A taqman fluorescent quantitative PCR detection method for aeromonas veronii

Abstract

The application relates to a TaqMan fluorescent quantitative PCR detection method of Aeromonas veronii, and the method is characterized in that a pair of specific primers for Aeromonas veronii is designed according to the whole genome sequence of Aeromonas veronii, and a TaqMan fluorescent quantitative PCR method capable of rapidly detecting the Aeromonas veronii type is established, the method has the characteristics of rapidness, convenience, good specificity and high sensitivity, can be widely applied to fish Aeromonas veronii detection, and has high application value in the aquaculture industry.

Description

Technical Field

[0001] This invention belongs to the field of molecular detection technology, specifically relating to a TaqMan real-time quantitative PCR detection method for Aeromonas versicolor. Background Technology

[0002] Aeromonas veronii belongs to the family Aeromonadaceae and the genus Aeromonas. It is a Gram-negative facultative anaerobic bacillus that is widely distributed in the environment, especially in freshwater and river water environments. It has strong adaptability to the environment and is highly infectious and has a high mortality rate in various regions.

[0003] In recent years, reports of Aeromonas verrucosum infection in fish have been increasing both domestically and internationally. Fish infected with Aeromonas verrucosum may exhibit symptoms such as skin ulcers or sores, organ hemorrhage and severe ascites, abdominal erythema or anal swelling, leading to diseases such as ulcerative infections, hemorrhagic septicemia, and erythematous lesions. Its prevalence has significantly increased, and it is now considered an important pathogen of fish ulcer syndrome. Potentially pathogenic Aeromonas verrucosum is frequently isolated from farmed fish and aquatic environments, indicating a potential risk to both fish and human health. Currently, detection methods for Aeromonas verrucosum mainly include enzyme-linked immunosorbent assay (ELISA), conventional PCR, multiplex PCR, molecular biology methods, and CRISPR / Cas system detection methods. These methods have drawbacks such as long detection times, susceptibility to sample contamination, and inaccurate quantification.

[0004] The TaqMan quantitative PCR method can monitor the amplification of the target gene in real time based on fluorescence signals and calculate the copy number of the target gene using a formula. This method is simple to operate, time-efficient, has good repeatability and accuracy, and high sensitivity. Furthermore, the amplification of the target gene is completed in a closed system, reducing the probability of sample contamination. Therefore, a TaqMan quantitative PCR method for the detection of Aeromonas verrucosa was established based on its specific DNA sequence. Summary of the Invention

[0005] To address the current technical problems of long detection time, easy sample contamination, and inaccurate quantification of Aeromonas versicolor, this invention provides a highly specific and sensitive TaqMan real-time PCR detection method for Aeromonas versicolor.

[0006] The present invention provides a primer set for the specific detection of Aeromonas verrucosa, the specific primer set comprising an upstream primer and a downstream primer, the upstream primer having a nucleotide sequence as shown in SEQ ID NO.1, and the downstream primer having a nucleotide sequence as shown in SEQ ID NO.2.

[0007] The present invention provides a specific probe for the specific detection of Aeromonas verrucosa, the specific probe having a nucleotide sequence as shown in SEQ ID NO.3; the 5' end of the specific probe has at least one fluorescently labeled reporter group, and the 3' end of the specific probe has at least one quenching group.

[0008] Furthermore, the fluorescently labeled reporter group includes FAM, and the quenching group includes TAMRA.

[0009] The present invention provides a kit for detecting or diagnosing Aeromonas verrucosa, the kit comprising the primer set and specific probes.

[0010] Furthermore, it also includes the reaction mixture, positive control, and negative control.

[0011] Furthermore, the reaction mixture contains Taq DNA polymerase, dNTPs, reaction buffer (TAKARA), and Mg. 2 The reaction solution containing the main components such as ⁺ is premixed.

[0012] This invention provides the use of the specific primer pairs, probes and / or kits in the preparation of products for the detection or diagnosis of Aeromonas verrucosa.

[0013] This invention provides a method for detecting the presence of Aeromonas versicolor in the environment, using the specific primer pairs, probes and / or kits to detect samples. This method is not for disease diagnosis or treatment purposes.

