu / μL for Aeromonas dacca bacterial suspension detection and 9.37 × 10⁻⁶ for nucleic acid detection. ‑4 With a concentration of ng / μL, it can rapidly and accurately detect Aeromonas dacarba in a variety of biological and environmental samples.

Description

Technical Field

[0001] This invention belongs to the field of molecular biology, specifically relating to a primer set, kit, method, and application for detecting Aeromonas dacca. Background Technology

[0002] Aeromonas dhakensis, distributed in aquatic environments, is one of the most important bacterial pathogens in aquatic animals, capable of infecting both vertebrates and invertebrates, and is also a common cause of human infection. Formerly known as *Aeromonas dhakensis*, it was first isolated in 2008 by Martínez-Murcia et al. from the skin of imported ornamental fish and the water in the fishpond at an aquarium in Portugal. Subsequently, its widespread clinical and environmental presence was observed. In 2013, Beaz-Hidalgo et al. determined that *Aeromonas hydrophila* subspecies *dhakensis* and *Aeromonas dhakensis* are the same species, and that *dhakensis* differs from other subspecies of *Aeromonas hydrophila*. Therefore, they merged *Aeromonas hydrophila* subspecies *dhakensis* and *Aeromonas dhakensis*, officially naming it *Aeromonas dhakensis*, a new species. Studies have shown that clinically isolated *Aeromonas dacarina* carries a large number of virulence genes, such as hlyA, GcaT, alt, act, exu, fla, lip, ahyB, and aerA. It exhibits extremely high virulence to hematologic cytology and also has toxic effects on intestinal cells. This species is closely homologous to *Aeromonas hydrophila* and *Aeromonas guinea pig*, with no significant differences in biochemical phenotypes. Clinically, *Aeromonas dacarina* is often mistakenly identified as either *Aeromonas hydrophila* or *Aeromonas guinea pig*. Therefore, rapid identification of *Aeromonas dacarina* has significant clinical value.

[0003] Currently, Aeromonas dacarina is mainly identified through molecular methods such as gene sequencing, including dual-gene sequencing of gyrB and rpoD, or tandem comparative analysis of 16S rRNA and gyrB. However, gene sequencing methods are time-consuming, costly, and technically demanding, making them unsuitable for large-scale clinical applications. Therefore, a rapid, accurate, and simple method for identifying Aeromonas dacarina is needed. Summary of the Invention

[0004] The purpose of this invention is to overcome the shortcomings of existing technologies. The inventors isolated Aeromonas dacarba strain 202108B1 from fish and performed whole-genome sequencing. Through comparative genomic analysis, molecular markers for Aeromonas dacarba were screened to identify sequence fragments common to Aeromonas dacarba that differ from other Aeromonas species. These fragments were compiled into a molecular marker sequence library. Based on this library, numerous primers were designed and tested to verify specificity and sensitivity. Ultimately, highly specific and sensitive molecular markers and primers for identifying Aeromonas dacarba were obtained. The specific technical solution is as follows:

[0005] A primer set for detecting Aeromonas dacca, the primer set comprising an upstream primer DK1F and a downstream primer DK1R, the nucleotide sequence of the upstream primer DK1F being shown in SEQ ID NO.1, and the nucleotide sequence of the downstream primer DK1R being shown in SEQ ID NO.2; the target gene of the primer set is DK1 of Aeromonas dacca, the nucleotide sequence of which is shown in SEQ ID NO.3.

[0006] The novel target DK1 used in this invention is a genetic marker that exists in *Aeromonas dacca* and exhibits a certain degree of interspecies conservation, enabling specific identification of *Aeromonas dacca*. Based on the novel target DK1, upstream primer DK1F and downstream primer DK1R were designed and synthesized, achieving a detection sensitivity of 1.38 cfu / μL for *Aeromonas dacca* bacterial suspensions and a nucleic acid detection sensitivity of 9.37 × 10⁻⁶. -4 ng / μL.

[0007] This invention, based on the novel target DK1 and primer set, not only differentiates *Aeromonas daca* from other genera such as *Pseudomonas*, *Salmonella*, bacilli, and pantothenic bacteria, but also distinguishes *Aeromonas* from other *Aeromonas* species such as *Aeromonas hydrophila*, *Aeromonas vernix*, *Aeromonas guinea pig*, and *Aeromonas temperate*, demonstrating high specificity. Furthermore, it can be directly applied to bacterial suspension detection without the need for DNA extraction, making it simple, easy to perform, and enabling rapid and accurate detection of *Aeromonas daca* in various biological and environmental samples.

