A primer set, kit, method and application for detecting aeromonas

Abstract

The application belongs to the field of molecular biology, and specifically discloses a primer set, a kit, a method and application for detecting Aeromonas. The primer set comprises an upstream primer AER-1F and a downstream primer AER-1R, the nucleotide sequence of the upstream primer AER-1F is shown as SEQ ID NO. 1, and the nucleotide sequence of the downstream primer AER-1R is shown as SEQ ID NO. 2. The application provides a new genetic marker AER-1 as a new target of Aeromonas, and designs a corresponding primer set. The detection method based on the primer set has high specificity and sensitivity, and the sensitivity for detecting Aeromonas nucleic acid can reach 7.415 x 10 ‑5 ng / μL, which can quickly and accurately detect Aeromonas bacteria including Aeromonas dhakai, and can analyze the polymorphic distribution abundance of Aeromonas in the environment.

Description

Technical Field

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

[0002] Aeromonas bacteria are common Gram-negative facultative anaerobic cocci, widely distributed in aquatic environments such as aquaculture. Some of these bacteria, such as *Aeromonas dhakensi*, *Aeromonas hydrophila*, *Aeromonas verrucosa*, and *Aeromonas guinea pigensis*, are pathogens in aquaculture and are closely related to healthy aquaculture. Existing methods, such as patent ZL201210460319.3, disclose the detection of Aeromonas in water, but do not include the identification of *Aeromonas dhakensi*. With the development of aquaculture diseases, the types of pathogens and the main prevalent pathogens are also changing, and existing methods are no longer sufficient to meet current needs.

[0003] Furthermore, existing methods are mainly limited to the detection of Aeromonas pathogens and cannot analyze the polymorphic distribution and abundance of individual Aeromonas bacteria in the environment. Aquatic environments often contain a variety of bacteria; for example, more than 30 species of Aeromonas have been identified in water, including common species such as Citrobacter, Enterobacter, Pseudomonas, Salmonella, Pantothenia, and Acinetobacter. The polymorphic distribution and abundance of Aeromonas pathogens in the environment is closely related to the occurrence of diseases. Therefore, establishing a method that can specifically identify Aeromonas bacteria from among numerous bacteria and analyze the polymorphic distribution and abundance of each Aeromonas genus, especially the major pathogens, in aquatic environments is of great guiding significance for monitoring the reproduction and epidemic patterns of this genus of pathogens and for timely prevention and control of diseases. Summary of the Invention

[0004] The purpose of this invention is to overcome the shortcomings of existing technologies. The inventors utilize *Aeromonas dacarinae* strain 202108B2 (Genbank NO. OQ283677), *Aeromonas hydrophila* strain Jxsks1 (Genbank NO. KJ150710.1), *Aeromonas vernix* FZ1N (Genbank NO. MF716716), *Aeromonas guinea pig* XG3N-2 (Genbank NO. MF716666), and *Aeromonas temperateus* YTXWN (Genbank NO. MF716666) isolated from fish. NO.OQ283651 was subjected to whole-genome sequencing. Through comparative genomic analysis, molecular markers for Aeromonas spp. were screened to identify sequence fragments with high common similarity but also differences, forming a molecular marker sequence library. Based on this library, numerous primers were designed for experimental validation, verifying specificity and sensitivity. Ultimately, molecular markers and primers with high specificity and sensitivity were obtained, capable of both identifying Aeromonas spp. bacteria and analyzing the polymorphic distribution and abundance of Aeromonas spp. bacteria. The specific technical solution is as follows:

[0005] A primer set for detecting Aeromonas hydrophila includes an upstream primer AER-1F and a downstream primer AER-1R. The nucleotide sequence of the upstream primer AER-1F is shown in SEQ ID NO.1, and the nucleotide sequence of the downstream primer AER-1R is shown in SEQ ID NO.2. The target gene of the primer set is AER-1 of Aeromonas hydrophila, and the nucleotide sequence of AER-1 is shown in SEQ ID NO.3.

[0006] The novel target AER-1 used in this invention is a genetic marker that exists in Aeromonas and exhibits a certain degree of conservation among genera and species. It can specifically identify Aeromonas dacca, Aeromonas vernix, Aeromonas hydrophila, Aeromonas guinea pigii, and Aeromonas temperate, and can also analyze the polymorphic distribution and abundance of Aeromonas bacteria in the environment. Based on the novel target AER-1, upstream primer AER-1F and downstream primer AER-1R were designed and synthesized, achieving a sensitivity of 7.415 × 10⁻⁶ for the detection of Aeromonas spp. DNA nucleic acid. -5 ng / μL.