[0014] Furthermore, the reaction system consisted of: 10 μL of Premix Ex Taq, 0.8 μL of 10 μM probe, 0.4 μL each of 10 μM forward and reverse primers, 0.4 μL of 50× ROX Reference Dye, 6 μL of ddH2O, and 2 μL of template, totaling 20 μL. The reaction program was: 94℃ for 30 s; 94℃ for 5 s; 58℃ for 30 s, for a total of 40 cycles.

[0015] This invention provides a method for distinguishing Aeromonas versicolor from Aeromonas hydrophila, Bacillus, Vibrio cholerae, Edwardsiella tarda, Citrobacter, or Enterococcus faecalis. The method uses the specific primer pairs, probes, and / or kits to detect environmental samples that may contain the above-mentioned bacteria. This method is not for disease diagnosis or treatment purposes.

[0016] In summary, the beneficial effects of this invention are:

[0017] 1. This invention establishes a rapid, specific, sensitive, simple, and highly specific TaqMan real-time PCR method for detecting Aeromonas vesiculosus. Based on the complete genome sequence of Aeromonas vesiculosus, new target sites were screened, a pair of specific primers for Aeromonas vesiculosus was designed, amplified the 171 bp target protein gene, and constructed a recombinant plasmid (…). Figure 1 Using this as a standard, a TaqMan real-time PCR detection method was established, and standard curves and amplification curves were obtained. The specificity, sensitivity and repeatability were then verified.

[0018] 2. The standard curve of this invention was established using TaqMan real-time quantitative PCR amplification with standard plasmids diluted 10-fold as templates. The results showed ( Figure 2 The standard curve Ct value and the standard plasmid concentration have a good linear relationship. The equation of the standard curve is y=-3.167x+37.663, and the correlation coefficient is 0.9932.

[0019] 3. Specific detection results of this invention: Using the established TaqMan real-time PCR method, plasmid standards, Aeromonas verrucosa, Aeromonas hydrophila, Bacillus, Vibrio cholerae, Edwardsiella tarda, Citrobacter, Enterococcus faecalis, and ddH2O were detected. The results showed that, except for plasmid standards and Aeromonas verrucosa, which showed amplification curves, other pathogens and negative controls did not show amplification curves. Figure 3 This indicates that the TaqMan real-time PCR method established in this study has high specificity.

[0020] 4. Sensitivity test results of this invention: The detection limit of TaqMan quantitative PCR for plasmid standards is 3.25 × 10⁻⁶. 0 The limit of detection for plasmid standards in conventional PCR is 3.25 × 10⁻⁶ copies / μL. 3 copy / μL ( Figure 4 TaqMan quantitative PCR has a sensitivity 1000 times that of conventional PCR, indicating that this method has high sensitivity.

[0021] 5. Repeatability test results of the present invention: Standards at different dilutions were tested by TaqMan real-time PCR, and intra-batch and inter-batch repeatability were calculated. The coefficient of variation was less than 2% (Table 1), indicating that the established TaqMan real-time PCR detection method has good repeatability.

[0022] 6. Clinical Sample Detection Results: Using the established TaqMan quantitative PCR and conventional PCR methods, 55 tissue samples from diseased eels in different regions of Hubei Province were simultaneously tested. TaqMan quantitative PCR results showed a positive rate of 83.6% (46 / 55) for *Aeromonas vesiculosus* and a negative rate of 16.4% (9 / 55). Conventional PCR results showed a positive rate of 74.5% (41 / 55) for *Aeromonas vesiculosus* and a negative rate of 25.5% (14 / 55). All positive samples were from diseased eels. Samples that were positive by conventional PCR also showed positive results by TaqMan quantitative PCR. This indicates that the established TaqMan quantitative PCR method is more sensitive and can be used for the detection of *Aeromonas vesiculosus* in clinical samples, further demonstrating the high specificity of this method. Attached Figure Description

[0023] To more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings used in the embodiments will be briefly introduced below. It should be understood that the following drawings only show some embodiments of the present invention and should not be regarded as a limitation on the scope. For those skilled in the art, other related drawings can be obtained based on these drawings without creative effort.

