Primer and method for detecting actinobacillus pleuropneumoniae and application
By designing highly specific primer pairs and a real-time quantitative PCR method, combined with fluorescent probes and photoelectric conduction technology, the problems of long detection time, susceptibility to contamination, and false positives in existing technologies for detecting Actinobacillus pleuropneumoniae have been solved, achieving highly sensitive and specific gene detection.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- BEIJING UNIV OF AGRI
- Filing Date
- 2026-01-26
- Publication Date
- 2026-04-17
AI Technical Summary
Existing methods for detecting Actinobacillus pleuropneumoniae in porcine pleuropneumoniae suffer from problems such as long processing time, susceptibility to contamination by other bacteria, poor specificity, and a tendency to produce false positives and nonspecific amplification.
A highly specific primer pair was designed for a real-time quantitative PCR method to detect Actinobacillus pleuropneumoniae and its virulence gene. The copy number was calculated by plotting a standard curve to determine whether the sample contains the target gene and virulence gene. The amplification process was monitored in real time by combining fluorescent probes and photoelectric conduction technology.
It significantly improves the accuracy and reliability of detection, achieving highly sensitive, specific and accurate gene detection, simplifying the operation process, and avoiding false positive results and environmental pollution.
Smart Images
Figure CN121874374A_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the field of microbial detection technology, specifically to a primer, method, and application for detecting Actinobacillus pleuropneumoniae in pigs. Background Technology
[0002] Actinobacillus pleuropneumoniae is the main pathogen causing infectious pleuropneumonia in pigs and is widely distributed in the global pig industry. This bacterium primarily parasitizes the respiratory tract of pigs, and its transmission routes are diverse. It can spread through droplets in close-range airborne transmission between pigs, and also indirectly through contaminated equipment and personnel, especially in high-density farming environments where the spread is extremely rapid. Initial symptoms in pigs infected with Actinobacillus pleuropneumoniae are mild, often manifesting as a slight cough and slight lethargy. Without timely intervention and treatment, the condition can rapidly deteriorate, developing into severe pleuropneumonia with typical symptoms such as high fever, severe cough, difficulty breathing, open-mouth breathing, and a dog-sitting posture. In severe cases, respiratory failure can lead to death. Autopsy reveals adhesions between the lungs and pleura, hemorrhagic consolidation and necrotic foci in the lungs, and a large amount of turbid exudate in the pleural cavity. Some infected pigs, even in a mild state, may become asymptomatic carriers, continuously shedding the virus and threatening the health of the herd.
[0003] In existing technologies, the identification methods for Actinobacillus pleuropneumoniae mainly include bacterial isolation and culture and biochemical identification. Bacterial isolation and culture require long-term cultivation on specific culture media, which is not only time-consuming but also susceptible to contamination by other bacteria, affecting the identification results. Biochemical identification relies on the metabolic reactions of bacteria to different substrates, which suffers from poor specificity and makes it difficult to accurately distinguish between similar bacterial species. Immunological detection methods such as ELISA, although relatively simple to operate, are prone to cross-reactivity and false positive results. Traditional PCR methods suffer from insufficient primer specificity during detection, which may lead to non-specific amplification and affect the accuracy of identification. Summary of the Invention
[0004] To address the aforementioned technical problems, this invention provides primers, a method, and applications for detecting Actinobacillus pleuropneumoniae in porcines.
[0005] A primer for detecting *Actinomyces pleuropneumoniae* and its virulence gene is disclosed. The nucleotide sequences of the primer for detecting *Actinomyces pleuropneumoniae* are shown in SEQ ID NO.1 and SEQ ID NO.2, and the target gene is... rec F gene; The nucleotide sequences of the primers used to detect the virulence gene of *Actinomyces pleuropneumoniae* are shown in SEQ ID NO.3 and SEQ ID NO.4. The virulence gene is... tbp B gene.
[0006] When using the primers provided by this invention for detection, the primers for amplifying the various detection genes do not interfere with each other, resulting in high specificity and accuracy.
[0007] A method for detecting Actinobacillus pleuropneumoniae using the primers described above, wherein a standard curve of the target gene is plotted with the logarithm of the copy number of the target gene of Actinobacillus pleuropneumoniae as the X-axis and the CT value as the Y-axis. Using the DNA of the sample to be tested as a template, PCR amplification is performed using primers for the target gene to obtain the CT value. The copy number is then calculated by substituting the CT value into the standard curve. The copy number is used to determine whether the sample contains the target gene. If the target gene is present, the sample to be tested contains Actinobacillus pleuropneumoniae.
