Multiplex PCR (Polymerase Chain Reaction) detection primer probe for riemerella anatipestifer, novel duck reovirus and muscovy duck reovirus and application of multiplex PCR detection primer probe

Abstract

The invention discloses a multiple PCR (Polymerase Chain Reaction) detection primer probe for riemerella anatipestifer, novel duck reovirus and muscovy duck reovirus and application of the multiple PCR detection primer probe, and belongs to the technical field of bacterium and virus detection. Specific primers and TaqMan probes are respectively designed for MDRV genes, NDRV genes and RA genes, and the TaqMan fluorescent quantitative RT-PCR method for simultaneously detecting MDRV, NDRV and RA in the same system is established by optimizing reaction conditions. The method has the advantages of specificity, sensitivity and high efficiency, and lays a theoretical foundation for early detection and timely prevention and control of MDRV, NDRV and RA.

Description

Technical Field

[0001] The present invention relates to the technical field of bacterial and viral detection, and particularly relates to a multiplex PCR detection primer probe for Riemerella anatipestifer, novel duck reovirus and Muscovy duck reovirus and its application. Background Art

[0002] Muscovy Duck Reovirus Disease is an infectious disease mainly harmful to Muscovy ducks caused by Muscovy Duck Reovirus (MDRV). Its characteristics are that necrotic foci appear in organs such as the liver and spleen of Muscovy ducks, the growth of young Muscovy ducks is hindered, and symptoms such as listlessness, dyspnea and diarrhea often occur. The virus spreads widely in areas where Muscovy ducks are intensively farmed, and can be vertically transmitted through breeding eggs and horizontally transmitted through contact with contaminated environments, feeds, water sources, etc. In some Muscovy duck farms, once prevention and control measures are not in place, the virus is extremely likely to spread among the duck flock, resulting in an increase in the mortality rate of Muscovy ducks and a significant decline in growth performance, seriously affecting the economic benefits of Muscovy duck farming and bringing great challenges to the stable development of the Muscovy duck farming industry.

[0003] Riemerellaanatipestifer Disease is a common infectious disease of ducks caused by Riemerella anatipestifer (RA). It mainly affects young ducks. The typical symptoms of diseased ducks include fibrinous pericarditis, perihepatitis and airsacculitis. Diseased ducks show symptoms such as slow movement, lethargy, reluctance to eat, coughing, running at the nose, etc. Some ducks also show joint swelling and lameness. Riemerella anatipestifer is more likely to spread in breeding environments with a large density of duck flocks, poor ventilation in duck houses and poor sanitary conditions, and can infect ducks through respiratory and digestive tracts. Its incidence rate is relatively high in duck farms. If not treated and prevented in time and effectively, it will cause a large number of deaths in the duck flock, slow weight gain and reduced feed conversion rate, bringing a heavy economic burden to the duck farming industry and being one of the key diseases to be prevented and controlled in the process of duck farming.

[0004] New-type duck reovirus disease (NDRVD) is a new disease characterized by liver hemorrhage and necrosis and spleen enlargement and necrosis caused by New-type duck reovirus (NDRV). It can occur in various breeds of ducks. Clinically, it mainly shows a decrease in the hatching rate of young ducks, immunosuppression and growth disorders, etc. Since the first report by Chen Shaoying et al., the new-type duck reovirus infection has been widely distributed in China and has become a seriously harmful infectious disease in the duck farming industry in recent years.

