TaqMan fluorescent quantitative PCR (polymerase chain reaction) detection method for aeromonas veronii

Abstract

The invention relates to an aeromonas veronii TaqMan fluorescent quantitative PCR (Polymerase Chain Reaction) detection method, a pair of specific primers for aeromonas veronii is designed according to an aeromonas veronii whole genome sequence, and a TaqMan fluorescent quantitative PCR method capable of rapidly detecting aeromonas veronii types is established, and has the characteristics of rapidness, simplicity, convenience, good specificity and high sensitivity. The method can be widely applied to fish aeromonas veronii detection, and has a high application value in the aquaculture industry.

Description

Technical Field

[0001] The present invention belongs to the technical field of molecular detection, and particularly relates to a TaqMan fluorescence quantitative PCR detection method for Aeromonas veronii. Background Art

[0002] Aeromonas veronii belongs to the family Aeromonadaceae and the genus Aeromonas. It is a Gram-negative facultative anaerobic bacillus, which is widely present in the environment, especially in environments such as fresh water and river water. It has strong adaptability to the environment and has high infectivity and high mortality in various regions.

[0003] In recent years, reports on Aeromonas veronii infecting fish at home and abroad have increased year by year. When fish are infected with Aeromonas veronii, symptoms such as skin ulcers or sores, organ hemorrhage, severe ascites, abdominal erythema or anal swelling may occur, leading to diseases such as ulcerative infection, hemorrhagic septicemia, and red swelling disease. Its prevalence has increased significantly, and it has been regarded as an important pathogen of fish animal ulcer syndrome. Potential pathogenic Aeromonas veronii are often isolated from farmed fish and aquatic environments, indicating potential risks to both fish and human health. Currently, the detection methods for Aeromonas veronii mainly include enzyme-linked immunosorbent assay (ELISA), conventional PCR detection, multiplex PCR detection, molecular biology methods, CRISPR / Cas system detection methods, etc. There are disadvantages such as long detection time, easy sample contamination, and inaccurate quantification.

[0004] The TaqMan fluorescence quantitative PCR detection method can monitor the amplification of the target gene in real time according to the fluorescence signal, and can calculate the copy number of the target gene according to the formula. This method is simple to operate, time-consuming, has good repeatability and accuracy, and high sensitivity. Moreover, the amplification of the target gene is completed in a closed system, reducing the probability of sample contamination. Therefore, according to the specific DNA sequence of Aeromonas veronii, its TaqMan fluorescence quantitative PCR detection method has been established. Summary of the Invention

[0005] In order to solve the technical problems of long detection time, easy sample contamination, and inaccurate quantification for Aeromonas veronii at present, the present invention provides a TaqMan fluorescence quantitative PCR detection method for Aeromonas veronii with strong specificity and high sensitivity.

[0006] The present invention provides a primer set for specifically detecting Aeromonas veronii. The specific primer set includes an upstream primer and a downstream primer. The upstream primer has the nucleotide sequence shown in SEQ ID NO.1, and the downstream primer has the nucleotide sequence shown in SEQ ID NO.2.

[0007] The present invention provides a specific probe for specifically detecting Aeromonas veronii, and the specific probe has a nucleotide sequence as shown in SEQ ID NO.3; at least one fluorescent-labeled reporter group is provided at the 5'-end of the specific probe, and at least one quenching group is provided at the 3'-end of the specific probe.

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

[0009] The present invention provides a kit for detecting or diagnosing Aeromonas veronii, and the kit includes the primer pair and the specific probe.

[0010] Furthermore, it further includes a reaction mixture, a positive control, and a negative control.

[0011] Furthermore, the reaction mixture contains a reaction solution in which main components such as Taq DNA polymerase, dNTPs, a reaction buffer (from TAKARA), and Mg2+ are pre-mixed.

[0012] The present invention provides the application of the specific primer pair, the probe, and / or the kit in the preparation of products for detecting or diagnosing Aeromonas veronii.

[0013] The present invention provides a method for detecting the presence of Aeromonas veronii in the environment, and the sample is detected by using the specific primer pair, the probe, and / or the kit, and the method is for non-disease diagnosis or treatment purposes.