[0024] Figure 1 This is a plasmid standard prepared by TaqMan real-time PCR in this invention. The concentration is 102.3 ng / μL, and the calculated value of the plasmid standard is 3.25 × 10⁻⁶. 10 Copy / μL. Where M: DL2000 DNA Marker; 1: Aeromonas vera; 2: ddH2O.

[0025] Figure 2 The standard curve (a) and amplification curve (b) of TaqMan real-time PCR in this invention are shown.

[0026] Figure 3 These are the specific results of TaqMan quantitative PCR in this invention. Wherein: 1: plasmid standard; 2: Aeromonas vera; 3: Aeromonas hydrophila; 4: Bacillus; 5: Vibrio cholerae; 6: Edwardsiella tarda; 7: Citrobacter; 8: Enterococcus faecalis; 9: ddH2O.

[0027] Figure 4 The results show the sensitivity test results of TaqMan real-time PCR (a) and conventional PCR (b) of this invention. M: DL2000 DNA Marker; 1-8: 3.25 × 10⁻⁸ 7 Copies / μL ~ 3.25 × 10⁻⁶ 0 Copy / μL. Detailed Implementation

[0028] The following embodiments are only used to more clearly illustrate the technical solutions of the present invention, and are therefore merely examples and should not be used to limit the scope of protection of the present invention. It should be noted that, unless otherwise stated, the technical or scientific terms used in this application should have the ordinary meaning understood by those skilled in the art. Unless specifically stated, the reagents, methods, and equipment used in this invention are conventional reagents, methods, and equipment in this technical field. Unless specifically stated, the reagents and materials used in the following embodiments are commercially available.

[0029] Example

[0030] A TaqMan real-time quantitative PCR method for detecting Aeromonas villus subtilis includes the following steps:

[0031] S1. Preparation of specific primers and probes: Based on the complete genome sequence of Aeromonas vesiculosus, new specific targets were selected, and specific primers (F1, R1) and probe (P1) for Aeromonas vesiculosus were designed and sequence alignment analysis was performed by BLAST. Among them, the upstream primer F1: 5'-TGATCATGGTGCGACAATTCCT-3' (SEQ ID NO.1), the downstream primer R1: 5'-TTCCACTCGGTCTCTTTATCACT-3' (SEQ ID NO.2), and the amplified nucleic acid fragment size was 171bp; probe P1: FAM-TCAAATCACGCTCCGCCTT-TAMRA (SEQ ID NO.3).

[0032] S2. Preparation of recombinant plasmid standards and DNA extraction; Inoculation of eel kidney cells with Aeromonas villi, collection of cell debris, and extraction of DNA using a bacterial genomic DNA extraction kit (Tiangen Biotech Co., Ltd.). Specifically, this includes: S201, discarding the cell suspension, adding an appropriate amount of PBS, scraping cells with a cell scraper and placing them into centrifuge tubes; S202, centrifuging at 5000 rpm for 5 min, discarding the supernatant, and collecting cell debris.

[0033] S3. PCR amplification: PCR amplification was performed on the extracted DNA template using specific primers. The amplification conditions were 94℃ for 5 min; 94℃ for 30 s, 58℃ for 30 s, 72℃ for 30 s, for a total of 30 cycles; and 72℃ for 7 min.

[0034] S4. Amplification and Detection Processing: Process the PCR products and sequence the recombinant plasmids that are identified as positive. Specifically, this includes: S401. After PCR products are subjected to 1% agarose gel electrophoresis, the PCR products are purified and recovered according to the instructions of the ordinary agarose gel DNA recovery kit, ligated with the pMD18-T vector, transformed into DH5α competent cells, and inoculated onto ampicillin-resistant LB plates; S402. After overnight incubation at 37°C, single colonies are picked for bacterial culture PCR identification; S403. Plasmids are extracted from the bacterial cultures that show positive PCR results and identified, and the recombinant plasmids that are identified as positive are sequenced.

