Multiplex PCR (Polymerase Chain Reaction) detection primer combination and kit for detecting bovine respiratory diseases

Abstract

The invention discloses a multiple PCR (Polymerase Chain Reaction) primer group and a detection kit for simultaneously detecting four pathogens, and also discloses a method for carrying out multiple PCR detection by utilizing the primer group and application of the primer group. Sensitivity test results show that the sensitivity of the mycoplasma bovis, mannheimia haemolytica, pasteurella multocida and mycobacterium bovis detected by PCR has a wide detection range, and the sensitivity can be detected in samples in a certain range. Specific detection results show that only mycoplasma bovis, mannheimia haemolytica, pasteurella multocida and mycobacterium bovis can be well amplified, and other bacteria cannot be amplified, so that the primer group has good specificity.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of molecular biology, and particularly relates to a multiplex PCR detection primer combination and kit for detecting bovine respiratory disease. BACKGROUND

[0002] Mycoplasma bovis (Mb), Mannheimia haemolytica (Mh), Pasteurella multocida (Pm) and Mycobacterium bovis (Tb) are the main bacterial pathogenic factors causing bovine respiratory disease, which can infect cattle at different growth stages, and often cause co-infection or secondary infection.

[0003] The current mainstream multiplex PCR detection technology for the four pathogenic bacteria is designed based on the principle of specific gene sequence amplification of pathogenic bacteria. Many detections are designed for 16S rDNA primers, and the 16S rDNA of different bacteria has high homology, which is easy to cause false positive. From the existing research, the design of some specific primers has not been widely verified, and there may be a risk of non-specific amplification. In addition, the multiplex PCR detection method used in some research reports lacks systematic verification and evaluation of key indicators such as specificity and sensitivity of the detection system, which to some extent limits the accuracy and reliability of the detection results.

[0004] Traditional methods for identifying bacteria and viruses are complicated, complex and time-consuming. In recent years, immunology and molecular biology technologies have been continuously improved, such as real-time fluorescent quantitative PCR, enzyme-linked immunosorbent assay, etc. These technologies have certain limitations in detection time and sensitivity. Compared with PCR technology, it has high sensitivity, strong specificity and simple operation. However, there are few studies on simultaneous detection of Mycoplasma bovis, Mannheimia haemolytica, Pasteurella multocida and Mycobacterium bovis. Therefore, it is of great practical significance and application value to develop a multiplex PCR detection method that can simultaneously detect Mycoplasma bovis, Mannheimia haemolytica, Pasteurella multocida and Mycobacterium bovis. SUMMARY

[0005] The technical problem to be solved by the present application is to provide a multiplex PCR system for simultaneously detecting Mycoplasma bovis, Mannheimia haemolytica, Pasteurella multocida and Mycobacterium bovis, and the amplification product can be detected by agarose gel electrophoresis technology.

[0006] The present application is realized by the following technical solutions:

[0007] A multiplex PCR primer set for simultaneously detecting four pathogens, the four pathogens being Mycoplasma bovif, Mannheimia haemolytica, Pasteurella multocida and Mycobacterium bovis, the primer set consisting of the following primers:

[0008] MycBov-ftsH-pF: 5'-CGCAGGTGAAGCCAATGTTC-3'(SEQ ID NO. 1), MycBov-ftsH-pR: 5'-ACCAGGACGGGTTAATGCAG-3'(SEQ ID NO. 2);

[0009] ManHae-pepS6-pF: 5'-TGGACTATCAATACTTCCGCGA-3'(SEQ ID NO. 3), ManHae-pepS6-pR: 5'-TACCTGCATTGGTGACTGCG-3'(SEQ ID NO. 4);

[0010] PasMul-lon-pF: 5'-GAGATCCGGCATCAGCGTTA-3'(SEQ ID NO. 5),

[0011] PasMul-lon-pR: 5'-AATCACCACCCACTTCGGTC-3'(SEQ ID NO. 6);

[0012] MtbBov-pks2-pF: 5'-GGTGTGGTTGTCACGCTAGA-3'(SEQ ID NO. 7), MtbBov-pks2-pR: 5'-GCTGATACACCGCGATCTGA-3'(SEQ ID NO. 8).

[0013] A multiplex PCR detection kit for simultaneously detecting four pathogens, the kit comprising the primer set.