[0014] Furthermore, the reaction system is: 10 μL of Premix Ex Taq, 0.8 μL of 10 μM probe, 0.4 μL each of 10 μM upstream and downstream primers, 0.4 μL of 50×ROX Reference Dye, ddH 2 O 6 μL, 2 μL of template, totaling 20 μL; the reaction program is: 30 s at 94 °C; 5 s at 94 °C and 30 s at 58 °C, for a total of 40 cycles.

[0015] The present invention provides a method for distinguishing Aeromonas veronii from Aeromonas hydrophila, Bacillus, Vibrio cholerae, Edwardsiella tarda, Citrobacter, or Enterococcus faecalis, and the environmental sample that may contain the above bacteria is detected by using the specific primer pair, the probe, and / or the kit, and the method is for non-disease diagnosis or treatment purposes.

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

[0017] 1. The present invention establishes a TaqMan fluorescence quantitative PCR method for quickly, specifically, sensitively, simply, and with good specificity detecting Aeromonas veronii. According to the whole genome sequence of Aeromonas veronii, new targets are screened, a pair of specific primers for Aeromonas veronii are designed, the target protein gene of 171 bp is amplified, and a recombinant plasmid ( Figure 1 ) is constructed. Using it as a standard product, a TaqMan fluorescence quantitative PCR detection method is established, a standard curve and an amplification curve are obtained, and the specificity, sensitivity, and repeatability are verified.

[0018] 2. The establishment of the standard curve of the present invention uses the standard plasmid diluted in a 10-fold gradient as a template for TaqMan fluorescence quantitative PCR amplification. The results show ( Figure 2 ) that there is a good linear relationship between the Ct value of the standard curve and the concentration of the standard plasmid. The equation of the standard curve is y = -3.167x + 37.663, and the correlation coefficient is 0.9932.

[0019] 3. The specific detection results of the present invention; using the established TaqMan fluorescence quantitative PCR method to detect plasmid standard products, Aeromonas veronii, Aeromonas hydrophila, Bacillus, Vibrio cholerae, Edwardsiella tarda, Citrobacter, Enterococcus faecalis, and ddH 2 O respectively. The results show that except for the plasmid standard product and Aeromonas veronii showing amplification curves, other pathogenic bacteria and negative controls have no amplification curves ( Figure 3 ), indicating that the TaqMan fluorescence quantitative PCR method established in this study has strong specificity.

[0020] 4. The sensitivity detection results of the present invention; the detection limit of TaqMan fluorescence quantitative PCR for plasmid standard products is 5.4×10 1 copies / μL, and the detection limit of conventional PCR for plasmid standard products is 5.4×10 4 copies / μL ( Figure 4 ). The sensitivity of TaqMan fluorescence quantitative PCR is 1000 times that of conventional PCR, indicating that the sensitivity of this method is relatively high.

[0021] 5. The repeatability detection results of the present invention; TaqMan fluorescence quantitative PCR detection is performed on standard products with different dilution degrees, and the repeatability within batches and between batches is calculated. The coefficient of variation is less than 2% (Table 1), indicating that the established TaqMan fluorescence quantitative PCR detection method has good repeatability.

[0022] 6. Detection results of clinical samples: Using the established TaqMan fluorescence quantitative PCR and conventional PCR to simultaneously detect 55 tissue samples of diseased rice field eels from different regions in Hubei Province. The results of TaqMan fluorescence quantitative PCR showed that the positive rate of Aeromonas veronii was 83.6% (46 / 55), and the negative rate was 16.4% (9 / 55). The results of conventional PCR showed that the positive rate of Aeromonas veronii was 74.5% (41 / 55), and the negative rate was 25.5% (14 / 55). All the detected positive samples were samples of diseased rice field eels, and the detection results of TaqMan fluorescence quantitative PCR for the samples positive by conventional PCR were all positive. It indicates that the established TaqMan fluorescence quantitative PCR detection method is more sensitive and can be used for the detection of Aeromonas veronii in clinical samples, and further shows that the method has strong specificity. Description of the Drawings

[0023] To more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings required for use 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 thus should not be regarded as limiting the scope. For those of ordinary skill in the art, other related drawings can be obtained based on these drawings without creative efforts.