[0008] Preferably, the number of copies is on the order of 10. 2 It was determined that a target gene was present.
[0009] A method for detecting the virulence gene of Actinobacillus pleuropneumoniae using the primers described above, wherein a standard curve of the virulence gene is plotted with the logarithm of the copy number of the virulence gene of Actinobacillus pleuropneumoniae as the X-axis and the CT value as the Y-axis. Using the DNA of the sample to be tested as a template, PCR amplification was performed using the primers of the virulence gene to obtain the CT value. The copy number was then calculated by substituting the CT value into the standard curve. The copy number was used to determine whether the sample contained the virulence gene.
[0010] Preferably, the number of copies is on the order of 10. 2 It was identified as a toxic gene.
[0011] The application of the primers described therein in the identification of Actinobacillus pleuropneumoniae.
[0012] Preferably, during identification, the copy number is obtained using PCR, and the copy number is on the order of ≥10. 2 It was identified as Actinobacillus pleuropneumoniae.
[0013] The application of the primers described therein in the detection of virulence genes in Actinobacillus pleuropneumoniae.
[0014] Compared with the prior art, the beneficial effects of the present invention are as follows: This invention optimizes primer design and reaction system to ensure that primers for amplifying various detection genes do not interfere with each other, thus significantly improving the accuracy and reliability of detection. Attached Figure Description
[0015] Figure 1 for rec The amplification curves of different concentrations of the F gene, from left to right, are 10... 7 ng / μL, 10 6 ng / μL, 10 5 ng / μL, 104 ng / μL, 10 3 ng / μL, 10 2 The concentration was measured in ng / μL, and each concentration was tested three times for repeatability, with the median value taken.
[0016] Figure 2 For standard Staphylococcus aureus strains rec The F gene was tested, and the experiment was repeated three times.
[0017] Figure 3 For standard Escherichia coli strains rec The F gene was tested, and the experiment was repeated three times.
[0018] Figure 4 For sample 1 rec The F gene detection showed that the CT value of sample 1 was 16.77, and the repeatability of the three experiments was good.
[0019] Figure 5 For sample 2 rec The F gene detection showed that the CT value of sample 2 was 14.74, and the repeatability of the three experiments was good.
[0020] Figure 6 For sample 3 rec The F gene detection showed that the CT value of sample 3 was 18.13, and the repeatability of the three experiments was good.
[0021] Figure 7 For sample 4 rec The F gene detection showed that the CT value of sample 4 was 27.98, and the repeatability of the three experiments was good.
[0022] Figure 8 for rec The melting curve summary of the F gene test shows a single peak and an appropriate Tm value range.
[0023] Figure 9 for tbp Amplification curves of different concentrations of gene B, from left to right: 10... 7 ng / μLl, 10 6 ng / μL, 10 5 ng / μL, 10 4 ng / μL, 10 3 ng / μL, 10 2 The concentration was measured in ng / μL, and each concentration was tested three times for repeatability, with the median value taken.
[0024] Figure 10 For sample 1 tbp The B gene test, after three repeated experiments, yielded a CT value of 16.56.
[0025] Figure 11 For sample 2 tbp The B gene test, after three repeated experiments, yielded a CT value of 13.85.
[0026] Figure 12 For sample 3 tbp The B gene test, after three repeated experiments, yielded a CT value of 17.68.
[0027] Figure 13 For sample 4 tbp The B gene test, after three repeated experiments, yielded a CT value of 27.47.
[0028] Figure 14 For standard Escherichia coli strains tbp The B gene-specific test, after three repeated experiments, yielded a CT value of 30.30.
[0029] Figure 15 For standard Staphylococcus aureus strains tbp The B gene-specific test, after three repeated experiments, yielded a CT value of 28.80.
[0030] Figure 16 for tbp The melting curve summary of the B gene test shows a single peak and an appropriate Tm value range. Detailed Implementation
[0031] The specific embodiments of the present invention are described in detail below, but it should be understood that the scope of protection of the present invention is not limited to the specific embodiments. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without inventive effort are within the scope of protection of the present invention. Unless otherwise specified, the experimental methods described in the embodiments of the present invention are conventional methods.