[0008] The primer set described above can be used to prepare products for detecting Aeromonas dacarba, such as identification reagents or kits. Based on this, the present invention also provides a kit for detecting Aeromonas dacarba, comprising the primer set described above, as well as Taq DNA polymerase and ddH2O. In some preferred embodiments, positive and negative controls are also included.

[0009] Furthermore, this invention also provides a method for detecting Aeromonas dacarba, comprising the following steps: (1) extracting DNA from a sample and performing PCR amplification using the primer set described above; (2) detecting the PCR amplification product by gel electrophoresis (preferably agarose gel electrophoresis), and determining the presence of Aeromonas dacarba in the sample based on the bands after electrophoresis. This method can be used for rapid and accurate detection of Aeromonas dacarba in water bodies or animal tissues (mainly aquatic animals).

[0010] In the above method, the PCR reaction conditions are as follows: pre-denaturation at 94℃ for 4 min, followed by denaturation at 94℃ for 30 s, annealing at 65℃ for 30 s, extension at 72℃ for 1 min, for 35 cycles, and then a final extension at 72℃ for 7 min. The inventors discovered that a clear positive band is obtained when the annealing temperature is 65℃, while other temperatures result in mismatched band sizes, disordered and unclear bands, or no bands. Therefore, the preferred annealing temperature in this invention is 65℃.

[0011] The corresponding PCR reaction system is 25 μL, including 12.5 μL of 2×PCR Mix, 1 μL of upstream primer DK1F, 1 μL of downstream primer DK1R, 8.5 μL of ddH2O, and 2 μL of template.

[0012] The beneficial effects of this invention are as follows: This invention provides a novel genetic marker, DK1, as a new target for Aeromonas dacca, and designs a corresponding primer set. The detection method proposed based on this primer set has high specificity and high sensitivity. The sensitivity for detecting Aeromonas dacca bacterial culture can reach 1.38 cfu / μL, and the sensitivity for nucleic acid detection can reach 9.37 × 10⁻⁶. -4 With a concentration of ng / μL, it can rapidly and accurately detect Aeromonas dacarba in a variety of biological and environmental samples. Attached Figure Description

[0013] Figure 1 The image shows the amplification effect of primer pair DK1F / DK1R at different temperatures;

[0014] Figure 2 The image shown is a graph of primer specificity detection results;

[0015] Figure 3 The image shows colony diagrams on plates with different concentration gradients.

[0016] Figure 4 The diagram shows the PCR reaction conditions for bacterial culture.

[0017] Figure 5 The image shown is a graph of the bacterial culture PCR sensitivity detection results;

[0018] Figure 6 The image shown is a graph of nucleic acid PCR sensitivity detection results;

[0019] Figure 7 The image shown is a graph of the tissue sample test results. Detailed Implementation

[0020] The following will provide a clear and complete description of the concept and technical effects of the present invention in conjunction with the embodiments and accompanying drawings, so as to fully understand the purpose, solution and effects of the present invention.

[0021] The DNA extraction kit used in the following examples is the TaKaRaMiniBEST Bacteria Genomic DNA Extraction Kit Ver. 3.0 from Takara Bio Engineering (Dalian) Co., Ltd., and the PCR reaction reagent is the DreamTaq PCR mix from Thermo Fisher Scientific (China) Co., Ltd.

[0022] Example 1:

[0023] A primer set for detecting Aeromonas dacca, comprising an upstream primer DK1F and a downstream primer DK1R, wherein the nucleotide sequence of the upstream primer DK1F is shown in SEQ ID NO.1 (5'-CCCCGTGAGCAAACG-3') and the nucleotide sequence of the downstream primer DK1R is shown in SEQ ID NO.2 (5'-ACTTGCCCGCCTCACCC-3'); the target gene of the primer set is DK1 of Aeromonas dacca, and the nucleotide sequence of DK1 is shown in SEQ ID NO.3.

[0024] Example 2:

[0025] Based on the primer set of Example 1, a primer annealing temperature screening experiment was conducted, as detailed below:

[0026] (1) Annealing temperature setting. Annealing temperatures were screened using a PCR instrument, selecting four temperature gradients: 55℃, 57℃, 63℃, and 65℃, to determine the optimal temperatures for detecting *Aeromonas dacca* using primer pairs DK1F (5'-CCCCGTGAGCAAACG-3') and DK1R (5'-ACTTGCCC GCCTCACCC-3'). Six strains of bacteria, including *Aeromonas hydrophila*, *Aeromonas vesiculosus*, *Aeromonas temperate*, and *Aeromonas vulgaris*, were used as negative controls. Strain information is shown in Table 1.