[0024] Figure 1 The plasmid standard product prepared for TaqMan fluorescence quantitative PCR in the present invention. The concentration is 102.3 ng / μL, and after calculation, the plasmid standard product is 5.4×10 11 copies / μL. Where M: DL2000 DNA Marker; 1: Aeromonas veronii; 2: ddH 2 O.

[0025] Figure 2 The standard curve (a) and amplification curve (b) of TaqMan fluorescence quantitative PCR in the present invention.

[0026] Figure 3 The specificity result of TaqMan fluorescence quantitative PCR in the present invention. Where 1: plasmid standard product; 2: Aeromonas veronii; 3: Aeromonas hydrophila; 4: Bacillus; 5: Vibrio cholerae; 6: Edwardsiella tarda; 7: Citrobacter; 8: Enterococcus faecalis; 9: ddH 2 O.

[0027] Figure 4 The sensitivity test results of TaqMan fluorescence quantitative PCR (a) and conventional PCR (b) in the present invention. M: DL2000 DNA Marker; 1 - 8: 5.4×10 8 copies / μL to 5.4×10 1 copies / μL. Specific Embodiments

[0028] The following examples are only used to illustrate the technical solutions of the present invention more clearly. Therefore, they are only examples and cannot be used to limit the protection scope of the present invention. It should be noted that unless otherwise specified, the technical terms or scientific terms used in this application should be the common meanings understood by those skilled in the art to which the present invention belongs. Unless otherwise specified, the reagents, methods, and equipment used in the present invention are conventional reagents, methods, and equipment in this technical field. Unless otherwise specified, the reagents and materials used in the following examples are all commercially available.

[0029] Example

[0030] A TaqMan fluorescence quantitative PCR detection method for Aeromonas veronii includes the following steps:

[0031] S1. Prepare specific primers and probes; according to the whole genome sequence of Aeromonas veronii, a new specific target was selected, and specific primers (F1, R1) and a probe (P1) for Aeromonas veronii were designed by ourselves, and sequence alignment analysis was carried out by BLAST. Among them, the upstream primer F1 of the specific primer: 5'-TG ATCATGGTGCGACAATTCCT-3' (SEQ ID NO.1), the downstream primer R1 of the specific primer: 5'-TTCCACTCGGTCTCTTTATCACT-3' (SEQ ID NO.2), and the size of the amplified nucleic acid fragment is 171bp; the probe P1: FAM-TCAAATCACGCTCCGCCTT-TAMRA (SEQ ID NO.3).

[0032] S2. Prepare a recombinant plasmid standard and extract DNA; inoculate Aeromonas veronii into yellow eel kidney cells, collect cell debris, and extract DNA using a bacterial genomic DNA extraction kit (Tiangen Biotech Co., Ltd.). Specifically, it includes: S201. Pour out the cell suspension, add an appropriate amount of PBS, and scrape the cells with a cell scraper into a centrifuge tube; S202. Centrifuge at 5000 rpm for 5 min, pour out the supernatant, and collect the cell debris.

[0033] S3. PCR amplification; use specific primers to perform PCR amplification on the extracted DNA template. The amplification conditions are 94°C for 5 min; 94°C for 30 s, 58°C for 30 s, 72°C for 30 s, for a total of 30 cycles; 72°C for 7 min.

[0034] S4. Amplification and detection processing: Process the PCR products and sequence the recombinant plasmids identified as positive, specifically including: S401. After the PCR products are electrophoresed on 1% agarose gel, purify and recover the PCR products according to the instruction manual of the ordinary agarose gel DNA recovery kit, ligate them with the pMD18-T vector, transform DH5α competent cells, and inoculate them on an LB plate with ampicillin resistance; S402. After culturing overnight at 37°C, pick single colonies for bacterial liquid PCR identification; S403. Extract and identify the plasmids from the bacterial liquid with positive PCR detection results, and sequence the recombinant plasmids identified as positive.

