Primer pair for rapidly identifying pasteurella multocida in diseased material as well as application and identification method of primer pair

Abstract

The invention provides a primer pair for rapidly identifying pasteurella multocida in a diseased material as well as application and an identification method of the primer pair. The primer pair for rapidly identifying the pasteurella multocida in the diseased material is obtained through PCR (Polymerase Chain Reaction) amplification by taking an outer membrane protein ompH gene of which the nucleotide sequence is SEQ ID NO.1 as a template, and can be used for accurately and rapidly identifying whether the diseased material contains the pasteurella multocida or not. In addition, the method for rapidly identifying the pasteurella multocida in the diseased material by adopting the primer pair is simple and convenient to operate, the operation process is saved compared with the traditional process, a plurality of bacterial colonies can be identified at one time, and the method is more economical and efficient.

Description

Technical Field

[0001] The invention relates to the technical field of veterinary microbiology, and in particular to a primer pair for rapidly identifying avian Pasteurella in diseased materials, and an application and an identification method thereof. Background Art

[0002] Fowl cholera, also known as avian hemorrhagic septicemia and avian Pasteurella multocida, is a chronic or acute contact, septicemic infectious disease caused by Pasteurella multocida (hereinafter referred to as avian Pasteurella) that mainly harms poultry and wild birds. Due to the characteristics of rapid onset, rapid death, and strong infectivity of the disease, and the fact that healthy-looking chickens and wild waterfowl may carry avian Pasteurella, fowl cholera has become prevalent in poultry farming in recent years. In order to avoid increasing economic losses in the farming industry, timely identification and discrimination of the pathogenic bacteria avian Pasteurella from dead animal diseased materials is of great significance for preventing and controlling the outbreak of fowl cholera.

[0003] The existing technology for identifying avian Pasteurella from diseased materials generally includes the following steps: 1) collecting tissues / organs of the diseased materials to be tested from dead animals; 2) taking bacteria from the diseased materials by streaking and isolating through aseptic operation; 3) picking suspected Pasteurella strains for purification and culture and staining and microscopic examination; 3) extracting nucleic acids from the purified strains; 4) determining whether it is avian Pasteurella through PCR identification, etc. However, in actual operation, a variety of bacteria will be cultivated on the plate after the diseased materials are inoculated in step 2. Picking out avian Pasteurella from these bacteria requires a lot of separation, purification, culture, staining and microscopic examination, nucleic acid extraction and PCR identification, which will lead to problems such as long experimental time, low efficiency, and waste of experimental consumables. Delays in the identification process are likely to cause significant economic losses. Summary of the invention

[0004] In view of the deficiencies in the prior art, the present invention discloses a method for rapidly identifying poultry Pasteurella in diseased materials, which can rapidly identify whether poultry Pasteurella exists in diseased materials. The identification method is efficient and accurate, and has important promotion and application value.

[0005] In order to achieve the above technical objectives, on the one hand, the present invention proposes a primer pair for rapidly identifying avian Pasteurella in diseased materials, wherein the primer pair is obtained by PCR amplification using the outer membrane protein ompH gene with the nucleotide sequence of SEQ ID NO.1 as a template.

[0006] The research and development team of the present invention found through a large amount of research and development experimental data that the outer membrane protein ompH gene and the protein it encodes play an important role in the pathogenic mechanism of Pasteurella multocida. The technical solution of the present invention uses the ompH gene as a template to design primer pairs, which can accurately, quickly and efficiently identify whether the diseased material contains avian Pasteurella.

[0007] In a further example of the present invention, the nucleotide sequence of the primer pair is explored and optimized. Optionally, the nucleotide sequence of the primer pair is PM-ompH-F2: 5'-CAGCAACTTCAGCAAAC-3', PM-ompH-R2: 5'-CCAAAACCTACAGCGAC-3', and the optimization process is shown in the implementation of the present invention, and the technical effect of the primer pair for rapid identification of whether avian Pasteurella is contained in the diseased material is verified.

