Serotyping method for Mannhemia haemolitica, serotyping PCR kit and primer pair

Abstract

This invention provides a serotyping method, a serotyping PCR kit, and a primer pair for Mannhemia haemolitica, enabling simple and rapid serotyping of the species. [Solution] For the 12 serotypes of Mannhemia haemolitica (A1, A2, A5, A6, A7, A8, A9, A12, A13, A14, A16, A17), 11 specific capsule-coding genes are targeted, with serotypes A14 and A16 having homologous capsule-coding genes.

Description

Technical Field

[0001] The present invention relates to a method for serotyping Mannheimia haemolytica by genetic techniques, a serotyping PCR kit, and a primer pair that can be used in the kit.

Background Art

[0002] Bovine respiratory disease complex (BRDC) represented by pneumonia is a disease that occurs in association with stress sensitization due to long-distance transportation or changes in the rearing environment and co-infection with bacteria and viruses. When suffering from BRDC, growth rate and meat quality decline may be observed, and death may also occur, which is a factor causing serious economic damage and is a problem in farms around the world.

[0003] Pathogens causing BRDC include Mannheimia haemolytica (Mh), Mycoplasma bovis, bovine herpesvirus 1, bovine RSV, bovine parainfluenza virus type 3, bovine viral diarrhea virus, and adenovirus type 7. In particular, cases involving Mh are known to have a high fatality rate. Mh, a bacterium belonging to the family Pasteurellaceae, is a resident bacterium in the upper respiratory tract of cattle, but it grows in the lower respiratory tract along with stress sensitization and causes respiratory symptoms. The capsule expressed by Mh has diversity, and 12 serotypes (A1, A2, A5, A6, A7, A8, A9, A12, A13, A14, A16, A17) that can be immunologically discriminated have been confirmed so far. It is also known that Mh has different toxicity depending on the serotype and that changes occur in the prevalent serotype of Mh.

[0004] From these facts, in order to consider the prevention and countermeasures of BRDC, identification and serotyping of Mh are required at livestock farms and research institutions. For the above-mentioned immunological serotype-specific tests, immune sera prepared using each serotype strain (antigen) are required, and a lot of labor is required for the preparation of the immune sera. Therefore, the development of new test methods is demanded.

[0005] In microorganisms in general, genetic testing methods have been developed to replace immunological serotyping, identifying genes or base sequences specific to each serotype and detecting them specifically. The PCR testing method described in Non-Patent Literature 1, through comparative analysis of the Mh genome, identifies unique genes related to capsular polysaccharide synthesis that distinguish between the three major serotypes (A1, A2, and A6) known as circulating Mh serotypes out of 12 types, thereby enabling the determination of serotype strains A1, A2, and A6. [Prior art documents] [Non-patent literature]

[0006] [Non-Patent Document 1] Klima CL, Zaheer R, Briggs RE, McAllister TA. A multiplex PCR assay for molecular capsular serotyping of Mannheimia haemolytica serotypes 1, 2, and 6. J Microbiol Methods. 2017 Aug;139:155 160 [Overview of the project] [Problems that the invention aims to solve]

[0007] However, the PCR testing method described in Non-Patent Document 1 can only determine three serotypes (A1, A2, A6) and cannot determine Mh belonging to other serotypes, thus having the problem of being unable to detect their emergence or outbreaks.

[0008] This invention has been made in view of these problems, and aims to provide a method for determining the serotype of Mannhemia haemolitica, a serotyping PCR kit, and a primer pair that can determine the serotype of Mannhemia haemolitica in a simple and rapid manner. [Means for solving the problem]

[0009] To solve the aforementioned problems, the serotype determination method of the present invention is: This is a serotyping method for determining the serotype of Mannhemia haemolitica. This approach is characterized by targeting 11 specific capsular coding genes for 12 serotypes of Mannhemia haemolitica (A1, A2, A5, A6, A7, A8, A9, A12, A13, A14, A16, and A17), with serotypes A14 and A16 being homologous capsular coding genes. This feature allows for simple and rapid serotyping of Mannhemia haemolitica by targeting 11 specific capsule-coding genes, with serotypes A14 and A16 being homologous capsule-coding genes, for each of the 12 serotypes of Mannhemia haemolitica.