[0014] The application also provides a method for multiplex PCR detection using the primer set, the PCR detection method system being: 1 μL of template of the sample to be detected, 10 μL of 2x Taq Master Mix, 1 μL of each of the four pairs of above-mentioned upstream and downstream primers, and enzyme-free water being supplemented to 20 μL, and PCR amplification being performed; the reaction procedure being: 94℃ pre-denaturation for 5 min; 98℃ denaturation for 10 s, 63.2℃ annealing for 30 s, 72℃ extension for 20 s, 30 cycles of amplification, 72℃ total extension for 5 min, and the amplification product being stored at 4℃; the PCR product is detected by agarose gel electrophoresis, and the target bands of Mycoplasma bovis, Mannheimia haemolytica, Pasteurella multocida and Mycobacterium bovis are 319 bp, 390 bp, 528 bp and 274 bp, respectively.

[0015] The application also provides the use of the primer set, which is for non-disease detection and treatment purposes, and the use method is to perform PCR reaction by using the PCR primer set, and to perform agarose gel electrophoresis on the reaction product, and the target bands of the Mycoplasma bovis, Mannheimia haemolytica, Pasteurella multocida and Mycobacterium tuberculosis are 319bp, 390bp, 528bp and 274bp respectively.

[0016] The detection sensitivity and specificity of the multiplex PCR amplification system are experimentally tested, and the sensitivity test result shows that the sensitivity of the PCR for detecting the Mycoplasma bovis, Mannheimia haemolytica, Pasteurella multocida and Mycobacterium tuberculosis has a wide detection range, and samples within a certain range can be detected. The specificity detection result shows that the Mycoplasma bovis, Mannheimia haemolytica, Pasteurella multocida and Mycobacterium tuberculosis, Salmonella, Escherichia coli, Staphylococcus aureus, Streptococcus agalactiae, bovine coronavirus, bovine respiratory syncytial virus, bovine parainfluenza virus type 3 and bovine adenovirus type 3 are detected by using the method, and only the Mycoplasma bovis, Mannheimia haemolytica, Pasteurella multocida and Mycobacterium tuberculosis can be well amplified, and other bacteria cannot be amplified, so the primer set has good specificity. BRIEF DESCRIPTION OF DRAWINGS

[0017] The technical solutions of the application will be further described below with reference to the drawings:

[0018] Figure 1 The annealing temperature optimization result graph of the multiplex PCR amplification reaction provided for the embodiment 2 is shown in the figure;

[0019] In A, lanes 1-12 take Mb as a template, and lanes 13-24 take Mh as a template; in B, lanes 1-12 take Pm as a template, and lanes 13-24 take Tb as a template.

[0020] Figure 2 The extension time optimization result graph of the multiplex PCR amplification reaction provided for the embodiment 2 is shown in the figure;

[0021] A, B, C and D are extension times of 10s, 20s, 30s and 40s respectively, M: DL2000 DNA Maker, 1: Mb, 2: Mh, 3: Pm, 4: Tb, 5: Mb, Mh, Pm and Tb are mixed samples in equal proportions, 6: mixed samples in equal proportions diluted by 10 times, 7: mixed samples in equal proportions diluted by 100 times, and 8: negative control.

[0022] Figure 3 The primer concentration optimization result graph of the multiplex PCR amplification reaction provided for the embodiment 2 is shown in the figure;

[0023] A, B, C, D primer dosage is 1 μL, 0.8 μL, 0.6 μL, 0.4 μL (10 μmol / L) respectively; M: DL2000 DNA Maker, 1: Mh, 2: Mh, 3: Pm, 4: Tb, 5: Mb, Mh, Pm, Tb, etc. mixed sample, 6: mixed 10-fold dilution sample, 7: mixed 100-fold dilution sample, 8: negative control.

[0024] Figure 4 The figure of specific test results of the primer combination of the multiplex PCR detection of Example 3.

[0025] Figure 5 The figure of sensitivity test results of the primer combination of the multiplex PCR detection of Example 4. DETAILED DESCRIPTION

[0026] The technical solutions in the embodiments of the present application will be described clearly and completely in combination with the accompanying drawings in the embodiments of the present application. Obviously, the described embodiments are only part of the embodiments of the present application, rather than all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative labor fall within the scope of protection of the present application.