[0008] On the other hand, the present invention provides the use of the above primer pair in rapid identification of avian Pasteurella in diseased materials.

[0009] Furthermore, the application is to use the primer pair to perform colony PCR. Compared with the existing technology for identifying avian Pasteurella, the use of the primer pair for colony PCR can reduce the difficulty of operation and improve the identification efficiency and reduce the cost under the premise of accurate identification.

[0010] Furthermore, the band of the product obtained by colony PCR using the primers is between 750bp and 1000bp. In the actual application of colony PCR using the above primer pair, it can be judged whether the sampled diseased material contains avian Pasteurella based on the product band obtained by PCR, which is conducive to timely formulating appropriate poultry breeding and avian cholera prevention and control strategies.

[0011] On the other hand, the present invention proposes a method for rapidly identifying avian Pasteurella in diseased materials using the above primer pair, the method comprising the steps of: (1) inoculating the diseased material aseptically on a TSA plate for culture; (2) scratching the target colony to obtain a PCR reaction system, and using the primer pair for PCR amplification; (3) analyzing the PCR amplification product. The present invention uses the above primer pair to perform colony PCR on the diseased material, which omits the steps of strain purification and culture, staining and microscopic examination, and nucleic acid extraction compared to the existing technology for identifying avian Pasteurella. The process is simpler and more efficient, and the amount of consumables used is effectively reduced; in addition, the technical solution of the present invention can identify multiple colonies at one time, which is relatively shorter in time and has higher separation efficiency, and achieves the technical effect of rapidly separating and identifying whether the diseased material contains avian Pasteurella.

[0012] In a further example of the present invention, the source of the diseased material, the specific control conditions of colony PCR, and the standards for analyzing and judging PCR products are explored and optimized. An optional embodiment of the present invention shows the specific process of using the above primer pairs to quickly identify avian Pasteurella in diseased materials.

[0013] Optionally, the diseased material is taken from the liver, heart or lung of a dead animal.

[0014] Optionally, the PCR reaction system includes 2×Taq Plus Master Mix 12.5-25 μL, upstream primer PM-ompH-F2 1-2 μL, downstream primer PM-ompH-R2 1-2 μL, ddH2O 8-16 μL and target strain.

[0015] Optionally, the PCR program is: pre-denaturation at 94-98°C for 3-5 min; denaturation at 94-98°C for 30s-1min, annealing at 50-60°C for 15-45s, extension at 68-75°C for 20s-1min, and cycle for 30-40 times; and extension at 68-75°C for another 8-15 min.

[0016] Optionally, the step (3) comprises: observing the PCR product by gel electrophoresis; if the PCR product has a band between 750-1000 bp on the electrophoresis map, it indicates that the diseased material contains avian Pasteurella; if the PCR product is not between 750-1000 bp or has no band, it indicates that the diseased material does not contain avian Pasteurella.

[0017] Compared with the prior art, the invention has the following beneficial effects: the primer pair of the invention is obtained by PCR amplification using the outer membrane protein ompH gene as a template, and can accurately and quickly identify whether the diseased material contains avian Pasteurella. In addition, the method of quickly identifying avian Pasteurella in diseased materials using the primer pair of the invention is simple to operate, saves operating procedures compared to traditional processes, and can identify multiple colonies at one time, which is more economical and efficient. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] The accompanying drawings constituting a part of the present invention are used to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute an improper limitation of the present invention. In the accompanying drawings:

[0019] Figure 1 The agarose gel electrophoresis result of the colony PCR shown in Example 3 of the present invention is shown, wherein lane M is the DNA amplification result of DL2000 (positive control), lane 8 is sterile water (negative control), and lanes 1-6 are the DNA amplification results of the target colonies suspected of avian Pasteurella that were streaked.

[0020] Specific implementation party

[0021] In order to facilitate the understanding of the present invention, the present invention will be described more comprehensively below, and preferred embodiments of the present invention are given. However, it should be understood that these embodiments are only used for more detailed description and should not be understood as limiting the present invention in any form, i.e., not intended to limit the scope of protection of the present invention.