[0010] This method is characterized by its use of polymorphisms in the recN gene to identify Mannhemia hemorrhagicia. This characteristic allows for the simple and rapid identification and serotyping of Mannhemia haemolitica by utilizing polymorphisms in the Mannhemia haemolitica-specific recN gene, which are different from the capsule-coding gene targeted for serotyping.

[0011] The present invention provides a PCR kit for determining the serotype of Mannhemia haemolitica. This is a multiplex PCR kit for determining the serotype of Mannhemia haemolitica. It features 11 specific primer pairs targeting the capsule-coding genes of 12 serotypes of Mannhemia haemolitica (A1, A2, A5, A6, A7, A8, A9, A12, A13, A14, A16, and A17), with serotypes A14 and A16 sharing a common primer pair targeting homologous capsule-coding genes. This characteristic allows for the simultaneous amplification of different target genes by multiplex PCR using 11 primer pairs that are highly specific to and non-interfering with 11 capsule-coding genes that target 12 serotypes of Mannhemia haemolitica. Therefore, serotyping of Mannhemia haemolitica can be performed simply and quickly.

[0012] It is further characterized by having one type of primer pair specific to Mannhemia hemorrhagica, utilizing polymorphisms in the recN gene. This characteristic allows for the simultaneous amplification of target genes by serotyping primer pairs and identification primer pairs using multiplex PCR with 11 primer pairs for serotyping and 1 primer pair for identification that are specific to Mannhemia haemolitica and do not interfere with each other. Therefore, the identification and serotyping of Mannhemia haemolitica can be performed simply and quickly.

[0013] The primer pair of the present invention is These are 11 specific primer pairs targeting the capsule-coding genes of 12 serotypes of Mannhemia haemolitica (A1, A2, A5, A6, A7, A8, A9, A12, A13, A14, A16, and A17), and are characterized by the fact that serotypes A14 and A16 share a common primer pair targeting homologous capsule-coding genes. This characteristic allows for the simultaneous amplification of different target genes by multiplex PCR using 11 primer pairs that are highly specific to and non-interfering with 11 capsule-coding genes that target 12 serotypes of Mannhemia haemolitica. Therefore, serotyping of Mannhemia haemolitica can be performed simply and quickly.

[0014] The system is characterized by having 12 primer pairs, consisting of the 11 primer pairs mentioned above plus one primer pair specific to Mannhemia hemorrhagica that utilizes polymorphisms in the recN gene. This characteristic allows for the simultaneous amplification of target genes by serotyping primer pairs and identification primer pairs using multiplex PCR with 11 primer pairs for serotyping and 1 primer pair for identification that are specific to Mannhemia haemolitica and do not interfere with each other. Therefore, the identification and serotyping of Mannhemia haemolitica can be performed simply and quickly. [Brief explanation of the drawing]

[0015] [Figure 1] This figure shows the capsule-coding genes and marker genes for all serotypes of Mannhemia haemolitica (A14 and A16 are common). [Figure 2] This figure shows the capsule-coding gene groups of serotypes A14 and A16 of Mannhemia haemolitica. [Figure 3] This figure shows the composition of 11 primer pairs used in the Mannhemia haemolitica serotyping PCR kit in the embodiments of the present invention, as well as the primer sequence length, G+C content, and PCR product size for each primer pair. [Figure 4] (a) is a figure showing the diversity of primer annealing regions on the recN gene of Mannhemia species, and (b) is a figure showing in a table the composition of the identification primer pair used in the Mannhemia haemolitica serotyping PCR kit in the example, and the sequence length of the primers, G+C content, and PCR product size of the primer pair. [Figure 5] This figure shows the primer pair configurations of the Mannhemia haemolitica serotyping PCR kits (two types, Kit A and Kit B) used in the examples, and the results of specificity confirmation experiments for each primer pair using serotype reference strains (results of electrophoresis of each primer amplified by PCR). [Figure 6] (a) is a table showing the results of a specificity confirmation experiment targeting the Mannhemia haemolitica (Mh) strain, and (b) is a table showing the results of a specificity confirmation experiment targeting Mannhemia strains other than Mannhemia haemolitica.

Mode for Carrying Out the Invention

[0016] The mode for carrying out the method for serotype determination of Mannheimia haemolytica, the serotype determination PCR kit and the primer pair according to the present invention will be described below based on examples.