[0007] The detection method based on the novel target AER-1 and primer set of this invention can not only identify Aeromonas in the environment, but also effectively distinguish Aeromonas bacteria from other genera such as Acinetobacter, Pseudomonas, Salmonella, Bacillus, and Pantotheca. It can also analyze the polymorphic distribution and abundance of Aeromonas bacteria such as Aeromonas hydrophila, Aeromonas vernix, Aeromonas guinea pig, Aeromonas temperate, and Aeromonas daca in the environment, and has the characteristics of high sensitivity and strong specificity.

[0008] Based on this, the present invention also provides a method for analyzing the abundance of Aeromonas polymorphism in water, comprising the following steps: extracting total DNA from water samples, amplifying the DNA using the primer set described above, constructing a library from the amplified products, and comparing the amplified products with the AER-1 sequence using high-throughput sequencing to obtain the distribution abundance of Aeromonas in the water samples.

[0009] Furthermore, the primer set described above can be used in the preparation of products for detecting Aeromonas, such as identification reagents or kits. Based on this, the present invention also provides a kit for detecting Aeromonas, 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.

[0010] Furthermore, this invention also provides a method for detecting Aeromonas, 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 in the sample based on the bands after electrophoresis. This method can be used for rapid and accurate detection of Aeromonas in water or animal tissues (mainly aquatic animals).

[0011] 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 57℃ 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 when the annealing temperature is 57℃, the bands are the clearest and thickest, indicating the maximum amplification; while when the 62℃ condition is selected, almost no bands are observed. Therefore, the preferred annealing temperature of this invention is 57℃.

[0012] The corresponding PCR reaction system is 25 μL, including 12.5 μL of 2×PCR Mix, 1 μL of upstream primer AER-1F, 1 μL of downstream primer AER-1R, 9.5 μL of ddH2O, and 1 μL of template.

[0013] The beneficial effects of this invention are as follows: This invention provides a novel genetic marker, AER-1, as a new target for Aeromonas hydrophila, and designs a corresponding primer set. The detection method proposed based on this primer set has high specificity and high sensitivity, and its sensitivity for detecting Aeromonas hydrophila DNA nucleic acid can reach 7.415 × 10⁻⁶. -5With a concentration of ng / μL, this kit enables rapid and accurate detection of Aeromonas bacteria in a variety of biological and environmental samples. Furthermore, based on the novel genetic marker AER-1, the kit and detection method can analyze the polymorphic distribution and abundance of Aeromonas species in the environment, such as *Aeromonas daca*, *Aeromonas hydrophila*, *Aeromonas versicolor*, *Aeromonas guinea pigensis*, and *Aeromonas temperate*. Each Aeromonas genus can be well distinguished, and cluster analysis shows that each species clusters into its own clade. Attached Figure Description

[0014] Figure 1 The image shows the annealing temperature screening chart for primer pairs AER-1F / AER-1R.

[0015] Figure 2 The image shown is a graph of primer specificity detection results;

[0016] Figure 3 The image shows the sensitivity detection of the primer pair AER-1F / AER-1R;

[0017] Figure 4 The image shown is a sequence alignment analysis diagram;

[0018] Figure 5 The diagram shown is a genetic distance analysis chart of Aeromonas hydrophila.

[0019] Figure 6 The diagram shown is a cluster analysis chart of Aeromonas hydrophila.

[0020] Figure 7 The following is a flowchart of the sample collection process;

[0021] Figure 8 The diagram shows the bacterial species composition based on the 16S rRNA gene sequence.

[0022] Figure 9 The image shows the polymorphism distribution of the AER-1 gene marker sequence in Aeromonas hydrophila. Detailed Implementation

[0023] 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.

[0024] 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.

[0025] Example 1:

[0026] A primer set for detecting Aeromonas and analyzing Aeromonas polymorphism is disclosed. The primer set includes an upstream primer AER-1F and a downstream primer AER-1R. The nucleotide sequence of the upstream primer AER-1F is shown in SEQ ID NO.1 (5'-GCCGGCTTCAAGCATTTC-3'), and the nucleotide sequence of the downstream primer AER-1R is shown in SEQ ID NO.2 (5'-GGACCTTCAGGAGTCTCGA-3'). The target gene of the primer set is Aeromonas AER-1, and the nucleotide sequence of AER-1 is shown in SEQ ID NO.3.

[0027] Example 2:

[0028] Based on the primer set of Example 1, a primer annealing temperature screening experiment was conducted, as detailed below:

[0029] (1) Annealing temperature setting. Annealing temperature was screened using a PCR instrument. Reference primer design reference temperatures were used, and six temperature gradients of 57℃, 58℃, 59℃, 60℃, 61℃, and 62℃ were designed to detect the suitable temperatures for Aeromonas hydrophila (taking Aeromonas dacca strain 202108B3 as an example) by primer pairs AER-1F (5'-GCCGGCTTCAAGCATTTC-3') and AER-1R (5'-GGACCTTCAGGAGTCTCGA-3').