[0005] Since the above-mentioned MDRV, NDRV and RA have a great impact on duck breeding, and the current technologies for detecting duck viruses include ordinary PCR, RT-PCR and various PCRs. On the one hand, the detection differences of different viruses are relatively large, the mutual influences of virus genes in the same system are also different, and the detection difficulty will increase correspondingly with the increase in the number of virus species. On the other hand, the sensitivity of detecting a single one of the above viruses by traditional methods in the existing technologies is relatively low, which is not conducive to early detection and prevention and control. In addition, there is no existing technology that simultaneously uses multiplex PCR to efficiently detect the above three viruses. Therefore, providing a method for efficiently and sensitively detecting the above three viruses by multiplex PCR is of great significance for early detection, timely discovery and prevention and control. Summary of the Invention

[0006] The object of the present invention is to provide a multiplex PCR detection primer probe for Riemerella anatipestifer, novel duck reovirus and Muscovy duck reovirus, so as to solve the problems existing in the above-mentioned existing technologies. The multiplex PCR detection method constructed by using this primer probe can specifically, sensitively and efficiently detect RA, NDRV and MDRV, and the minimum detection concentration of the three viruses is 10 2 copies / μL, which is of great significance for early detection, timely discovery of RA, NDRV and MDRV infections and timely prevention and treatment.

[0007] To achieve the above object, the present invention provides the following solutions:

[0008] The present invention provides a multiplex PCR detection primer probe for Riemerella anatipestifer, novel duck reovirus and Muscovy duck reovirus, and the primer probe is:

[0009] RA forward primer: 5’-CCTCACGGAGAACCTATCCC-3’;

[0010] RA reverse primer: 5’-ACAGCGGCTAGTGTTACCTT-3’;

[0011] RA probe primer: 5’-ACTCAGCGAATGTACCGAAGGCTCT-3’;

[0012] NDRV forward primer: 5’-CGTGGATGGTCAAAGTCGTG-3’;

[0013] NDRV reverse primer: 5’-CCAACGTGGGTAGTCTCCTC-3’;

[0014] NDRV probe primer: 5’-TCGCACTCCGCGGGCTCCAT-3’;

[0015] MDRV forward primer: 5’-CGGGAAAGCCTGCTAAGTTC-3’;

[0016] MDRV reverse primer: 5’-ACGTCATGGTCCCAATTGTC-3’;

[0017] MDRV probe primer: 5’-AGTCGGCATAACTGCTACCAACGGT-3’.

[0018] Optionally, the 5’ ends of the RA probe primer, NDRV probe primer and MDRV probe primer are all modified with a fluorescent group, and the 3’ ends are all modified with a quenching group. In the present invention, the 5’ ends of the RA probe primer, NDRV probe primer and MDRV probe primer are respectively modified with FAM, CY5 and HEX, and the 3’ ends are respectively modified with BHQ1, BHQ2 and BHQ1.

[0019] The present invention also provides a multiplex PCR detection kit for Riemerella anatipestifer, novel duck reovirus and Muscovy duck reovirus, comprising the primer probes as described above.

[0020] The present invention also provides the application of the primer probes as described above in the preparation of a multiplex PCR detection kit for detecting Riemerella anatipestifer, novel duck reovirus and Muscovy duck reovirus.

[0021] The present invention also provides a multiplex PCR detection method for Riemerella anatipestifer, novel duck reovirus and Muscovy duck reovirus for non-diagnostic purposes, comprising the following steps:

[0022] Using the DNA or RNA of the sample to be tested as a template, performing multiplex PCR amplification with the primer probes as described above, and judging whether the sample to be tested contains Riemerella anatipestifer, novel duck reovirus or Muscovy duck reovirus by detecting the presence or absence of an amplification curve.

[0023] Optionally, the reaction system for the multiplex PCR amplification includes: 2×Q3 probe qPCR Probe MasterMix 10 μL, RA forward primer 0.5 μL, RA reverse primer 0.5 μL, RA probe primer 0.8 μL, NDRV forward primer 0.3 μL, NDRV reverse primer 0.3 μL, NDRV probe primer 0.8 μL, MDRV forward primer 0.4 μL, MDRV reverse primer 0.4 μL, MDRV probe primer 0.4 μL and template 1.0 μL, and ddH2O is added to make up the total volume to 20 μL.