Examples

[0017] The inventors obtained genomic information from 12 Mh serotype reference strains and identified the gene groups encoding the capsules targeted by 12 serotypes. As a result of homology analysis, each was found to possess 3 to 5 specific genes, and it was found that serotypes A14 and A16 possess homologous capsule-encoding gene groups (see Fig. 2). For each serotype, a method for serotype determination of Mannheimia haemolytica targeting 11 specific capsule-encoding genes (marker genes, see Fig. 1) with serotypes A14 and A16 as homologous capsule-encoding genes was established. Further, based on the above serotype determination method, the inventors considered the primer sequence length, G (guanine) + C (cytosine) content, and PCR product size in order to exhibit the specificity of the primer pair and the stability of PCR, and prepared a multiplex PCR kit as a serotype determination PCR kit for Mannheimia haemolytica comprising 11 primer pairs for the capsule-encoding genes specific to each serotype. Since serotypes A14 and A16 possess homologous capsule-encoding genes, a common primer pair is used for serotype determination.

[0018] Hereinafter, the serotype determination PCR kit for Mannheimia haemolytica of this example may also be referred to as "this serotype determination PCR kit".

[0019] This serotype determination PCR kit is a multiplex PCR kit comprising 11 primer pairs (common for serotypes 14 and 16) for the capsule-encoding genes specific to 12 serotypes.

[0020] Figure 3 shows the configurations of 11 primer pairs, as well as the primer sequence length, G+C content, and PCR product size for each primer pair. By setting the primer sequence length to 19-21 nucleotides, a sequence pattern specific to the target gene can be obtained. Furthermore, by setting the G+C content to 50-52.6%, differences in primer Tm values ​​can be minimized, enabling efficient annealing. Finally, by setting the PCR product size to 132-990 bp, amplification efficiency can be increased.

[0021] Furthermore, in addition to the 11 primer pairs for the capsule-coding genes specific to the 12 Mh serotypes mentioned above, this serotyping PCR kit also includes one additional Mh-specific primer pair utilizing polymorphisms in the recN gene. The diversity of primer annealing regions on the recN gene of Mannhemia species is shown in Figure 4(a).

[0022] Furthermore, in order to simultaneously identify Mh and determine serotyping using multiplex PCR in this serotyping PCR kit, the primer pair for identification must be designed with the same considerations as the primer pair for serotyping. Therefore, the primer pair for identification was created as an Mh-specific primer pair using polymorphisms in the recN gene.

[0023] Figure 4(b) shows the primer pair for identification, as well as the sequence length of the primers in that pair, the G+C content, and the size of the PCR product.

[0024] Here, as the serotyping PCR kit, a multiplex PCR kit was constructed containing 11 primer pairs for serotyping and 1 primer pair for identification, as described above, and Mh identification and serotyping were performed. Specifically, as shown in Figure 5, two types of multiplex PCR kits were constructed: Kit A, which contains 6 primer pairs corresponding to serotypes A1, A9, A8, A2, A6, and A12, and an identification primer; and Kit B, which contains 5 primer pairs corresponding to serotypes A17, A5, A7, A13, and A14 / A16, and an identification primer. Using 11 Mh serotype reference strains (excluding serotype A17) and other strains belonging to the genus Mannhemia, the presence or absence of amplification by PCR and the specificity of individual primer pairs and multiplex PCR kits were confirmed. The thermal reaction conditions for PCR (20 seconds at 94°C, 20 seconds at 58°C, and 30 seconds at 72°C, for a total of 25 cycles) were the same for Kit A and Kit B.

[0025] First, Figure 5 shows the results of electrophoresis performed on DNA fragments amplified by PCR using Kit A and Kit B for 11 Mh serotype reference strains, excluding serotype A17. The Mh serotype reference strains used here are the genome sequencing strains used to identify the capsule-coding gene group.

[0026] As shown in Figure 5, for all Mh serotype reference strains amplified by PCR using Kit A and Kit B, a band appeared at a position corresponding to 591 bp, confirming that Mh could be identified by the identification primers. Furthermore, for all Mh serotype reference strains, a band appeared at a position corresponding to the PCR product size set by the corresponding primer pair (A14 and A16 are at the same position), confirming that the Mh serotype could be accurately determined.