[0030] (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 AER-1F, 1 μL of downstream primer AER-1R, 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.

[0031] The results are as follows Figure 1 As shown, by Figure 1It was found that Aeromonas dacca DNA could be amplified into a clear positive band of 719 bp at temperatures of 57℃, 58℃, 59℃, 60℃, and 61℃. However, the band was the clearest and thickest at 57℃, indicating the highest amplification. Almost no band was observed at 62℃. These results indicate that primer pair AER-1F / AER-1R amplifies Aeromonas dacca most effectively at an annealing temperature of 57℃. Therefore, 57℃ was chosen as the annealing temperature for primer pair AER-1F / AER-1R. The lane numbers represent the annealing temperature in °C, and lane M represents the DL2000 Marker.

[0032] Example 3:

[0033] Based on the primer set from Example 1, Aeromonas-specific detection was performed, as detailed below:

[0034] (1) Selection of test and control strains. Twenty-one Aeromonas strains were selected, including 10 Aeromonas hydrophila, 6 Aeromonas verrucosa, 2 Aeromonas daca, 1 Aeromonas temperate, and 1 Aeromonas guinea pig strain as test strains. Twenty-five other common environmental bacteria, including Citrobacter, Enterobacter, Pseudomonas, Salmonella, Pantothenia, and Acinetobacter, were used as control strains. Bacterial DNA was extracted using a kit, and AER-1F / AER-1R primers were used as templates for specific detection. 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 1.

[0035] Table 1

[0036]

[0037]

[0038] (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 AER-1F, 1 μL downstream primer AER-1R, 9.5 μL dd H2O, and 1 μL DNA template. The PCR reaction conditions were: 94℃ pre-denaturation for 4 min, followed by 94℃ denaturation for 30 s, 57℃ 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.

[0039] The results are as follows Figure 2 As shown, by Figure 2It can be seen that all 21 Aeromonas bacteria strains amplified a clear band of 719 bp, while none of the 25 non-Aeromonas bacteria strains amplified a band, indicating that the primer pair AER-1F / AER-1R amplification of Aeromonas bacteria is highly specific. The lanes correspond to the strain numbers in Table 1, with lane M representing the DL2000 Marker.

[0040] Example 4:

[0041] Based on the primer set from Example 1, nucleic acid sample sensitivity was tested, as detailed below:

[0042] (1) Preparation of bacterial culture gradient concentrations. Aeromonas DNA extracted using the kit was measured to be 7.415 ng / μL using a micro-spectrophotometer (as shown in Table 2). After 10-fold serial dilutions, 10... -1 10 -2 Continue diluting to 10 -5 The concentration gradients were as follows: original DNA concentration was 7.415 ng / μL, 10 -1 The gradient is 7.415 × 10⁻⁶. -1 ng / μL, 10 -2 The gradient is 7.415 × 10⁻⁶. - 2 ng / μL, 10 -3 The gradient is 7.415 × 10⁻⁶. -3 ng / μL, 10 -4 The gradient is 7.415 × 10⁻⁶. -4 ng / μL, 10 -5 The gradient is 7.415 × 10⁻⁶. -5 ng / μL.

[0043] Table 2

[0044] 1 blank -0.007 0 0 0.6 0.3 DNA 2 Aeromonas DNA 7.415 0.148 0.096 1.54 0.8 DNA

[0045] (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 AER-1F, 1 μL downstream primer AER-1R, 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, 57℃ 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.

[0046] The results are as follows Figure 3 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 3 It can be seen that 10 -4 Gradient concentrations and above can show very clear bands, 10 -5 Even with gradient concentrations, faint bands can still be observed; therefore, the sensitivity of this primer for detecting nucleic acids is 7.415 × 10⁻⁶. - 5 ng / μL.

[0047] Example 5:

[0048] Based on the primer set of Example 1, *Aeromonas dacca* strain 202108B1, *Aeromonas hydrophila* strain Jxsks1 (KJ150710.1), *Aeromonas vernix* FZ1N, *Aeromonas guinea pig* XG3N-2, and *Aeromonas temperate* YTXWN were amplified. The amplified products were sent to Shanghai Sangon Biotech Co., Ltd. for sequencing. The sequencing primer pair was AER-1F / AER-1R. The sequences were aligned and clustered using MEGA6 software. The results are as follows: Figures 4-6 As shown.