[0022] Unless otherwise defined, the technical terms used in the following examples have the same meanings as those generally understood by those skilled in the art to which the present invention belongs. The test reagents used in the following examples, unless otherwise specified, are all conventional biochemical reagents; the experimental methods, unless otherwise specified, are all conventional methods.

[0023] In the present invention, "further" and "further optionally" are used for descriptive purposes to indicate differences in content, but should not be understood as limiting the scope of protection of the present invention; "optionally" and "optional" mean optional, that is, any one of the two parallel schemes of "yes" or "no". If multiple "options" appear in a technical solution, unless otherwise specified and there is no contradiction or mutual restriction, each "optional" is independent.

[0024] In the present invention, when it comes to numerical intervals (i.e., numerical ranges), unless otherwise specified, the optional numerical distribution is considered continuous within the above numerical interval and includes the two numerical endpoints (i.e., the minimum value and the maximum value) of the numerical range, and each numerical value between the two numerical endpoints. In addition, when multiple ranges are provided to describe features or characteristics, these ranges can be combined. In other words, unless otherwise specified, the ranges disclosed herein should be understood to include any and all subranges included therein.

[0025] The temperature parameters in the present invention, if not specifically limited, are allowed to be either constant temperature treatment or to vary within a certain temperature range. It should be understood that the constant temperature treatment allows the temperature to fluctuate within the precision range controlled by the instrument. Fluctuations within the range of ±5°C, ±4°C, ±3°C, ±2°C, and ±1°C are allowed.

[0026] The term "primer" of the present invention refers to an oligonucleotide, whether it is naturally present in a purified restriction digest or produced synthetically, which can act as a starting point for synthesis when placed under conditions that induce synthesis of primer extension products complementary to a nucleic acid chain (e.g., in the presence of nucleotides and an inducing agent such as a DNA polymerase and at a suitable temperature and pH). Typically, a primer pair refers to two primers that do not have base sequences that are complementary to each other.

[0027] Example 1

[0028] A primer pair for rapidly identifying avian Pasteurella in diseased materials is obtained by PCR amplification using the outer membrane protein ompH gene with the nucleotide sequence of SEQ ID NO.1 as a template.

[0029] Optionally, the nucleotide sequence of the primer pair is PM-ompH-F2: 5'-CAGCAACTTCAGCAAAC-3', PM-ompH-R2: 5'-CCAAAACCTACAGCGAC-3'.

[0030] This example shows the screening process of the primer pairs of the present invention: the research and development team of the present invention found through a large amount of research and development experimental data that the outer membrane protein OmpH gene and the protein encoded by it play a key role in the pathogenic mechanism of avian Pasteurella, so they tried to design primer pairs for identifying avian Pasteurella using the ompH gene as a template.

[0031] The specific primer pair design process includes: obtaining the ompH gene sequence of Pasteurella multocida strain X-73 outer membrane protein (ompH) gene, complete cds from NCBI, and using the Primer Premier5 software to design primers. Optionally, the principles followed by the research and development team in the primer pair design process include: ① giving priority to primer pairs with high rating values ​​designed by the system; ② the hairpin, dimer, and falsepriming of the best primer pair are None; ③ the primer length is generally 18-22bp and the bp difference between the two primers is preferably less than or equal to 2; ④ preferably no continuous identical bases, such as TTT, GGG, etc.; ⑤ when Found appears, the absolute value of ΔG is less than 10.

[0032] Following the above principles, the primer pair for rapid identification of avian Pasteurella in diseased materials of the present invention is obtained: the upstream primer PM-ompH-F2: 5'-CAGCAACTTCAGCAAAC-3'; the downstream primer PM-ompH-R2 is a reverse primer, and its nucleotide sequence is: 5'-CCAAAACCTACAGCGAC-3'.

