Nested PCR primer set, kit, and application for detecting porcine infectious pleuropneumonia.

Abstract

This invention relates to the field of molecular biology, specifically to a nested PCR primer set, kit, and application for detecting porcine infectious pleuropneumonia. The kit, developed by establishing a nested PCR amplification primer set, can be used for the in vitro diagnosis of porcine infectious pleuropneumonia. It exhibits high sensitivity, detecting up to 0.355 pg / µL, which is nearly 100 times higher than that of conventional PCR (35.5 pg / µL). It also demonstrates strong specificity, overcoming the problems of low sensitivity and poor specificity associated with conventional PCR. Compared to existing indirect immunofluorescence and serological detection methods, it offers lower cost and a shorter detection cycle.

Description

Technical Field

[0001] This invention relates to the field of molecular biology technology, specifically to a nested PCR primer set, kit, and application for detecting porcine infectious pleuropneumonia. Background Technology

[0002] Porcine infectious pleuropneumonia (PVP) is a common contagious disease in swine farming. Pigs of all ages are susceptible to PVP. The disease is primarily transmitted through contact and respiratory routes, with infected and carrier pigs serving as the main sources of infection. PVP often occurs in damp, rainy, and hot / humid weather, and is easily confused with swine enzootic pneumonia and swine pneumonia in clinical diagnosis. Treatment is challenging, with a high mortality rate, and even after recovery, pigs often experience slowed growth. In recent years, my country's livestock industry has developed rapidly, with a continuously increasing number of pig farmers. Outbreaks of infectious swine diseases can cause significant economic losses to farmers and seriously affect the healthy development of animal husbandry. Therefore, it is necessary to strengthen research on the diagnosis and treatment of PVP.

[0003] Currently, laboratory diagnosis of porcine contagious pleuropneumonia mainly employs direct microscopy, bacterial isolation and identification, serological diagnosis, indirect immunofluorescence, and molecular biological diagnosis. Direct microscopy and bacterial isolation and identification have low accuracy rates in diagnosing porcine contagious pleuropneumonia, while serological diagnosis and indirect immunofluorescence are cumbersome and costly. Molecular biology is a commonly used method for detecting porcine contagious pleuropneumonia, but often the template concentration is low, resulting in unclear bands. Therefore, improving the sensitivity of molecular biology is essential. Summary of the Invention

[0004] The purpose of this invention is to provide a nested PCR primer set, a kit, and its application for detecting porcine infectious pleuropneumonia. The kit made from this primer set can be used for in vitro diagnosis of porcine infectious pleuropneumonia. It has high sensitivity, can detect up to 0.355 pg / uL, which is nearly 100 times higher than that of ordinary PCR (35.5 pg / uL). It also has strong specificity, overcoming the problems of low sensitivity and poor specificity of ordinary PCR. The method of use is simple, the detection cycle is short, and the cost is low, making it suitable for routine laboratory testing.

[0005] To achieve the above objectives, the present invention provides a nested PCR primer set for detecting porcine infectious pleuropneumonia, comprising the following two pairs of primers:

[0006] P1-APP-F: 5'-AGCCGTAACAAACGGCGATA-3';

[0007] P2-APP-R: 5'-TTGGCTACCATTGACCGCTT-3';

[0008] P3-APP-F: 5'-AGCAAGTCGCCCAAGCTATT-3';

[0009] P4-APP-R: 5'-GTAGGAGTACCTGCATCCGC-3'.

[0010] Furthermore, in the technical solution described above, the primers are derived from *Actinobacillus pleuropneumoniae* (porcine infectious pleuropneumoniae Actinobacillus). Actinobacillus pleuropneumonia Specifically, the primers screened in this invention are designed based on the 16SRNA gene sequence of Actinobacillus pleuropneumoniae in porcines. Through nested design, the specificity of the primers can be significantly improved.

[0011] Furthermore, in the above-described technical solution, P3-APP-F and P4-APP-R are primers designed within the amplification fragments of P1-APP-F and P2-APP-R.

[0012] The present invention also provides a kit comprising the above-described primer set.

[0013] Further, the above-described technical solution includes (1) the two pairs of primers mentioned above; (2) the premixed solution; and (3) the negative control.

[0014] In the above-described technical solution, the concentration of the primer is further 25 pmol / L.

[0015] In the above-described technical solution, the premixed liquid is 2 × HiFiTaq high-fidelity premix; the negative control is ddH2O.