[0035] S5. Optimization of reaction conditions and establishment of a standard curve: Use a micro-spectrophotometer to measure the concentration of the recombinant plasmid. Convert it into the corresponding copy number according to the following formula: Copy number / μL = (ng / μL × 10 -9 ) × (6.02 × 10 23 ) / (DNA length × 660). Use the recombinant plasmid standard product with 10-fold serial dilution (10 -1 ~10 -8 , that is, 5.4 × 10 10 copies / μL~5.4 × 10 3 copies / μL) as the template, and perform TaqMan fluorescence quantitative PCR detection established. After the reaction, collect data and draw a standard curve. The 20 μL reaction system is: 10 μL of Premix Ex Taq, 0.8 μL of probe (concentration: 10 μM), 0.4 μL each of upstream and downstream primers (concentration: 10 μM), 0.4 μL of ROX Reference Dye (50×) (TAKARA), 6 μL of ddH 2 O, and 2 μL of template. The reaction program is: 30 s at 94°C; 5 s at 94°C and 30 s at 58°C, for a total of 40 cycles.

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

[0037] S6. Specificity detection: Use the genomic DNA of different species (Aeromonas veronii, Aeromonas hydrophila, Bacillus sp., Vibrio cholerae, Edwardsiella tarda, Citrobacter sp., Enterococcus faecalis) as templates, and use the TaqMan fluorescence quantitative PCR method for Aeromonas veronii established in this study for detection. Use ddH 2 O as the negative control to evaluate the specificity of this method.

[0038] The results showed that the established TaqMan fluorescence quantitative PCR method was used to detect plasmid standards, Aeromonas veronii, Aeromonas hydrophila, Bacillus sp., Vibrio cholerae, Edwardsiella tarda, Citrobacter sp., Enterococcus faecalis, and ddH 2 O. Except for the plasmid standard and Aeromonas veronii showing amplification curves, no amplification curves were observed for other pathogens and the negative control ( Figure 3 ), indicating that the TaqMan fluorescence quantitative PCR method established in this study had strong specificity.

[0039] S7. Sensitivity detection: The recombinant plasmid standard was serially diluted 10-fold (10 -3 ~10 -10 ) and used as templates respectively. With ddH 2 O as the negative control, the TaqMan fluorescence quantitative PCR method established in this study was used for detection. Since there is currently no recommended detection method for this pathogen, conventional PCR reported in the literature was used to detect the above plasmid standards simultaneously in this experiment to compare the sensitivities of the two methods. Among them, the 8 concentrations of the plasmid standard were 5.4×10 8 copies / μL, 5.4×10 7 copies / μL, 5.4×10 6 copies / μL, 5.4×10 5 copies / μL, 5.4×10 4 copies / μL, 5.4×10 3 copies / μL, 5.4×10 2 copies / μL, 5.4×10 1 copies / μL.

[0040] The results showed that the detection limit of TaqMan fluorescence quantitative PCR for the plasmid standard was 5.4×10 1 copies / μL, and the detection limit of conventional PCR for the plasmid standard was 5.4×10 4 copies / μL ( Figure 4 ). The sensitivity of TaqMan fluorescence quantitative PCR was 1000 times that of conventional PCR, indicating that this method had high sensitivity.

[0041] S8. Repeatability detection: The plasmid standard serially diluted 10-fold (10 -2 ~10 -5 ) was used as the template, and the TaqMan fluorescence quantitative PCR method established in this study was used to detect the within-run repeatability of this method. Each dilution was detected 3 times; the plasmid standards of the above 4 dilutions were used as templates, and this method was used for detection every 3 days for 3 consecutive times. Each dilution was set with 3 replicates each time to detect the between-run repeatability of this method. The average threshold values within the group and between groups were calculated based on the Ct values. The standard deviation (s) and coefficient of variation (CV) were used to evaluate the repeatability of the method. Among them, the four concentrations of the plasmid standard were 5.4×10 9 copies / μL, 5.4×10 8 copies / μL, 5.4×10 7 copies / μL, and 5.4×10 6 copies / μL.

[0042] The results showed that TaqMan fluorescence quantitative PCR was performed on the standard products with different dilutions, and the repeatability within and between batches was calculated. The coefficients of variation were all less than 2% (Table 1), indicating that the established TaqMan fluorescence quantitative PCR detection method had good repeatability.