[0024] Optionally, the reaction program for the multiplex PCR amplification is: pre-denaturation at 95°C for 30 sec; denaturation at 95°C for 10 sec, annealing at 60°C for 30 sec, for 40 cycles.

[0025] Optionally, if specific curves of the target gene can be amplified using all three primer-probes of RA, NDRV, and MDRV, the sample to be tested contains Riemerella anatipestifer, novel duck reovirus, and Muscovy duck reovirus; if specific curves of the target gene of only one or two viruses are amplified using the three primer-probes of RA, NDRV, and MDRV, the sample to be tested contains only the corresponding one or two viruses. For example, if only the target gene of RA is amplified to produce a specific curve, the sample to be tested contains RA but does not contain NDRV and MDRV. If only the target genes of RA and NDRV are amplified to produce specific curves, the sample to be tested contains RA and NDRV but does not contain MDRV. The same applies to others.

[0026] The present invention discloses the following technical effects:

[0027] The present invention respectively designs specific primers and TaqMan probes for the genes of RA, NDRV, and MDRV, and through optimizing the reaction conditions, a TaqMan fluorescence quantitative RT-PCR method for simultaneously detecting MDRV, NDRV, and RA in the same system is established. This method has the advantages of specificity, sensitivity, and high efficiency. The minimum detection concentrations of RA, NDRV, and MDRV are all 10 2 copies / μL, which is of great significance for early detection and timely prevention and treatment of RA, NDRV, and MDRV infections. Description of the Drawings

[0028] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required to be used in the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can be obtained based on these drawings.

[0029] Figure 1 For the detection results of the specificity test;

[0030] Figure 2 For the amplification curves after amplification of different concentration standards; in the figure, the curves of the same color correspond to the concentrations of the virus standard solutions from left to right in sequence as 10 9 、10 8 、10 7 、10 6 、10 5 、10 4 、10 3 、10 2 copies / μL;

[0031] Figure 3Schematic diagram of the PCR amplification setup;

[0032] Figure 4 is the standard curve of MDRV;

[0033] Figure 5 is the standard curve of NDRV;

[0034] Figure 6 is the standard curve of RA;

[0035] Figure 7 The curves in the figure represent the concentrations of RA standard solutions from left to right, which are 10 9 , 10 8 , 10 7 , 10 6 , 10 5 , 10 4 , 10 3 , 10 2 copies / μL;

[0036] Figure 8 The curves in the figure represent the concentrations of RA standard solutions from left to right, which are 10 9 , 10 8 , 10 7 , 10 6 , 10 5 , 10 4 , 10 3 , 10 2 copies / μL;

[0037] Figure 9 The curves in the figure represent the concentrations of RA standard solutions from left to right, which are 10 9 , 10 8 , 10 7 , 10 6 , 10 5 , 10 4 , 10 3 , 10 2 copies / μL. DETAILED DESCRIPTION

[0038] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0039] It should be understood that the terms used in the present invention are only for describing specific embodiments and are not intended to limit the present invention. Additionally, for the numerical ranges in the present invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any intermediate value within any stated value or stated range, as well as each smaller range between any other stated value or intermediate value within the stated range, is also included in the present invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0040] Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which the present invention pertains. Although the present invention only describes preferred methods and materials, any methods and materials similar or equivalent to those described herein can also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials related to the documents. In case of conflict with any incorporated document, the content of this specification shall prevail.

[0041] Without departing from the scope or spirit of the present invention, various improvements and changes can be made to the specific embodiments of the present invention specification, which are obvious to those skilled in the art. Other embodiments obtained from the specification of the present invention are obvious to those skilled in the art. The specification and examples of the present invention are merely exemplary.

[0042] Regarding the use of "comprising", "including", "having", "containing", etc. herein, they are all open-ended terms, meaning including but not limited to.