[0032] Example 1 target genes rec F and virulence genes tbp The detection method for B includes the following steps: One strain of Actinobacillus pleuropneumoniae FX2, identified by sequencing, was used (preserved in Laboratory 410, College of Animal Science and Technology, Beijing University of Agriculture, isolated from lung tissue of diseased pigs in farms in the Beijing-Tianjin-Hebei region).
[0033] (1) DNA extraction Bacterial DNA was extracted from Actinobacillus pleuropneumoniae FX2 according to the instructions of the bacterial genomic DNA extraction kit.
[0034] (2) Design of primer sets for multiple virulence genes of Actinobacillus pleuropneumoniae in PCR Based on the target genes of Actinobacillus pleuropneumoniae strain in GenBank rec F and virulence genes tbp The gene sequence of B was used to design specific primers that meet the requirements of quantitative real-time PCR amplification using Primer 5 software. The primers are shown in Table 1.
[0035] Table 1: Primer Information (3) DNA amplification The target gene was amplified by PCR using the DNA of Actinobacillus pleuropneumoniae extracted in step (1) as a template and the primers designed in step (2). The PCR reaction system is shown in Table 2 and the PCR reaction procedure is shown in Table 3. The amplified target fragment was purified using a gel extraction kit.
[0036] Table 2 PCR reaction system Table 3 PCR reaction procedure (4) Constructing standard plasmids The PCR products were recovered and purified, and TA cloning was performed. Positive plasmids were extracted according to the instructions of the plasmid mini-extraction kit and identified by PCR. A standard plasmid was constructed and sequenced to obtain the positive bacterial strain.
[0037] (5) Construction of standard curve (5.1) Inoculate the positive strain in (4) into LB medium containing ampicillin resistance, and shake overnight at 37°C and 200 r / min.
[0038] (5.2) Extract the plasmid, digest it with a single enzyme, and then recover the digestion product.
[0039] (5.3) Determine the concentration of the recombinant plasmid, and then perform serial dilutions of the recombinant plasmid, selecting 10... 7 ng / μL, 10 6 ng / μL, 10 5 ng / μL, 10 4 ng / μL, 10 3 ng / μL, 10 2 Six positive plasmids at concentrations of ng / μL were used as templates, with corresponding copy numbers of 1.6 × 10⁻⁶. 6 copies / μL, 1.6×10 5 copies / μL, 1.6×10 4 copies / μL, 1.6×10 3 copies / μL, 1.6×10 2copies / μL, 1.6×10 1 The sample was amplified by real-time PCR using copies / μL of the sample, with each template repeated three times. ddH2O was used as a negative control. Figure 1 and Figure 9 They are respectively rec F gene and tbp Amplification curves of gene B at different concentrations were plotted to represent the target gene. rec F and virulence genes tbp The standard curve for gene B (using the logarithm of copy number as x and CT value as y, calculating the linear regression equation), and the correlation R after regression analysis. 2 >0.99, rec F and tbp The standard curve for B is y = -3.8379x + 35.441 (R²). 2 =0.9931) and y=-3.881x+34.892(R) 2 =0.9982), the calculated copy number is 1*10 X copies / μL (copy count ≤ 10) 2 All were determined to be without the target gene.
[0040] The preparation of the reaction system for quantitative real-time PCR (total volume 20 μL) is as follows: Table 4. Real-time PCR reaction system The amplification conditions for quantitative real-time PCR were set as follows: 94℃ for 30s; 94℃ for 10s, 60℃ for 12s, 72℃ for 30s, repeated 45 times. Fluorescence signals were detected at a single point at 72℃ to obtain the amplification curve and CT value. The copy number was calculated using the CT value in a linear regression equation to determine whether the target gene and virulence gene were amplified. This allows for qualitative and quantitative determination of whether the sample contains the corresponding target gene and virulence gene. (The detection range is defined based on the CT value of negative samples).
[0041] (6) Verify that the amplified fragment is the target fragment. Melting curves: 95℃ 0s, 65℃ 15s, 95℃ 0s, continuous signal detection, based on whether the melting curve has a single peak and is in the appropriate Tm region, to ensure the accuracy of the amplification curve.
[0042] Verification Experiment 1. rec F gene testing scope definition Standard Escherichia coli strains and standard Staphylococcus aureus strains were tested. rec F gene-specific experiment, results as follows Figure 2 and Figure 3 As shown.