[0035] S5. Optimize reaction conditions and establish a standard curve; determine the concentration of the recombinant plasmid using a micro spectrophotometer. Convert the corresponding copy number using the following formula: Copy number / μL = (ng / μL × 10⁻⁶) -9 )×(6.02×10 23 ) / (DNA length × 660). Diluted 10-fold (10 -1 ~10 -8 That is, 3.25 × 10 9 Copies / μL ~ 3.25 × 10⁻⁶ 2 A standard recombinant plasmid (copy / μL) was used as the template, and TaqMan real-time PCR was employed for detection. Data were collected after the reaction, and a standard curve was plotted. The 20 μL reaction mixture consisted of: 10 μL Premix Ex Taq, 0.8 μL probe (10 μM), 0.4 μL each of forward and reverse primers (10 μM), 0.4 μL ROX Reference Dye (50×) (TAKARA), 6 μL ddH2O, and 2 μL template. The reaction program was: 94℃ for 30 s; 94℃ for 5 s, 58℃ for 30 s, for a total of 40 cycles.

[0036] The results show ( Figure 2 The standard curve Ct value and the standard plasmid concentration have a good linear relationship. The equation of the standard curve is y=-3.167x+37.663, and the correlation coefficient is 0.9932.

[0037] S6. Specificity detection: Using genomic DNA of different species (Aeromonas versicolor, Aeromonas hydrophila, Bacillus, Vibrio cholerae, Edwardsiella tarda, Citrobacter, Enterococcus faecalis) as templates, the TaqMan real-time PCR method for Aeromonas versicolor established in this study was used for detection, with ddH2O as a negative control to evaluate the specificity of the method.

[0038] The results showed that, using the established TaqMan quantitative PCR method to detect plasmid standards, Aeromonas verrucosa, Aeromonas hydrophila, Bacillus, Vibrio cholerae, Edwardsiella tarda, Citrobacter, Enterococcus faecalis, and ddH2O, only plasmid standards and Aeromonas verrucosa showed amplification curves; the other pathogens and negative controls did not show amplification curves. Figure 3 This indicates that the TaqMan real-time PCR method established in this study has high specificity.

[0039] S7. Sensitivity test; serially dilute the recombinant plasmid standard 10-fold (10... -3 ~10 -10 The plasmids were then used as templates, with ddH2O as a negative control. The TaqMan quantitative PCR method established in this study was used for detection. Since there is currently no recommended detection method for this pathogen, this experiment also used conventional PCR as reported in the literature to simultaneously detect the above plasmid standards and compare the sensitivity of the two methods. The eight plasmid standards had a concentration of 3.25 × 10⁸. 7 Copies / μL, 3.25 × 10 6 Copies / μL, 3.25 × 10 5 Copies / μL, 3.25 × 10 4 Copies / μL, 3.25 × 10 3 Copies / μL, 3.25 × 10 2 Copies / μL, 3.25 × 10 1 Copies / μL, 3.25 × 10 0 Copy / μL.

[0040] The results showed that the detection limit of TaqMan real-time PCR for plasmid standards was 3.25 × 10⁻⁶. 0 The limit of detection for plasmid standards in conventional PCR is 3.25 × 10⁻⁶ copies / μL. 3 copy / μL ( Figure 4 TaqMan quantitative PCR has a sensitivity 1000 times that of conventional PCR, indicating that this method has high sensitivity.

[0041] S8. Repeatability test; diluted 10-fold (10 -2 ~10 -5 Using plasmid standards as templates, the intra-assay reproducibility of the method was tested using the TaqMan real-time PCR method established in this study, with each dilution tested in triplicate. For inter-assay reproducibility, plasmid standards of the above four dilutions were used as templates, and the method was tested every three days for three consecutive tests, with three replicates for each dilution. The mean thresholds within and between groups (Ct values) were calculated. The repeatability of the method was evaluated by measuring the standard deviation (s) and coefficient of variation (CV). Four plasmid standards were used, each with a concentration of 3.25 × 10⁻⁶. 8 Copies / μL, 3.25 × 10 7 Copies / μL, 3.25 × 10 6 Copies / μL, 3.25 × 10 5 Copy / μL.