[0027] Example 1 primer design

[0028] According to the ftsH gene of Mycoplasma bovis, the pepS6 gene of Mannheimia haemolytica, the lon gene of Pasteurella multocida and the pks2 gene of Mycobacterium bovis tuberculosis published by GenBank, specific primers of Mycoplasma bovis, Mannheimia haemolytica, Pasteurella multocida and Mycobacterium bovis tuberculosis are designed respectively, and are synthesized by Jingke Biotechnology (Beijing) Co., Ltd.

[0029] Table 1 related specific primer combination information is as follows:

[0030]

[0031] Example 2 optimization of multiplex PCR amplification reaction system and conditions

[0032] 1. Annealing temperature optimization

[0033] Figure 1 The figure of annealing temperature optimization results of the multiplex PCR amplification reaction provided in the present embodiment.

[0034] M stands for DL2000 DNA Maker. Lanes 1-12 in A used Mb as the template, and lanes 13-24 used Mh as the template. Lanes 1-12 in B used Pm as the template, and lanes 13-24 used Tb as the template. Annealing temperatures were 45°C, 46.8°C, 48.6°C, 50.4°C, 52.3°C, 54.1°C, 55.9°C, 57.7°C, 59.5°C, 61.3°C, 63.2°C, and 65°C, respectively.

[0035] 2. Extension time optimization

[0036] Figure 2 Figure 1 shows the optimization results of the extension time for the multiplex PCR amplification reaction provided in this example. A, B, C, and D represent extension times of 10s, 20s, 30s, and 40s, respectively. M: DL2000 DNA Maker; 1: Mb; 2: Mh; 3: Pm; 4: Tb; 5: Mb, Mh, Pm, and Tb mixed in equal proportions; 6: 10-fold diluted sample mixed in equal proportions; 7: 100-fold diluted sample mixed in equal proportions; 8: Negative control.

[0037] 3. Primer Concentration Optimization

[0038] Figure 3 This is a graph showing the primer concentration optimization results for the multiplex PCR amplification reaction provided in this example. Primer dosages for A, B, C, and D were 1 μL, 0.8 μL, 0.6 μL, and 0.4 μL (10 μmol / L), respectively. M: DL2000 DNA Maker, 1: Mb, 2: Mh, 3: Pm, 4: Tb, 5: Mb, Mh, Pm, and Tb mixed in equal proportions, 6: 10-fold diluted sample mixed in equal proportions, 7: 100-fold diluted sample mixed in equal proportions, 8: Negative control

[0039] From the above Figures 1-3 Through annealing temperature gradient test, extension time optimization test, and primer concentration optimization test, the optimal reaction conditions for the multiple PCR amplification reaction of the present invention were obtained as follows: pre-denaturation at 94°C for 5 min; denaturation at 98°C for 10 s, annealing at 63.2°C for 30 s, extension at 72°C for 20 s, amplification for 30 cycles, and extension at 72°C for 5 min.

[0040] Example 3 specificity test:

[0041] Specific primers for the four pathogens, 2×Taq Master Mix, and ddH2O were added to 12 reaction tubes, and the extracted genomic DNA of Mycoplasma bovis, Mannheimia haemolytica, Pasteurella multocida, Mycobacterium bovis tuberculosis, Salmonella, Escherichia coli, Staphylococcus aureus, Streptococcus agalactiae, bovine coronavirus, bovine respiratory syncytial virus, bovine parainfluenza virus type 3, and bovine adenovirus type 3 were added for amplification to test the specificity of the multiplex PCR method.

[0042] Figure 4 This is a graph showing the specificity test results of the multiplex PCR detection primer combination provided in this example.

[0043] Figure 4 M in the middle represents DL2000 DNA Maker. Lanes 1-12 represent Mycoplasma bovis, Mannheimia haemolytica, Pasteurella multocida, Mycobacterium bovis tuberculosis, Salmonella, Escherichia coli, Staphylococcus aureus, Streptococcus agalactiae, bovine coronavirus, bovine respiratory syncytial virus, bovine parainfluenza virus type 3, and bovine adenovirus type 3. Lane 13 is a blank control.

[0044] Depend on Figure 4 It can be seen that the multiplex PCR can amplify the target bands for Mycoplasma bovis, Mannheimia haemolytica, Pasteurella multocida, and Mycobacterium bovis, and there are no other nonspecific reactions. The amplification results for the genomes of Salmonella, Escherichia coli, Staphylococcus aureus, Streptococcus agalactiae, bovine coronavirus, bovine respiratory syncytial virus, bovine parainfluenza virus type 3, and bovine adenovirus type 3 are negative, indicating that the primers have good specificity.