[0043] Figure 2 The results showed that the sample was a standard Staphylococcus aureus strain, and the average cycle number after three replicates was 28.91. Substituting these values into the calculation, the copy number was 50.22 copies / μL. Based on this result, it can be concluded that this strain does not contain... rec The F gene was used to demonstrate the specificity of this method. Figure 3 The results showed that the sample was a standard E. coli strain, and the average cycle number after three replicate experiments was 30.91. Substituting these values into the calculation, the copy number was 15.13 copies / μL. Based on this result, it can be concluded that this strain does not contain [the specific bacteria mentioned earlier]. rec The F gene was used to demonstrate the specificity of this method.
[0044] 2. Four samples were obtained from the suspected swine pleuropneumoniae-infected lung lesions through bacterial isolation and culture, and were designated as Sample 1, Sample 2, Sample 3 and Sample 4. The four samples were identified and confirmed to be swine pleuropneumoniae-infected, while Sample 4 was not swine pleuropneumoniae-infected.
[0045] (1) Bacterial genomes were extracted from four samples using a bacterial genome extraction kit (purchased from Tiangen Biotech). The extracted bacterial genomes were used as templates in a fluorescent PCR reaction system to detect CT values, verifying the effectiveness of the method of the present invention. The results are as follows: Figures 4-13 .
[0046] Figure 4 The results showed that sample 1 had an average cycle number of 16.77 after three repeated experiments, and fluorescence intensity was detectable. Substituting these values into the calculation, the copy number was 73265.59 copies / μL. Based on this result, sample 1 contains... rec The F gene identified it as Actinobacillus pleuropneumoniae.
[0047] Figure 5 The results showed that sample 2 had an average cycle number of 14.74 after three repeated experiments, and fluorescence intensity was detectable. Substituting these values into the calculation, the copy number was 247648.20 copies / μL. Based on this result, it can be concluded that sample 2 contains... rec The F gene identified it as Actinobacillus pleuropneumoniae.
[0048] Figure 6 The results showed that sample 3 had an average cycle number of 18.13 after three repeated experiments, and fluorescence intensity was detectable. Substituting these values into the calculation, the copy number was 32334.87 copies / μL. Based on this result, it can be concluded that sample 3 contains... rec The F gene identified it as Actinobacillus pleuropneumoniae.
[0049] Figure 7The results showed that sample 4 had an average cycle number of 27.98 after three repeated experiments. Due to the high CT value and the absence of a clear amplification curve, the calculated copy number was 88.08 copies / μL. Based on this result, it can be concluded that the sample does not contain [the desired amplification]. rec F gene, sample 4 is not Actinobacillus pleuropneumoniae.
[0050] Figure 8 for recF The melting curve summary graph of the gene test shows a single peak, and the Tm value range is appropriate.
[0051] The above results demonstrate the accuracy of the identification method of the present invention.
[0052] (2) For the four samples tbp The B gene was tested.
[0053] Figure 10 The results showed that sample 1 had an average cycle number of 16.56 after three repeated experiments, and fluorescence intensity was detectable. Substituting these values into the calculation, the copy number was 53013.17 copies / μL. Based on this result, sample 1 contains... tbp Gene B can be identified as a virulence gene.
[0054] Figure 11 The results showed that sample 2 had an average cycle number of 13.85 after three repeated experiments, and fluorescence intensity was detectable. Substituting these values into the calculation, the copy number was 264,118.31 copies / μL. Based on this result, it can be concluded that sample 2 contains... tbp Gene B can be identified as a virulence gene.
[0055] Figure 12 The results showed that sample 3 had an average cycle number of 17.68 after three repeated experiments, and fluorescence intensity was detectable. Substituting these values into the calculation, the copy number was 27169.43 copies / μL. Based on this result, sample 3 contains... tbp Gene B can be identified as a virulence gene.
[0056] Figure 13 The results showed that the average cycle number of sample 4 after three repeated experiments was 27.47, and the calculated copy number was 81.89 copies / μL. Based on this result, it can be concluded that the sample does not contain [the substance mentioned earlier]. tbp B gene.
[0057] Figure 16 for tbp The melting curve summary of the B gene test shows a single peak and an appropriate Tm value range.
[0058] The above results demonstrate the accuracy of the method for detecting virulence genes in this invention.
[0059] 3. tbp B gene specificity experiment Standard Escherichia coli strains and standard Staphylococcus aureus strains were tested. tbp B gene-specific experiment, results as follows Figure 14 and Figure 15 As shown.