[0042] The results showed that the coefficients of variation for intra- and inter-batch repeatability of the standards at different dilutions using TaqMan quantitative PCR were all less than 2% (Table 1), indicating that the established TaqMan quantitative PCR detection method has good repeatability.

[0043] Table 1. Results of TaqMan qPCR repeatability tests

[0044]

[0045] S9. Clinical Sample Detection: DNA was extracted from clinical samples using a tissue DNA extraction kit. Fifty-five tissue samples from diseased eels from different regions of Hubei Province were simultaneously detected using the TaqMan quantitative real-time PCR method established in this study and conventional PCR. The results are shown in Table 2. TaqMan quantitative real-time PCR showed a positive rate of 83.6% (46 / 55) and a negative rate of 16.4% (9 / 55) for Aeromonas vera. Conventional PCR showed a positive rate of 74.5% (41 / 55) and a negative rate of 25.5% (14 / 55) for Aeromonas vera. All positive samples were from diseased eels. Samples that were positive by conventional PCR were also positive by TaqMan quantitative real-time PCR. This indicates that the established TaqMan quantitative real-time PCR method is more sensitive and can be used for the detection of Aeromonas vera in clinical samples, further demonstrating the high specificity of this method.

[0046] Table 2. Results of clinical sample testing using TaqMan qPCR

[0047]

[0048] In summary, this invention provides a set of specific primers for detecting Aeromonas versicolor and establishes a rapid, simple, highly sensitive, and specific TaqMan real-time PCR method for detecting Aeromonas versicolor serotypes. This method can be applied to the detection of Aeromonas versicolor in fish and has broad application prospects and high application value in aquaculture.

[0049] The above detailed embodiments describe the implementation of the present invention; however, the present invention is not limited to the specific details described in the above embodiments. Within the scope of the claims and technical concept of the present invention, various simple modifications and changes can be made to the technical solution of the present invention, and these simple modifications all fall within the protection scope of the present invention.

Claims

1. A primer set and probe combination for the specific detection of Aeromonas verrucosa, characterized in that, The primer set comprises an upstream primer and a downstream primer, the nucleotide sequence of the upstream primer is shown as SEQ ID NO. 1, and the nucleotide sequence of the downstream primer is shown as SEQ ID NO. 2; the nucleotide sequence of the probe is shown as SEQ ID NO. 3, the 5' end of the probe has at least one fluorescent labeling reporter group, and the 3' end of the probe has at least one quenching group.

2. The primer set and probe combination of claim 1, wherein, The fluorescent labeling reporter group comprises FAM, and the quenching group comprises TAMRA.

3. A kit for detecting or diagnosing Veillonella parvula, comprising, The kit comprises the primer set and the probe combination of claim 1.

4. The kit of claim 3, wherein The kit further comprises a reaction mixture, a positive control and a negative control.

5. The kit of claim 4, wherein The reaction mixture comprises: Taq DNA polymerase, dNTPs, reaction buffer, .

6. Use of the primer set and the probe combination of any one of claims 1-2 or the kit of any one of claims 3-5 in the preparation of a product for detecting or diagnosing A. wautersii.

7. A method of detecting Aeromonas veronii, characterized by, The sample is detected by using the primer set and the probe combination of any one of claims 1-2 or the kit of any one of claims 3-5, and the method is not for the purpose of disease diagnosis or treatment.

8. The method of claim 7, wherein, The reaction system is as follows: Premix Ex Taq 10 μL, 10 μM probe 0.8 μL, 10 μM upstream and downstream primers each 0.4 μL, 50× ROX Reference Dye 0.4 μL, ddH2O 6 μL, template 2 μL, and a total of 20 μL; the reaction procedure is as follows: 94℃ for 30 s; 94℃ for 5 s, 58℃ for 30 s, a total of 40 cycles.

9. A method of differentiating Aeromonas veronii from Aeromonas hydrophila, Bacillus, Vibrio cholera, Edwardsiella tarda, Citrobacter or Enterococcus faecalis, characterized in that, The sample possibly containing the above-mentioned bacteria is detected by using the primer set and the probe combination of any one of claims 1-2 or the kit of any one of claims 3-5, and the method is not for the purpose of disease diagnosis or treatment.

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