[0016] The technical solution described above further includes a conventional PCR amplification system and a nested PCR amplification system. The reaction program for the conventional PCR amplification system is: 95℃ pre-denaturation for 5 min, 95℃ denaturation for 45 s, 56.5℃ annealing for 35 s, 72℃ extension for 30 s, for a total of 35 cycles, and a final extension at 72℃ for 10 min. The reaction program for the nested PCR amplification system is: 95℃ pre-denaturation for 5 min, 95℃ denaturation for 45 s, 58℃ annealing for 35 s, 72℃ extension for 30 s, for a total of 35 cycles, and a final extension at 72℃ for 10 min.

[0017] Furthermore, in the technical solution described above, the detection limit of the kit is 0.355 pg / μL. The detection limit of conventional PCR is approximately 35.5 pg / μL, while the detection limit using the kit of this invention can reach 0.355 pg / μL, which is nearly 100 times higher in sensitivity than conventional PCR, indicating that the kit of this invention has high sensitivity and strong specificity.

[0018] The present invention also provides the application of the above-described kit in the in vitro detection of porcine infectious pleuropneumonia.

[0019] The beneficial effects of this invention are:

[0020] This invention employs nested PCR for two-stage PCR amplification, which improves the sensitivity of porcine infectious pleuropneumonia detection by 100 times compared to conventional PCR, while also exhibiting strong specificity and repeatability. It overcomes the problems of low sensitivity and poor specificity of conventional PCR detection. Compared with existing indirect immunofluorescence PCR, serological and other detection methods, it has lower cost and shorter detection cycle, making it suitable for routine laboratory testing.

[0021] This invention uses nested PCR primers to create a kit that offers high detection efficiency, convenience, and cost savings. When applied to the in vitro detection of porcine infectious pleuropneumonia, the detection limit is only 0.355 pg / μL, enabling effective and rapid detection of early-stage porcine infectious pleuropneumonia. Using this kit allows for early detection and treatment, thus playing an effective preventive role. Attached Figure Description

[0022] Figure 1 This is an electrophoresis image of Actinobacillus pleuropneumoniae amplified by primers P1 and P2 in Example 2 of the present invention.

[0023] Figure 2 This is an electrophoresis image of Actinobacillus pleuropneumoniae amplified by primers P3, P4 and P5, P6 in Example 2 of the present invention.

[0024] Figure 3 This is an electrophoresis image of Actinobacillus pleuropneumoniae amplified by ordinary PCR primers in the sensitivity verification of Example 3 of the present invention;

[0025] Figure 4 This is an electrophoresis image of Actinobacillus pleuropneumoniae amplified by nested PCR primers in the sensitivity verification of Example 3 of the present invention.

[0026] Figure 5 This is an electrophoresis image of the specific amplification of Actinobacillus pleuropneumoniae in porcine infectious pleuropneumoniae verified by nested PCR primers in Example 3 of the present invention; wherein, M is the negative control, 1 is Actinobacillus pleuropneumoniae in porcine infectious pleuropneumoniae, 2 is Staphylococcus, 3 is Pasteurella, 4 is Escherichia coli, 5 is Streptococcus, and 6 is Haemophilus parasuis.

[0027] Figure 6 This is a repeat electrophoresis image of Actinobacillus pleuropneumoniae in porcine infectious pleuropneumoniae verified by nested PCR primers in Example 3 of the present invention; where M is the negative control; 1-8 are positive samples of Actinobacillus pleuropneumoniae in porcine infectious pleuropneumoniae.

[0028] The present invention is illustrated by the following embodiments, which are intended for illustrative purposes only and not to limit the invention in any way. Specific experimental methods not mentioned in the embodiments are generally performed according to conventional or obvious experimental methods.

[0029] The present invention will be further described below with reference to the accompanying drawings and embodiments. Detailed Implementation

[0030] The present invention will be explained below with reference to embodiments. Those skilled in the art will understand that the following embodiments are for illustrative purposes only and should not be considered as limiting the scope of the invention. Where specific techniques or conditions are not specified in the embodiments, they are performed according to the techniques / conditions described in literature or reference books in the field or according to the product instructions. Reagents or instruments used, unless otherwise specified, are all conventional products purchased through provincial bidding and procurement departments.

[0031] The above-described technical features of the present invention and the technical features specifically described below (such as in the embodiments) can be combined with each other to form new or preferred technical solutions.