[0027] Table 1

[0028]

[0029] (2) Reaction system. Using the bacterial DNA described above as a template, PCR was performed in a 25 μL volume, consisting of: 12.5 μL of 2×PCR Mix, 1 μL of upstream primer DK1F, 1 μL of downstream primer DK1R, 9.5 μL of ddH2O, and 1 μL of DNA template. The PCR reaction conditions were: 94℃ pre-denaturation for 4 min, followed by 94℃ denaturation for 30 s, annealing at the above different temperatures for 30 s, extension at 72℃ for 1 min, for 35 cycles, and then a final extension at 72℃ for 7 min. After the reaction, 5 μL of the PCR product was observed by 1.2% agarose gel electrophoresis.

[0030] The results are as follows Figure 1 As shown, by Figure 1 It was found that both *Aeromonas dacca* DNA strains amplified a clear positive band of 874 bp at each temperature (lanes 1-2). *Aeromonas dacca* also showed specific amplification at an annealing temperature of 65℃, while other *Aeromonas* strains showed blurred or no bands (lanes 3-8). This indicates that primer pair DK1F / DK1R amplifies *Aeromonas dacca* most effectively at an annealing temperature of 65℃. Therefore, 65℃ was chosen as the annealing temperature for primer pair DK1F / DK1R. The lane numbers correspond to the strain numbers in Table 1, with lane M representing the DL2000 Marker.

[0031] Example 3:

[0032] Based on the primer set from Example 1, specific detection of Aeromonas dacarba was performed, as detailed below:

[0033] (1) Selection of test and control strains. Two strains of Aeromonas daca were used as test bacteria, and 28 other Aeromonas species, such as Aeromonas hydrophila, Aeromonas vernix, Aeromonas guinea pig, Aeromonas temperate, and Citrobacter flexneri, were used as control strains. Bacterial DNA was extracted using a kit, and the bacterial DNA was used as a template for specific detection using DK1F / DK1R primers. All selected bacteria were preserved in the Aquatic Animal Nutrition and Disease Control Research Laboratory of Jiangxi Provincial Fisheries Research Institute. Bacterial information is shown in Table 2.

[0034] Table 2

[0035]

[0036] (2) Primer-specific PCR detection. The bacterial DNA described above was used as a template for PCR. The reaction volume was 25 μL, composed of: 12.5 μL 2×PCR Mix, 1 μL upstream primer DK1F, 1 μL downstream primer DK1R, 9.5 μL ddH2O, and 1 μL DNA template. The PCR reaction conditions were: 94℃ pre-denaturation for 4 min, followed by 94℃ denaturation for 30 s, 65℃ annealing for 30 s, and 72℃ extension for 1 min, for 35 cycles, followed by a final extension at 72℃ for 7 min. After the reaction, 5 μL of the PCR product was observed by 1.2% agarose gel electrophoresis.

[0037] The results are as follows Figure 2 As shown, by Figure 2It was found that only two Aeromonas dacca DNA strains amplified to produce a clear positive band of 874 bp (lanes 1-2). Other Aeromonas strains showed disordered or unclear bands, or no bands at all (lanes 3-12). No amplification bands were observed in the PCR products of other non-Aeromonas bacteria (lanes 13-30). This indicates that the primer pair DK1F / DK1R amplifies Aeromonas dacca with high specificity. The lane numbers correspond to the strain numbers in Table 2, with lane M representing the DL2000 Marker.

[0038] Example 4:

[0039] Based on the primer set from Example 1, sensitivity testing for bacteria in water was performed, as detailed below:

[0040] (1) Preparation of bacterial culture with gradient concentrations. Daka strain JXB1, stored at -80℃, was taken out of the freezer and streaked onto plates for 14 hours. Single colonies were picked and inoculated into liquid culture medium, and cultured overnight to obtain the stock solution. The stock solution was then serially diluted 10-fold, up to nine dilutions, each resulting in a 10-fold increase in concentration. 0 10 -1 10 -2 10 -3 10 -4 10 -5 10 -6 10 -7 10 -8 Take 10 -5 10 -6 10 -7 Three gradients of bacterial suspension were counted on 100 μL plates, with each gradient spread onto 3 plates. The results were 10. -5 The plate colonies are too dense, 10 -7 The plate had a low colony count, only 10. -6 Gradient plates have a moderate number of colonies, with a count of 10. -6 The average number of colonies from three plates of gradient bacterial suspension was 138 (e.g., ...). Figure 3 As shown, from left to right, they are 10. -5 10 -6 10 -7 (Three gradients). The converted concentrations of the bacterial culture are as follows: stock solution 10... 0 The concentration was 1.38 × 10⁻⁶. 9 cfu / mL, 10 -1 The concentration was 1.38 × 10⁻⁶. 8 cfu / mL, 10 -2 The concentration was 1.38 × 10⁻⁶. 7 cfu / mL, 10 -3 The concentration was 1.38 × 10⁻⁶. 6 cfu / mL, 10 -4The concentration was 1.38 × 10⁻⁶. 5 cfu / mL, 10 -5 The concentration was 1.38 × 10⁻⁶. 4 cfu / mL, 10 -6 The concentration was 1.38 × 10⁻⁶. 3 cfu / mL, 10 -7 The concentration was 1.38 × 10⁻⁶. 2 cfu / mL, 10 -8 The concentration was 1.38 × 10⁻⁶. 1 cfu / mL.