[0033] Furthermore, the research and development team performed BLAST on the upstream and downstream primers of the present invention in NCBI, and the comparison results showed 100% similarity with avian Pasteurella, confirming that the primer pair of the present invention can specifically recognize and bind to the DNA sequence of avian Pasteurella, has high specificity, accuracy and effectiveness, and is suitable for avian Pasteurella detection.

[0034] The physicochemical characteristics and biological characteristics of the primer pair are further disclosed in this embodiment. Among them, the primer length of PM-ompH-F2: CAGCAACTTCAGCAAAC is about 17 bp, the Tm value is about 51.58°C, and the GC content is about 47.06%; the primer length of PM-ompH-R2: CCAAAACCTACAGCGAC is about 17 bp, the Tm value is about 52.67°C, and the GC content is about 52.94%, and the PCR product length of the primer pair is about 895 bp.

[0035] The nucleic acid molecule sequence involved in the present invention includes: the outer membrane protein ompH gene sequence of Pasteurella multocida such as SEQ ID NO.1.

[0036] SEQ ID NO.1:

[0037]

[0038] Example 2

[0039] A method for rapidly identifying avian Pasteurella in diseased materials using the primer pair, the method comprising the steps of: (1) aseptically inoculating the diseased materials on a TSA plate for culturing; (2) streaking target colonies to obtain a PCR reaction system, and using the primer pair for PCR amplification; and (3) analyzing the PCR amplification products.

[0040] In this embodiment, there is no restriction on the sampling location of the bacteria to be detected in the diseased material. Optionally, the diseased material is taken from the liver, heart or lung of a dead animal, which can improve the accuracy of avian Pasteurella sampling. In the actual identification process, technicians in this field can make a choice according to their needs.

[0041] In this embodiment, the method for aseptically inoculating the diseased material on the TSA plate is not limited, and direct inoculation method, dilution inoculation method, streak inoculation method, coating inoculation method, etc. can be selected, and those skilled in the art can select according to their needs.

[0042] Optionally, in step (1), the diseased material is aseptically inoculated on a TSA plate and then cultured in a 37° C. incubator for 16-24 hours to obtain colonies.

[0043] Optionally, the PCR reaction system in the step (2) includes 2×Taq Plus Master Mix 12.5-25 μL, upstream primer PM-ompH-F2 1-2 μL, downstream primer PM-ompH-R2 1-2 μL, ddH2O 8-16 μL and the target strain. Optimizing the PCR reaction system is beneficial to improving the PCR reaction efficiency and enhancing the accuracy of the PCR detection results; further optionally, the PCR reaction system is a 25-50 uL reaction system.

[0044] After a lot of exploratory experiments, the research and development team of the present invention has optimized the operating conditions of the above PCR reaction. Optionally, the PCR program is: 94-98℃ pre-denaturation for 3-5min; 94-98℃ denaturation for 30s-1min, 50-60℃ annealing for 15-45s, 68-75℃ extension for 20s-1min, 30-40 cycles; 68-75℃ extension for 8-15min, which helps to enhance the specificity of the PCR reaction and promote the efficient PCR.

[0045] Further optionally, in an optional example of the present embodiment, the PCR program is: pre-denaturation at 94°C for 5 min, setting 1 cycle; denaturation at 94°C for 45 s, annealing at 58°C for 30 s, extension at 72°C for 30 s, setting 35 cycles; extension at 72°C for 10 min, setting 1 cycle.

[0046] Optionally, step (3) includes: observing the PCR product by gel electrophoresis; if the PCR product has a band between 750-1000 bp on the electrophoresis map, it indicates that the diseased material contains avian Pasteurella; if the PCR product is not between 750-1000 bp or has no band, it indicates that the diseased material does not contain avian Pasteurella, thereby quickly judging whether the detected diseased material contains avian Pasteurella.

[0047] Example 3

[0048] Based on the method for rapid identification of avian Pasteurella in diseased materials shown in Example 2, this example shows the identification process of detecting whether avian Pasteurella is contained in diseased materials under specific working conditions. It should be noted that this implementation is only an example display and does not limit the scope of protection of the present invention.