[0049] The results show that ( Figure 4 , Figure 5 The sequences of different bacterial species exhibit high conservation, allowing for amplification using common primers, yet they also possess certain differences, which can be distinguished by cluster analysis, with each species clustering into a separate clade. Figure 6 Using this primer pair to analyze Aeromonas satisfies both the identification function and the diversity analysis function of Aeromonas.

[0050] Example 6:

[0051] Based on the primer set from Example 1, polymorphism analysis of Aeromonas spp. in water was performed, as detailed below:

[0052] (1) Sample Collection. 300 mL of aquaculture water was collected from the pond. The water was first filtered using medium-speed filter paper with a pore size of 30-50 micrometers to remove plankton, and then vacuum filtered using a 0.22 micrometer aquatic microporous membrane (Tianjin Jinteng). The membrane was stored at -80℃ for later use. The sample collection process is detailed below. Figure 7 .

[0053] (2) The filter membrane was directly sent to a biotechnology company for total environmental DNA extraction and microbial diversity analysis. Bacterial 16S rRNA was amplified and a library constructed using primers 338F_806R. High-throughput sequencing and 16S RNA sequence alignment were then performed to analyze the bacterial species composition using a pie chart (e.g., ...). Figure 8 (As shown).

[0054] (3) The extracted DNA was amplified using primer pair AER-1F / AER-1R. The amplified products were used to construct a library, and high-throughput sequencing was performed to compare the DNA with the AER-1 genetic marker of Aeromonas strains. A pie chart analysis of the compositional polymorphism of Aeromonas spp. was then conducted (e.g.,...). Figure 9 (As shown).

[0055] The results showed that conventional 16S rRNA population analysis ( Figure 8 The bacterial species composition in the environment was as follows: uncultured bacteria (Aurantimicrobium unclassified) accounted for 20.41%, uncultured Flavobacterium uncultured accounted for 18.89%, unclassified Polynucleobacter accounted for 12.38%, unclassified Acinetobacter accounted for 8.18%, Flavobacterium sp. THG-DN6.19 accounted for 7.39%, unclassified Mycobacterium accounted for 6.64%, Acinetobacter johnsonii accounted for 4.87%, Rhizobiales accounted for 4.38%, and Limnohabita accounted for 4.38%. Aeromonas spp. accounted for 2.85%, planktophila versatilis for 1.57%, Escherichia coli for 1.15%, and other species with a content of less than 1% accounted for 11.49%. The abundance and distribution of Aeromonas spp. in the environment could not be determined by 16S rRNA polymorphism analysis.

[0056] Polymorphism analysis of Aeromonas hydrophila in water was performed using primer pairs AER-1F / AER-1R. The results showed that ( Figure 9Aeromonas veronii accounted for 39.72%, Aeromonas hydrophila and Aeromonas dhakensis accounted for 14.54%, Aeromonas sobria accounted for 3.90%, Aeromonas caviae accounted for 1.77%, and other species accounted for 29.43%.

[0057] 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 hydrophila, characterized in that, The primer set includes an upstream primer AER-1F and a downstream primer AER-1R. The nucleotide sequence of the upstream primer AER-1F is shown in SEQ ID NO.1, and the nucleotide sequence of the downstream primer AER-1R is shown in SEQ ID NO.

2. The target gene of the primer set is Aeromonas AER-1, and the nucleotide sequence of AER-1 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 hydrophila.

3. A kit for detecting Aeromonas hydrophila, characterized in that, It includes the primer set as described in claim 1, and also includes Taq DNA polymerase and ddH2O.

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

5. A method for detecting Aeromonas hydrophila 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 in the sample was determined based on the bands after electrophoresis.

6. The method according to claim 5, characterized in that, The PCR reaction conditions were as follows: 94℃ pre-denaturation for 4 min, followed by 94℃ denaturation for 30 s, 57℃ annealing for 30 s, 72℃ extension for 1 min, 35 cycles, and then a final extension reaction at 72℃ for 7 min.

7. The method according to claim 5, characterized in that, The PCR reaction system is 25 μL, including 12.5 μL of 2×PCRMix, 1 μL of the upstream primer AER-1F, 1 μL of the downstream primer AER-1R, 9.5 μL of ddH2O, and 1 μL of template.

8. The use of the method according to any one of claims 5 to 7 in the detection of Aeromonas in water or animal tissues.

9. The application of the primer set described in claim 1 in the analysis of polymorphism abundance of Aeromonas spp. in water.

10. A method for analyzing the abundance of polymorphism in Aeromonas spp. in water, characterized in that, Includes the following steps: Total DNA was extracted from the water sample and amplified using the primer set described in claim 1. The amplified product was used to construct a library and compared with the AER-1 by high-throughput sequencing to obtain the distribution abundance of Aeromonas spp. in the water sample.

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