[0041] (2) Colony PCR. Using the serially diluted bacterial cultures described above as templates, colony PCR was performed. The blank control template was ddH2O. The reaction volume was 25 μL, composed of the following: 12.5 μL of 2×PCR Mix, 1 μL of upstream primer DK1F, 1 μL of downstream primer DK1R, 8.5 μL of ddH2O, and 2 μL of bacterial template. PCR reaction conditions were as follows: Figure 4 As shown, the specific reaction was as follows: pre-denaturation at 95℃ for 7 min, followed by denaturation at 94℃ for 30 s, annealing at 65℃ for 30 s, extension at 72℃ for 1 min, for 35 cycles, and then a final extension at 72℃ for 7 min. After the reaction, 5 μL of the PCR product was observed by 1.2% agarose gel electrophoresis.

[0042] The results are as follows Figure 5 (Lane 1 is the negative control, lane 2 is 10) 0 Lane 3 is 10 -1 Lane 4 is 10 -2 Lane 5 is 10 -3 Lane 6 is 10 -4 Lane 7 is 10 -5 8 lanes are 10 -6 Lane 9 is 10 -7 10 lanes for swimming -8 M is DL2000Marker), by Figure 5 It can be seen that the lowest gradient dilution to 10 -6 That is, a clear band can be detected at a concentration of 1.38 cfu / μL, indicating that the primer has a sensitivity of 1.38 cfu / μL for detecting bacteria in water.

[0043] Example 5:

[0044] Based on the primer set from Example 1, nucleic acid sample sensitivity was tested, as detailed below:

[0045] (1) Preparation of nucleic acid sample gradient concentrations. DNA from Aeromonas dacca strain 202108B1, extracted using a kit, was determined to be 9.37 ng / μL using a micro-spectrophotometer (as shown in Table 3). After 10-fold serial dilutions, 10... -1 10 -2 Continue diluting to 10 -5 The concentration gradients were as follows: original DNA concentration was 9.37 ng / μL, 10 -1 The gradient is 9.37 × 10 -1 ng / μL, 10 -2 The gradient is 9.37 × 10 -2 ng / μL, 10 -3 The gradient is 9.37 × 10 -3 ng / μL, 10 -4 The gradient is 9.37 × 10 -4 ng / μL, 10 -5 The gradient is 9.37 × 10 -5 ng / μL.

[0046] Table 3

[0047] serial number Sample Name Nucleic acid concentration (ng / μl) A260 A280 A260 / A280 A260 / A230 Nucleic acid types 1 blank -0.008 0 0 3.63 0.87 DNA 2 202108B1 9.37 0.187 0.117 1.6 0.99 DNA

[0048] (2) Nucleic acid PCR. Take 10 of the above gradient... -1 ~10 -5 Diluted nucleic acid was used as a template, with ddH2O as the blank control template. PCR was performed in a 25 μL reaction volume, consisting of: 12.5 μL 2×PCR Mix, 1 μL upstream primer DK1F, 1 μL downstream primer DK1R, 8.5 μL ddH2O, and 2 μL DNA template. The PCR conditions were: 94℃ pre-denaturation for 4 min, followed by 94℃ denaturation for 30 s, 65℃ annealing for 30 s, and 72℃ extension for 1 min, for 35 cycles, and then a final extension at 72℃ for 7 min. After the reaction, 5 μL of the PCR product was observed by 1.2% agarose gel electrophoresis.