[0043] Example 1

[0044] In the present invention, primers and probes were designed successively for the target genes of MDRV, NDRV, and RA: MDRV-sigmaB (DQ643971.1), NDRV-sigmaB (GQ888710.1), RA-DTXR (EU541215.1), as shown in Tables 1 and 2 below, for a total of 6 pairs of primers and probes.

[0045] Table 1 Primer Information

[0046]

[0047]

[0048] Using the primer and probe information in Table 1 for fluorescence quantitative PCR, the fluorescence quantitative PCR experiment was carried out with reference to the instructions of 2×Q3 probe qPCR Probe Master Mix (product number: 22205-01) in the kit, and the PCR system and procedure are shown in Tables 2 and 3 below.

[0049] Table 2 qPCR Mixed Detection System

[0050]

[0051] Table 3 qPCR Program

[0052]

[0053] Judgment criterion: If the Ct value is less than or equal to 35, it is judged as positive; if it is greater than 35, it is judged as negative.

[0054] The experimental results show that amplification can be successfully carried out using the primers and probes in Table 1. The subsequent experiments were also completed using the primers and probes in Table 1.

[0055] Example 2 Specific Detection

[0056] Extract the total RNA of MDRV, NDRV, and goose astrovirus and transcribe it into cDNA using a reverse transcription kit. Extract the total DNA of RA, duck hepatitis B virus, Escherichia coli, and Salmonella for specific tests. The results are as Figure 1 shown.

[0057] The results show that only RA, NDRV, and MDRV can amplify specific curves, and no amplification was observed for other pathogens, indicating that the primers and probes involved in the present invention are specific.

[0058] Example 3 Standard Preparation

[0059] Use the forward and reverse primers of MDRV, NDRV, and RA respectively, and use the genomic DNA of MDRV, NDRV, and RA or the cDNA obtained by reverse transcription of RNA as a template for conventional PCR amplification, electrophoresis, and gel recovery. Connect the product to the T vector, transform it into Escherichia coli competent cells, extract the plasmid sample, and measure the concentration using Nanodrop.

[0060] Copy number calculation formula: (6.02×10 23 copies / mole)×(concentration) / (MWg / moL) = copies / mL;

[0061] That is, (6.02×10 23 )×(g / mL) / (DNA length×660) = copies / mL;

[0062] Or (6.02×10 23 )×(ng / μL×10 -9 ) / (DNA length×660) = copies / μL;

[0063] Finally, the standard product was serially diluted 10-fold to obtain 10 1 ~10 9 standard products with different concentrations.

[0064] Standard curve preparation:

[0065] Prepare 8 standard product gradients, which are 10 9 -10 2 copies / μL.

[0066] After amplification under the same amplification conditions as in Example 1, the PCR amplification settings are shown as Figure 2 shown, and the amplification curve is as Figure 3 shown, and the amplification efficiency is shown in Table 4, Figures 4 - 6 shown. The results show that the standard products prepared above can be used as control standards for the amplification detection of RA, NDRV, and MDRV.

[0067] Table 4 Primer amplification efficiency

[0068]

[0069] Example 4 Repeatability detection

[0070] The MDRV, RA, and NDRV positive control plasmid standard product solutions that were equally mixed after dilution in Step 2 were amplified in combination with the PCR reaction system and reaction program given in Example 1, and repeated tests were carried out, with each concentration repeated 3 times. The detection results are shown in Table 5.

[0071] Table 5 Repeatability detection results

[0072]

[0073] According to Table 5, it can be seen that the primer-probe group designed by the present invention has good repeatability, with a CV value within 2%, and has strong stability.

[0074] Example 5 Sensitivity detection

[0075] The RA, NDRV, and MDRV positive control plasmid standard product solutions (10 9 -10 2 copies / μL) diluted in Step 2 were amplified according to the PCR reaction system and reaction program given in the example to detect the sensitivity of the PCR system. The results are shown in Figures 7 - 9 shown.