[0027] Next, Figure 6 shows the results of confirming the specificity of individual primer pairs and multiplex PCR kits using 11 Mh serotype reference strains (excluding Mh serotype A17) and other strains belonging to the genus Mannhemia. In Figure 6, "+" indicates amplification of the Mh identification primer, and "-" indicates no amplification. For each serotype, "Serotype" indicates amplification of the corresponding primer pair, and "-" indicates no amplification. The Mh serotype reference strains used here consisted of 1 to 3 strains for each serotype, and included at least one genome sequencing strain.

[0028] As shown in Figure 6(a), for all samples of the 11 Mh serotype reference strains, the identification and serotyping of Mh were accurately performed by amplification of each primer. On the other hand, as shown in Figure 6(b), amplification of each primer did not occur for other strains belonging to the genus Mannhemia other than Mh, thus confirming the specificity of this serotyping PCR kit for Mh.

[0029] As explained above, this serotyping PCR kit enables widespread, simple, and rapid testing for Myrrh, demonstrating its effectiveness in Myrrh epidemic surveys and other related applications.

[0030] Although embodiments of the present invention have been described above with reference to the drawings, the specific configurations are not limited to these embodiments, and any changes or additions that do not depart from the spirit of the present invention are also included.

[0031] For example, in the above embodiment, the Mh serotyping PCR kit was configured as a multiplex PCR kit comprising 11 types of primer pairs for serotyping and 1 type of primer pair for identification, and an embodiment was described in which Mh identification and serotyping were performed simultaneously. However, the embodiment is not limited to this, and the Mh serotyping PCR kit may be configured as a multiplex PCR kit comprising 11 types of primer pairs for serotyping, and only Mh serotyping may be performed.

[0032] Furthermore, although the above-described examples mainly described serotyping using a PCR method with an Mh serotyping PCR kit, the method is not limited to this. Based on the Mh serotyping method of the present invention, serotyping may also be performed for the 12 Mh serotypes by gene identification targeting 11 specific capsular coding genes, with serotypes A14 and A16 being homologous capsular coding genes. [Industrial applicability]

[0033] The present invention offers industrial potential as a serotyping method, a serotyping PCR kit, and a primer pair usable with the kit, enabling simple and rapid serotyping of Mannhemia haemolitica (Mh), the cause of bovine respiratory disease syndrome (BRDC) in the livestock and veterinary fields. Furthermore, the serotyping PCR kit of the present invention is configured as a multiplex PCR kit comprising 11 primer pairs for serotyping and 1 primer pair for identification, enabling simple and rapid identification and serotyping of Mh, making it effective for Mh epidemic surveys and other applications. In addition, the present invention can be used for rapid identification and serotyping of isolates in isolation techniques, thus having a wide range of applications.

Claims

1. This is a serotyping method for determining the serotype of Mannhemia haemolitica. A method for determining the serotype of Mannhemia haemolitica, characterized by targeting 11 specific capsular coding genes, with serotypes A14 and A16 being homologous capsular coding genes, for the 12 serotypes A1, A2, A5, A6, A7, A8, A9, A12, A13, A14, A16, and A17 of Mannhemia haemolitica.

2. The method for determining the serotype of Mannhemia haemolitica according to claim 1, characterized by using polymorphisms of the recN gene to identify Mannhemia haemolitica.

3. This is a multiplex PCR kit for determining the serotype of Mannhemia haemolitica. A PCR kit for determining the serotype of Mannhemia haemolitica, characterized by having 11 specific primer pairs targeting the capsule-coding gene groups of 12 serotypes of Mannhemia haemolitica (A1, A2, A5, A6, A7, A8, A9, A12, A13, A14, A16, and A17), with serotypes A14 and A16 having a common primer pair targeting homologous capsule-coding gene groups.

4. The serotyping PCR kit for Mannhemia haemolitica according to claim 3, further comprising a single primer pair specific to Mannhemia haemolitica using polymorphisms of the recN gene.

5. This primer pair is characterized by having 11 specific primer pairs targeting the capsule-coding gene groups of 12 serotypes of Mannhemia haemolitica: A1, A2, A5, A6, A7, A8, A9, A12, A13, A14, A16, and A17, with serotypes A14 and A16 having a common primer pair for homologous capsule-coding gene groups.

6. The primer pair according to claim 5, characterized in that it consists of 12 types of primer pairs, which are obtained by adding one primer pair that is specific to Manhemia haemolitica using polymorphisms of the recN gene to the 11 types of primer pairs mentioned above.

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