[0049] Take the identification process of whether the diseased materials of a duck farm contain avian Pasteurella as an example. Specifically:

[0050] (1) The diseased materials are dead animal bodies / organs after dissection provided by the farm. The diseased hearts, livers and lungs are obtained after dissection. The diseased tissues and organs are then inoculated aseptically on TSA plates and cultured at 37°C for 24 hours to obtain colonies.

[0051] (2) Observe the colony morphology and take samples from the TSA plate that has been cultured for 24 hours in step (1). A person skilled in the art can use a sterile toothpick or other tool to pick, dip or scratch the colonies on the TSA plate as the target strain to prepare the PCR reaction system to be tested, so as to further determine whether it is avian Pasteurella by colony PCR.

[0052] It is worth noting that, in general, the colony morphology of avian Pasteurella is grayish white, round, slightly raised, moist, with a smooth surface and neatly edged dew-drop-like. Technical personnel in this field can preferably obtain colonies suspected of avian Pasteurella based on general judgment criteria, and can select target bacteria with a grayish white, round, slightly raised, moist, smooth surface and neatly edged dew-drop-like colony morphology for further testing.

[0053] (3) Prepare multiple PCR reaction systems for identification of avian Pasteurella: 12.5 μL of 2×Taq Plus Master Mix, 1 μL of upstream primer PM-ompH-F2, 1 μL of downstream primer PM-ompH-R2, 8 μL of ddH2O, and target strains to be detected; wherein the target strains to be detected are picked or scratched from the target colonies suspected of avian Pasteurella on the cultured plate. In this embodiment, a total of 6 target colonies were picked for subsequent identification.

[0054] (4) Set the PCR program: pre-denaturation at 94°C for 5 min, set 1 cycle; denaturation at 94°C for 45 s, annealing at 58°C for 30 s, and extension at 72°C for 30 s, set 35 cycles; extension at 72°C for 10 min, set 1 cycle.

[0055] (5) Perform agarose gel electrophoresis: 1.2% agarose gel, 7ul PCR product, 5ul Marker, Marker is DL2000; the electrophoresis instrument voltage is set to 150V, and the electrophoresis time is 30min.

[0056] (6) Analyze whether the samples and diseased materials contain avian Pasteurella based on the results of the gel imaging system. Figure 1 As shown, in the gel imaging system results, lane 8 is the negative control, lane M is DL2000, and lanes 1-6 are the DNA amplification results of different target bacteria selected in this example. Figure 1 The results shown are as follows: ① Combined with the electrophoresis results of the positive control and the negative control, it is reflected that the detection results of the primer system of the present invention are accurate; ② The bands of the products obtained by colony PCR in lanes 1-3 are between 750bp and 1000bp, and the target bands consistent with avian Pasteurella were obtained; ③ There are no bands in lanes 4-8.

[0057] The results show that the colonies picked from lanes 1-3 are avian Pasteurella, and the colonies picked from lanes 4-7 are not avian Pasteurella. According to the test results, it can be judged that the diseased material detected in this embodiment contains avian Pasteurella. Therefore, in the subsequent duck breeding process, attention should be paid to relevant isolation and prevention work to reduce economic losses in breeding.

[0058] It should be noted that when obtaining the target strain on the TSA plate, those skilled in the art prefer the suspected avian Pasteurella colonies as the target strains for further identification. In the actual disease material identification process, when all suspected avian Pasteurella colonies have been sampled and identified, and no avian Pasteurella is found, it is considered that the identified disease material does not contain avian Pasteurella.

[0059] Example 4

[0060] In order to further verify the accuracy of the method for rapid identification of avian Pasteurella in diseased materials of the present invention, the detection results of Example 3 were further verified in this example.