[0049] The results are as follows Figure 6 As shown (lane 1 is 10) -1 Lane 2 is 10 -2 Lane 3 is 10 -3 Lane 4 is 10 -4 Lane 5 is 10 -5 Lane 6 was the negative control, and M was the DL2000 Marker. Figure 6 It can be seen that 10 -4 Very clear bands can be observed above gradient concentrations, 10 -4 Gradient concentrations also produce bands; therefore, the sensitivity of this primer for detecting nucleic acids is 9.37 × 10⁻⁶.-4 ng / μL.

[0050] Example 6:

[0051] Based on the primer set of Example 1, sensitivity testing of tissue samples was performed, as detailed below:

[0052] (1) Infection experiment. Aeromonas dacca strain 202108B1 was taken from a -80℃ freezer, activated in nutrient broth, and cultured at 1.38 × 10⁻⁶ ppm. 8 Grass carp were infected with Dacarbazine CFU / mL via intraperitoneal injection, with grass carp injected with physiological saline serving as a negative control. After death from Dacarbazine infection, liver tissue was dissected and DNA was extracted as template DNA for the experimental group. The nucleic acid of Aeromonas deltaea strain 202108B1 served as a positive control. Simultaneously, liver DNA was extracted from grass carp injected with physiological saline as a negative control.

[0053] (2) PCR identification. DNA templates from the above experimental group, positive control group, and negative control group were used for PCR. The reaction volume was 25 μL, composed of: 12.5 μL 2×PCR Mix, 1 μL upstream primer DK1F, 1 μL downstream primer DK1R, 8.5 μL ddH2O, and 2 μL DNA template. The PCR reaction conditions were: 94℃ pre-denaturation for 4 min, followed by 94℃ denaturation for 30 s, 65℃ annealing for 30 s, and 72℃ extension for 1 min, for 35 cycles, followed by a final extension at 72℃ for 7 min. After the reaction, 5 μL of the PCR product was observed by 1.2% agarose gel electrophoresis.

[0054] The results are as follows Figure 7 As shown (lane 1 is the negative control, lane 2 is the positive control, lane 3 is the experimental group, M is the DL2000 Marker), by Figure 7 It can be seen that the positive control and experimental groups were able to amplify a clear band of 874 bp, while the negative control group had no band. The results indicate that the primer can effectively identify Aeromonas dacarina in tissues and can be used for the clinical diagnosis of Aeromonas dacarina bacterial infection.

[0055] The above description is merely a preferred embodiment of the present invention. The present invention is not limited to the above-described embodiments. Any embodiment that achieves the technical effects of the present invention using the same means should fall within the protection scope of the present invention. Within the protection scope of the present invention, various modifications and variations can be made to the technical solutions and / or implementation methods.

Claims

1. A primer set for detecting Aeromonas dacca, characterized in that, The primer set includes an upstream primer DK1F and a downstream primer DK1R. The nucleotide sequence of the upstream primer DK1F is shown in SEQ ID NO.1, and the nucleotide sequence of the downstream primer DK1R is shown in SEQ ID NO.

2. The target gene of the primer set is DK1 of Aeromonas daca, and the nucleotide sequence of DK1 is shown in SEQ ID NO.

3.

2. The application of the primer set according to claim 1 in the preparation of products for detecting Aeromonas dacca.

3. A kit for detecting Aeromonas dacca, characterized in that, Includes the primer set as described in claim 1.

4. The reagent kit according to claim 3, characterized in that, It also includes Taq DNA polymerase and ddH₂O.

5. The reagent kit according to claim 3, characterized in that, It also includes positive and negative controls.

6. A method for detecting Aeromonas dacarbamate for non-diagnostic or therapeutic purposes, characterized in that, Includes the following steps: (1) Extract DNA from the sample and perform PCR amplification using the primer set described in claim 1; (2) PCR amplification products were detected by gel electrophoresis, and the presence of Aeromonas dacarba in the sample was determined based on the bands after electrophoresis.

7. The method according to claim 6, characterized in that, The PCR reaction conditions were as follows: pre-denaturation at 94℃ for 4 min, followed by denaturation at 94℃ for 30 s, annealing at 65℃ for 30 s, extension at 72℃ for 1 min, 35 cycles, and then extension at 72℃ for 7 min.

8. The method according to claim 6, characterized in that, The PCR reaction system is 25 μL, including 12.5 μL of 2×PCRMix, 1 μL of the upstream primer DK1F, 1 μL of the downstream primer DK1R, 8.5 μL of ddH2O, and 2 μL of template.

9. The method according to claim 6, characterized in that, The detection was performed using agarose gel electrophoresis.

10. The use of the method according to any one of claims 6 to 9 in the detection of Aeromonas dacarba in water or animal tissues for non-diagnostic or therapeutic purposes.

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