[0045] Example 4 Sensitivity test

[0046] The extracted Mb, Mh, Pm, and Tb nucleic acids were used as templates for single specific PCR amplification. The target fragments were recovered according to the instructions of AxyPrep DNAGel Extraction Kit. TM -T Vector Cloning Kit instructions to clone the target fragment into pMD19 TM -T vector was transformed into E. coli DH5α competent cells, and the recombinant plasmid was extracted according to the instructions of AxyPrep Plasmid MiniPrep Kit. The recombinant plasmid was sent to Qingke Biotechnology (Beijing) Co., Ltd. for sequence analysis. The recombinant plasmids with correct sequencing results were used as positive standards and named as: pMD-T-ftsH, pMD-T-pepS6, pMD-T-lon, and pMD-T-pks2. The concentrations of the four prepared recombinant plasmid standards were adjusted to 1.0×10 10 After equal volume mixing, the samples were diluted 10-fold with ddH2O as templates and detected by the optimized multiplex PCR method. The minimum detection limit of the established multiplex PCR method for the recombinant plasmid standards was determined, and the detection sensitivity of the multiplex PCR method was evaluated.

[0047] Figure 5The sensitivity test results of the multiplex PCR detection primer combination provided in the present embodiment are shown in the figure. M: 2000 DNA Marker 1-10: Template concentration is 1.0 x 10 9 -10 0 copies / μL 11: Negative control

[0048] As can be seen from Figure 5 the multiplex PCR, the minimum detection concentration of Mycoplasma bovis, Mannheimia haemolytica, Pasteurella multocida and Mycobacterium bovis is 1.0 x 10 3 copies / μL.

Claims

1. A multiplex PCR primer set for simultaneous detection of four pathogens, characterized in that: The four pathogens are Mycoplasma bovis, Mannheimia haemolytica, Pasteurella multocida and Mycobacterium bovis, and the primer set consists of the following primers: MycBov-ftsH-pF: 5'-CGCAGGTGAAGCCAATGTTC-3' (SEQ ID NO. 1), MycBov-ftsH-pR: 5'-ACCAGGACGGGTTTAATGCAG-3' (SEQ ID NO. 2); ManHae-pepS6-pF: 5'-TGGACTATCAATACTTCCGCGA-3' (SEQ ID NO.3), ManHae-pepS6-pR: 5'-TACCTGCATTGGTGACTGCG-3' (SEQ ID NO. 4); PasMul-lon-pF: 5'-GAGATCCGGCATCAGCGTTA-3' (SEQ ID NO.5), PasMul-lon-pR: 5'-AATCACCACCCACTTCGGTC-3' (SEQ ID NO. 6); MtbBov-pks2-pF: 5'-GGTGTGGTGTTGTCACGCTAGA-3' (SEQ ID NO.7), MtbBov-pks2-pR: 5'-GCTGATACACCGCGATCTGA-3' (SEQ ID NO. 8).

2. A multiplex PCR detection kit for simultaneous detection of four pathogens, characterized in that: The kit comprises the primer set according to claim 1.

3. A method for multiplex PCR detection using the primer set according to claim 1, characterized in that: The PCR detection method comprises the following steps: 1 μL of the template of the sample to be tested, 10 μL of 2×Taq Master Mix, 1 μL of each of the four pairs of upstream and downstream primers, and enzyme-free water to make up to 20 μL for PCR amplification; the reaction procedure comprises: pre-denaturation at 94° C. for 5 minutes; denaturation at 98° C. for 10 seconds, annealing at 63.2° C. for 30 seconds, extension at 72° C. for 20 seconds, amplification for 30 cycles, and a total extension at 72° C. for 5 minutes; and storage of the amplified product at 4° C.; and detection by agarose gel electrophoresis of the PCR product. The target bands of Mycoplasma bovis, Mannheimia hemolytica, Pasteurella multocida, and Mycobacterium bovis are 319 bp, 390 bp, 528 bp, and 274 bp, respectively.

4. The use of the primer set according to claim 1, characterized in that: The application is for non-disease detection and treatment purposes. The application method is to use the PCR primer set to perform a PCR reaction, and perform agarose gel electrophoresis on the reaction products. The target bands of bovine Mycoplasma, hemolytic Mannheimia, Pasteurella multocida, and bovine Mycobacterium tuberculosis are 319bp, 390bp, 528bp, and 274bp, respectively.

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