[0060] Figure 14 The results showed that the sample was a standard E. coli strain, and the average cycle number after three replicates was 30.30. Substituting these values into the calculation, the copy number was 15.22 copies / μL. Based on this result, it can be concluded that this strain does not contain [the specific bacteria mentioned earlier]. tbp Gene B was used to demonstrate the specificity of this method.
[0061] Figure 15 The results showed that the sample was a standard Staphylococcus aureus strain, and the average cycle number after three replicates was 28.80. Substituting these values into the calculation, the copy number was 37.13 copies / μL. Based on this result, it can be concluded that this strain does not contain... tbp Gene B was used to demonstrate the specificity of this method.
[0062] In summary, the present invention has the following advantages: 1. High sensitivity: Can detect genes in single cells; 2. High specificity and precision: It combines the high specificity of DNA hybridization with the high precision of spectroscopy, and uses fluorescent probes and photoelectric conduction to directly detect changes in fluorescence signals during gene amplification to obtain quantitative results, which is equivalent to automatically completing Northern hybridization in the PCR reaction process; 3. Simple operation and fast speed: It integrates PCR amplification, product detection and quantitative analysis through computer and analysis software; 4. Safe and pollution-free: FQ-PCR performs PCR amplification and product analysis in a completely closed state, eliminating the environmental pollution and false positives caused by traditional open PCR testing. 5. Good repeatability of results observation: simultaneous online real-time observation, intuitive result judgment, and avoidance of interference from human factors.
[0063] It should be noted that when numerical ranges are mentioned in the claims of this invention, it should be understood that the two endpoints of each numerical range and any value between the two endpoints can be selected. To avoid redundancy, the present invention describes preferred embodiments.
[0064] Although preferred embodiments of the invention have been described, those skilled in the art, upon learning the basic inventive concept, can make other changes and modifications to these embodiments. Therefore, the appended claims are intended to be interpreted as including both the preferred embodiments and all changes and modifications falling within the scope of the invention.
[0065] Obviously, those skilled in the art can make various modifications and variations to this invention without departing from its spirit and scope. Therefore, if these modifications and variations fall within the scope of the claims of this invention and their equivalents, this invention also intends to include these modifications and variations.
Claims
1. A primer for detecting Actinobacillus pleuropneumoniae and its virulence gene, characterized in that, The nucleotide sequences of the primers for detecting Actinobacillus pleuropneumoniae are shown in SEQ ID NO.1 and SEQ ID NO.2, and the target gene is... rec F gene; The nucleotide sequences of the primers used to detect the virulence gene of *Actinomyces pleuropneumoniae* are shown in SEQ ID NO.3 and SEQ ID NO.
4. The virulence gene is... tbp B gene.
2. A method for detecting Actinobacillus pleuropneumoniae using the primers described in claim 1, characterized in that, A standard curve of the target gene was plotted with the logarithm of the copy number of the target gene of Actinobacillus pleuropneumoniae as the X-axis and the CT value as the Y-axis. Using the DNA of the sample to be tested as a template, PCR amplification is performed using primers for the target gene to obtain the CT value. The copy number is then calculated by substituting the CT value into the standard curve. The copy number is used to determine whether the sample contains the target gene. If the target gene is present, the sample to be tested contains Actinobacillus pleuropneumoniae.
3. The method according to claim 2, characterized in that, The number of copies is on the order of 10. 2 It was determined that a target gene was present.
4. A method for detecting the virulence gene of Actinobacillus pleuropneumoniae using the primers described in claim 1, characterized in that, A standard curve of the virulence gene was plotted with the logarithm of the copy number of the virulence gene of Actinobacillus pleuropneumoniae as the X-axis and the CT value as the Y-axis. Using the DNA of the sample to be tested as a template, PCR amplification was performed using the primers of the virulence gene to obtain the CT value. The copy number was then calculated by substituting the CT value into the standard curve. The copy number was used to determine whether the sample contained the virulence gene.
5. The method according to claim 4, characterized in that, The number of copies is on the order of 10. 2 It was identified as a toxic gene.
6. The application of the primers according to claim 1 in the identification of Actinobacillus pleuropneumoniae.
7. The application according to claim 6, characterized in that, During identification, the copy number is obtained using PCR, and the copy number is ≥10^6 copies / mL. 2 It was identified as Actinobacillus pleuropneumoniae.
8. The application of the primers according to claim 1 in the detection of virulence genes in Actinobacillus pleuropneumoniae.