[0043] Table 1 Results of the repeatability test of TaqMan qPCR

[0044]

[0045] S9. Detection of clinical samples; The DNA of clinical samples was extracted using a tissue DNA extraction kit. The established TaqMan fluorescence quantitative PCR and conventional PCR in this study were used to simultaneously detect 55 tissue samples of diseased rice field eels from different regions in Hubei Province, that is, the established TaqMan fluorescence quantitative PCR and conventional PCR were used to simultaneously detect 55 tissue samples of diseased rice field eels from different regions in Hubei Province. The results are shown in Table 2. The TaqMan fluorescence quantitative PCR results showed that the positive rate of Aeromonas veronii was 83.6% (46 / 55), and the negative rate was 16.4% (9 / 55). The conventional PCR results showed that the positive rate of Aeromonas veronii was 74.5% (41 / 55), and the negative rate was 25.5% (14 / 55). All the detected positive samples were samples of diseased rice field eels, and the detection results of TaqMan fluorescence quantitative PCR for the samples positive in conventional PCR were all positive. It was indicated that the established TaqMan fluorescence quantitative PCR detection method was more sensitive and could be used for the detection of Aeromonas veronii in clinical samples, and it further indicated that the method had strong specificity.

[0046] Table 2 Detection results of clinical samples by TaqMan qPCR

[0047]

[0048] In summary, the present invention provides a set of specific primers for detecting Aeromonas veronii, and establishes a TaqMan fluorescence quantitative PCR method for detecting Aeromonas veronii type, which is rapid, simple, highly sensitive and specific. It can be applied to the detection of Aeromonas veronii in fish and has broad application prospects and high application value in aquaculture.

[0049] The above specific embodiments have described the implementation of the present invention in detail. However, the present invention is not limited to the specific details in the above embodiments. Within the scope of the claims and the technical concept of the present invention, various simple modifications and changes can be made to the technical solutions of the present invention, and these simple variations all fall within the protection scope of the present invention.

Claims

1. A primer set for specific detection of Aeromonas veronii, characterized in that: The specific primer set includes an upstream primer and a downstream primer, the upstream primer has a nucleotide sequence as shown in SEQ ID NO.1, and the downstream primer has a nucleotide sequence as shown in SEQ ID NO.

2.

2. A specific probe for specific detection of Aeromonas veronii, characterized in that: The specific probe has a nucleotide sequence as shown in SEQ ID NO.3; the 5' end of the specific probe has at least one fluorescent labeling reporter group, and the 3' end of the specific probe has at least one quenching group.

3. The specific probe according to claim 2, characterized in that The fluorescent labeling reporter group includes FAM, and the quenching group includes TAMRA.

4. A kit for detecting or diagnosing Aeromonas veronii, characterized in that: The kit comprises the primer set according to claim 1 and the specific probe according to any one of claims 2-3.

5. The kit according to claim 4, characterized in that Also included are reaction mixes, positive controls, and negative controls.

6. The kit according to claim 5, characterized in that The reaction mixture comprises: Taq DNA polymerase, dNTPs, reaction buffer, Mg 2+ .

7. Use of the specific primer pair according to claim 1, the probe according to any one of claims 2-3 and / or the kit according to any one of claims 4-6 in the preparation of a product for detecting or diagnosing Aeromonas vernix.

8. A method for detecting Aeromonas veroni, characterized in that: The sample is detected using the specific primer pair of claim 1 and the probe described in any one of claims 2-3 or the kit described in any one of claims 4-6, and the method is for non-disease diagnosis or treatment purposes.

9. The method according to claim 8, characterized in that The reaction system was: Premix Ex Taq 10 μL, 10 μM probe 0.8 μL, 10 μM upstream and downstream primers 0.4 μL each, 50×ROX Reference Dye 0.4 μL, ddH2O 6 μL, template 2 μL, a total of 20 μL; the reaction program was: 94°C 30 s; 94°C 5 s, 58°C 30 s, for a total of 40 cycles.

10. A method for distinguishing Aeromonas veroni from Aeromonas hydrophila, Bacillus, Vibrio cholerae, Edwardsiella tarda, Citrobacter rodentium or Enterococcus faecalis, characterized in that: The specific primer pair of claim 1 and the probe described in any one of claims 2-3 or the kit described in claim 4 are used to detect environmental samples that may contain the above-mentioned bacteria, and the method is for non-disease diagnosis or treatment purposes.

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