[0032] Main test reagents and instruments

[0033] Reagents: Primers were synthesized by Shanghai Sangon Biotech Co., Ltd.; DL5000 DNA Marker was purchased from Dalian Takara Bio Inc.; DP302 bacterial genomic DNA extraction kit and plasmid miniprep kit were purchased from Beijing Tiangen Biotech Co., Ltd.; 2 × HiFiTaq high-fidelity premixed PCR reaction system was purchased from Beijing Sizhengbai Biotechnology Co., Ltd.

[0034] Instruments: Thermo Fisher Scientific microcentrifuge, Thermo Fisher Scientific ABI Veriti PCR instrument, Shanghai Tianneng gel imaging system, Shanghai Tianneng horizontal electrophoresis system, Nanodrop 2000, etc.

[0035] Pathological material: Positive sample of porcine infectious pleuropneumonia retained by the laboratory.

[0036] Example 1: Primer Screening

[0037] Download Actinobacillus pleuropneumoniae (a bacterium) from the NCBI website. Actinobacillus pleuropneumoniaThe 16SRNA gene sequence of APP (accession number: AF033085) was used to design three primer pairs for HPS using Primer 5.0 primer design software: P1, P2, P3, P4, P5, and P6. Primers P3, P4, P5, and P6 were designed within the amplified fragments of P1 and P2. These primers were then screened for nested PCR amplification. The primer sequences and the sizes of the target fragments are shown in Table 1. The primer sequences were synthesized by Shanghai Sangon Biotech.

[0038] Table 1. Primer sequences for nested PCR amplification of Actinobacillus contagious pleuropneumoniae in porcines.

[0039]

[0040] Example 2: PCR amplification

[0041] 1. Strain resuscitation

[0042] LB medium was selected, and 50 mL / L calf serum and 40 mg / kg NAD were added. Then, Actinobacillus pleuropneumoniae of pigs was inoculated with sterile cotton swabs and marked. The medium was then incubated in a 37°C incubator for 24 h and then stored in a 4°C refrigerator for later use.

[0043] 2. Conventional PCR amplification

[0044] Following the instructions of the Tiangen DP302 bacterial genomic DNA extraction kit, the genome of the above-mentioned Actinobacillus pleuropneumoniae was extracted and then stored at 4°C for later use.

[0045] Conventional PCR amplification was performed using primers P1 and P2 from Table 1 in Example 1. The amplification reaction system is as follows:

[0046] 12.5 μL of 2 × HiFiTaq high-fidelity premixed PCR reaction, 1.0 μL each of P1 and P2 primers (25 pmol / L), 2 μL of DNA template, and finally ddH2O to 25 μL.

[0047] The PCR reaction program was as follows: 95℃ pre-denaturation for 5 min, 95℃ denaturation for 45 s, 56.5℃ annealing for 35 s, 72℃ extension for 30 s, for a total of 35 cycles, and a final extension at 72℃ for 10 min.

[0048] After PCR, 5 μL of the PCR amplification product was electrophoresed on a 2% agarose gel for identification. The amplification results are as follows: Figure 1 As shown. From Figure 1 The results show that no band was amplified in the negative control, while the target fragment of 689 bp was amplified in the lane using the extracted Actinobacillus pleuropneumoniae genomic template DNA, indicating that the primer can be used for ordinary PCR amplification.

[0049] 3. Establishment of Nested PCR Amplification Method

[0050] The product amplified in step 2 was selected as the template for the second amplification. Primer pairs P3 and P4, and primer pairs P5 and P6 were used for the second amplification screening. The amplification reaction system is as follows:

[0051] 12.5 μL of 2 × HiFiTaq high-fidelity premixed PCR reaction, 1.0 μL each of the upstream and downstream primers (25 pmol / L) of the P3 and P4 primer pairs and the P5 and P6 primer pairs, 4 μL of the product amplified in the first conventional PCR step as template, and finally ddH2O to make up to 25 μL.

[0052] The PCR reaction program was as follows: 95℃ pre-denaturation for 5 min, 95℃ denaturation for 45 s, 58℃ annealing for 35 s, 72℃ extension for 30 s, for a total of 35 cycles, and a final extension at 72℃ for 10 min.

[0053] After PCR, 5 μL of the PCR amplification product was electrophoresed on a 2% agarose gel for identification. The amplification results are shown below. Figure 2 As shown. From Figure 2 The results show that the negative control showed no amplification, while the products amplified by primer pairs P3 and P4 had a clear target fragment of 214 bp, indicating that primer pairs P3 and P4 can be used for nested PCR amplification of Actinobacillus pleuropneumoniae in porcine infectious pleuropneumoniae. However, the amplification products of primer pairs P5 and P6 showed no amplification of the target fragment, indicating that primer pairs P5 and P6 are not suitable for nested PCR amplification of Actinobacillus pleuropneumoniae in porcine infectious pleuropneumoniae.