[0061] Specifically, the colony PCR products of lanes 1-3 in the gel imaging system results were sent to a sequencing company for sequencing verification; after the sequencing results were obtained, the Nucleotide BLAST tool on NCBI was used for sequencing comparison, and the results obtained confirmed that the Per.ident of Pasteurella multocida reached 99.88%-100%, verifying that the technical solution of the present invention can determine that the identified strain is avian Pasteurella based on the colony PCR products of lanes 1-3.

[0062] Therefore, the present invention adopts the colony PCR identification method of specific primer pairs, which can efficiently and accurately detect whether the diseased material contains avian Pasteurella. Compared with the traditional technology of identifying avian Pasteurella in diseased materials, this method omits the steps of strain purification and cultivation, staining microscopy and nucleic acid extraction, greatly simplifying the identification process; and the identification method of the present invention can identify multiple strains at one time, so as to provide a basis for timely judging whether there is a high concentration of avian Pasteurella in the diseased material, which is helpful to adjust disease prevention and control measures to reduce economic losses; the method of the present invention for quickly identifying avian Pasteurella in diseased materials has important promotion and application value.

[0063] It should be noted that the above contents are further detailed descriptions of the present invention in combination with specific implementation methods, and it cannot be determined that the specific implementation of the present invention is limited to these descriptions; the dimensional data of this embodiment does not limit the technical solution, but only shows one of the specific working conditions. For ordinary technicians in the technical field to which the present invention belongs, several simple improvements and modifications can be made without departing from the concept of the present invention, which should be regarded as falling within the scope of protection of the present invention.

Claims

1. A primer pair for rapid identification of avian Pasteurella in diseased materials, characterized in that: The primer pair is obtained by PCR amplification using the outer membrane protein ompH gene with the nucleotide sequence of SEQ ID NO.1 as a template.

2. The primer pair for rapid identification of avian Pasteurella in diseased materials according to claim 1, characterized in that: The nucleotide sequences of the primer pair are PM-ompH-F2: 5'-CAGCAACTTCAGCAAAC-3', PM-ompH-R2: 5'-CCAAAACCTACAGCGAC-3'.

3. Use of the primer pair described in claim 1 or 2 in rapid identification of avian Pasteurella in diseased materials.

4. The use according to claim 3, characterized in that: The application is to perform colony PCR using the primer pair.

5. The use according to claim 4, characterized in that: The band of the product obtained by colony PCR using the primers is between 750 bp and 1000 bp.

6. A method for rapidly identifying avian Pasteurella in diseased materials using the primer pair described in claim 1 or 2, characterized in that: The method comprises the following steps: (1) sterilely inoculating the diseased material on a TSA plate for culturing; (2) streaking the target colony to obtain a PCR reaction system, and using the primer pair for PCR amplification; and (3) analyzing the PCR amplification product.

7. The method for rapid identification of avian Pasteurella in diseased materials according to claim 6, characterized in that: The diseased material is taken from the liver, heart or lung of dead animals.

8. The method for rapid identification of avian Pasteurella in diseased materials according to claim 6, characterized in that: The PCR reaction system includes 2×Taq Plus Master Mix 12.5-25 μL, upstream primer PM-ompH-F2 1-2 μL, downstream primer PM-ompH-R2 1-2 μL, ddH2O 8-16 μL and target strain.

9. The method for rapid identification of avian Pasteurella in diseased materials according to claim 6, characterized in that: The PCR procedure is: pre-denaturation at 94-98°C for 3-5 min; denaturation at 94-98°C for 30s-1 min, annealing at 50-60°C for 15-45s, extension at 68-75°C for 20s-1 min, and cycle for 30-40 times; and extension at 68-75°C for 8-15 min.

10. The method for rapid identification of avian Pasteurella in diseased materials according to claim 6, characterized in that: The step (3) comprises: observing the PCR product by gel electrophoresis; if the PCR product has a band between 750-1000 bp on the electrophoresis map, it indicates that the diseased material contains avian Pasteurella; if the PCR product is not between 750-1000 bp or has no band, it indicates that the diseased material does not contain avian Pasteurella.

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