[0076] The results show that the lowest concentration detected by the RA fluorescence quantitative PCR detection method is 10 2 copies / μL, and the lowest concentration detected by the NDRV fluorescence quantitative PCR detection method is 10 2copies / μL, the lowest concentration detected by the MDRV fluorescence quantitative PCR detection method is 10 2 copies / μL.

[0077] The embodiments described above are only descriptions of the preferred embodiments of the present invention, and do not limit the scope of the present invention. Without departing from the design spirit of the present invention, various deformations and improvements made by those of ordinary skill in the art to the technical solutions of the present invention shall fall within the protection scope determined by the claims of the present invention.

Claims

1. A multiplex PCR detection primer probe for Riemerella anatipestifer, novel duck reovirus, and Muscovy duck reovirus, characterized in that, The primer probes are as follows: RA forward primer: 5’-CCTCACGGAGAACCTATCCC-3’; RA reverse primer: 5’-ACAGCGGCTAGTGTTACCTT-3’; RA probe primer: 5’-ACTCAGCGAATGTACCGAAGGCTCT-3’; NDRV forward primer: 5’-CGTGGATGGTCAAAGTCGTG-3’; NDRV reverse primer: 5’-CCAACGTGGGTAGTCTCCTC-3’; NDRV probe primer: 5’-TCGCACTCCGCGGGCTCCAT-3’; MDRV forward primer: 5’-CGGGAAAGCCTGCTAAGTTC-3’; MDRV reverse primer: 5’-ACGTCATGGTCCCAATTGTC-3’; MDRV probe primer: 5’-AGTCGGCATAACTGCTACCAACGGT-3’.

2. The primer-probe according to claim 1, wherein The 5’ ends of the RA probe primer, NDRV probe primer, and MDRV probe primer are all modified with a fluorescent group, and the 3’ ends are all modified with a quenching group.

3. A multiplex PCR detection kit for Riemerella anatipestifer, novel duck reovirus, and Muscovy duck reovirus, characterized in that, Comprising the primer probes described in claim 1 or 2.

4. Use of the primer probes described in claim 1 or 2 in the preparation of a multiplex PCR detection kit for detecting Riemerella anatipestifer, novel duck reovirus, and Muscovy duck reovirus.

5. A multiplex PCR detection method for Riemerella anatipestifer, novel duck reovirus, and Muscovy duck reovirus for non-diagnostic purposes, characterized in that, Comprising the following steps: Using the DNA or RNA of the sample to be tested as a template, performing multiplex PCR amplification with the primer probes described in claim 1 or 2, and detecting the presence or absence of an amplification curve to determine whether the sample to be tested contains Riemerella anatipestifer, novel duck reovirus, or Muscovy duck reovirus.

6. The method according to claim 5, wherein The reaction system for the multiplex PCR amplification includes: 2×Q3probe qPCR Probe Master Mix 10 μL, RA forward primer 0.5 μL, RA reverse primer 0.5 μL, RA probe primer 0.8 μL, NDRV forward primer 0.3 μL, NDRV reverse primer 0.3 μL, NDRV probe primer 0.8 μL, MDRV forward primer 0.4 μL, MDRV reverse primer 0.4 μL, MDRV probe primer 0.4 μL, and template 1.0 μL, supplemented with ddH2O to a total volume of 20 μL.

7. The method according to claim 5, characterized in that, The reaction program for the multiplex PCR amplification is: pre-denaturation at 95°C for 30 sec; denaturation at 95°C for 10 sec, annealing at 60°C for 30 sec, for 40 cycles.

8. The method according to claim 5, wherein If specific curves of the target genes can be amplified using all three primer probes of RA, NDRV, and MDRV, then the sample to be tested contains Riemerella anatipestifer, novel duck reovirus, and Muscovy duck reovirus; if specific curves of only one or two virus target genes are amplified using the three primer probes of RA, NDRV, and MDRV, then the sample to be tested contains only the corresponding one or two viruses.

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