[0054] Example 3: Verification Experiment

[0055] 1. Sensitivity Verification

[0056] The concentration of *Actinomyces pleuropneumoniae* genome extracted in step 2 of Example 2 was determined using a Nanodrop 2000, and the concentration was 355 ng / μL; sterile distilled water was used for 10-fold serial dilutions to 10 μL. -8 Using this as a template, amplification was performed according to steps 2 and 3, and its sensitivity was tested.

[0057] Based on the results of the conventional PCR amplification in step 2 of Example 2, as follows: Figure 3 As shown, from Figure 3 The results show that DNA diluted to 10 -4 Clear bands were visible, indicating a detection limit of 35.5 pg / µL; the results of nested PCR amplification established in step 3 of Example 2 are as follows. Figure 4 As shown, from Figure 4The results show that when DNA is diluted to 10... -6 Clear bands can be seen, with a detection limit of 0.355 pg / uL; the sensitivity is 100 times higher than that of ordinary PCR methods.

[0058] 2. Specificity test verification

[0059] Staphylococcus, Pasteurella, Escherichia coli, Streptococcus, and Haemophilus parasuis, all from our laboratory, were used to extract genomic DNA using the Tiangen DP302 bacterial genomic DNA extraction kit. The extracted DNA templates were then amplified using a pre-established nested PCR method. The amplification results are shown below. Figure 5 As shown. From Figure 5 The results show that, except for *Actinomyces suis*, which amplified the target fragment, no bands were amplified in the electrophoretic lanes of *Staphylococcus*, *Pasteurella*, *Escherichia coli*, *Streptococcus*, and *Haemophilus parasuis*. This indicates that the nested PCR primers for porcine infectious pleuropneumonia designed in this invention have high specificity.

[0060] 3. Repeatability test verification

[0061] Using the nested PCR established in Example 2, eight laboratory-preserved samples positive for Actinobacillus pleuropneumoniae were amplified. The amplification results are as follows: Figure 6 As shown. From Figure 6 The results show that all eight positive samples were positive, indicating that the method has good stability and repeatability.

[0062] In summary, the nested PCR kit established in this invention has high sensitivity, strong specificity, short detection cycle, and low cost, and can be effectively used for the in vitro detection of porcine infectious pleuropneumonia.

[0063] Finally, it should be emphasized that the above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention can have various changes and modifications. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A set of PCR primers for detecting porcine contagious pleuropneumonia, characterized by, Includes the following two pairs of primers: P1-APP-F: 5'-AGCCGTAACAAACGGCGATA-3'; P2-APP-R: 5'-TTGGCTACCATTGACCGCTT-3'; P3-APP-F: 5'-AGCAAGTCGCCCAAGCTATT-3'; P4-APP-R: 5'-GTAGGAGTACCTGCATCCGC-3'; The P3-APP-F and P4-APP-R primers are designed within the P1-APP-F and P2-APP-R amplification fragments.

2. A kit characterized in that, It includes the primer set as described in claim 1.

3. The kit of claim 2, wherein It includes (1) the two primer pairs as described in claim 1; (2) the premixed solution; and (3) the negative control.

4. The kit of claim 3, wherein The concentration of the primers was 25 pmol / L.

5. The kit of claim 3, wherein The premixed solution is a 2 × HiFiTaq high-fidelity premix; the negative control is ddH2O.

6. The kit of claim 3, wherein The system includes a conventional PCR amplification system and a nested PCR amplification system. The reaction program for the conventional PCR amplification system is as follows: 95℃ pre-denaturation for 5 min, 95℃ denaturation for 45 s, 56.5℃ annealing for 35 s, 72℃ extension for 30 s, for a total of 35 cycles, and a final extension at 72℃ for 10 min. The reaction program for the nested PCR amplification system is as follows: 95℃ pre-denaturation for 5 min, 95℃ denaturation for 45 s, 58℃ annealing for 35 s, 72℃ extension for 30 s, for a total of 35 cycles, and a final extension at 72℃ for 10 min.

7. The kit of claim 2, wherein The detection limit of the kit is 0.355 pg / μL.

8. The use of the kit according to any one of claims 2-7 in the in vitro detection of porcine infectious pleuropneumonia for non-disease diagnosis purposes.

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