PCR primers and a multiplex PCR kit for distinguishing atypical O serogroups of Escherichia coli.

Abstract

To provide clarification of details of atypical O serogroup judged as "O-serogroup untypable" and classification techniques for classifying the same.SOLUTION: Provided is a multiplex PCR kit given by anyone of from MP22 to MP25 to comprehensively classify O serogroups of pathogenic Escherichia coli. These MP22 to MP25 comprise 30 primers of specific sequences for atypical O serogroups to enable efficient detection and classification of pathogenic E. coli including atypical O serogroups.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] The present invention relates to PCR primers and the like for identifying atypical O serogroups. [Background technology]

[0002] Approximately 4,000 cases of infection caused by enterohemorrhagic Escherichia coli (EHEC) occur annually in Japan, and some patients die from complications such as hemolytic uremic syndrome and brain damage. For this reason, EHEC is considered one of the bacteria that cause food poisoning and requires the most attention, and is designated as a Category 3 bacterium (all cases must be reported) under the Infectious Diseases Control Law. When EHEC infection occurs, detailed classification of the isolates is necessary to determine whether it is a group infection, identify the source of contamination, and compare with past cases. Serological classification (O serogroup classification) targeting the bacterial somatic antigen (O antigen) is commonly used to subclassify E. coli.

[0003] O antigens are cell wall antigens in eubacteria. O antigens have carbohydrate structures, and even within the same species, the types of sugars that make up the O antigen and the order of their linkages vary. Taking advantage of the antigenicity of these different carbohydrate structures for each O serogroup, O serogroups are used to detect, identify, and classify eubacteria.

[0004] The same applies to E. coli, including enterohemorrhagic E. coli. For example, in the classification of O serogroups in enterohemorrhagic Escherichia coli, if two strains have the same O serogroup, it can be inferred that they are phylogenetically closely related, which is useful information for outbreak and epidemiological investigations.

[0005] Currently, the Danish State Serum Institute (SSI) has determined the O-serogroups of E. coli, from O1 to O188, and standard strains and antiserum reagents are commercially available. Furthermore, public health centers, local public health institutes, and the National Institute of Infectious Diseases in Japan classify all EHEC isolates into O-serogroups, which are used in outbreak and epidemiological investigations.

[0006] The gene region encoding the O antigen (O antigen coding gene cluster) exists on the chromosome of E. coli, and there is diversity in the genetic organization or base sequence that differs for each O serogroup. The inventors' research group has previously disclosed various technologies based on the base sequence information of the O antigen-encoding gene clusters of Escherichia coli typical O serogroup (O1 to O188) reference strains and Shigella typical serogroup reference strains. In other words, in the gene region encoding the O antigen of E. coli, there are gene pairs (wzy / wzx, wzm / wzt) involved in the transport and binding of O antigen glycans, and the inventors' research group has disclosed technology related to PCR primers that can target these gene pairs and identify E. coli O serogroups, as well as multiplex PCR kits (MP-1 to MP-20) that combine these primers (Patent Documents 1 and 2, Non-Patent Documents 1 and 2).

[0007] On the other hand, some EHEC isolates in Japan (approximately 10-40 cases per year) belong to the unclassifiable atypical O serogroup. In other words, atypical O serogroups are treated as O-serogroup untypable (OUT) because the O serogroup cannot be determined as a result of testing. Therefore, atypical O serogroups cannot be further classified, and information that can be used for prevalence and epidemiological investigations cannot be obtained. Furthermore, a large proportion of EHEC isolated from livestock and wild animals, as well as pathogenic E. coli other than EHEC, are identified as OUT, and the breakdown of this proportion is unclear. For these reasons, the existence of atypical O serogroups has been a bottleneck in the investigation and research of pathogenic E. coli, including EHEC. [Prior art documents] [Patent documents]

[0008] [Patent Document 1] Japanese Patent Application Laid-Open No. 2013-220032 [Patent Document 2] WO2014 / 157611 [Non-patent literature]

[0009] [Non-Patent Document 1] Iguchi A et al. 2015. Escherichia coli O-genotyping PCR: a comprehensive and practical platform for molecular O serogrouping. J Clin Microbiol 53 : 2427-2432. [Non-patent document 2] Iguchi A et al.,2020. Additional Og-Typing PCR Techniques Targeting Escherichia coli-Novel and Shigella-Unique O-Antigen Biosynthesis Gene Clusters. J Clin Microbiol.58 : e01493-20. DISCLOSURE OF THE INVENTION [Problem to be solved by the invention]

[0010] Against the background of the above circumstances, the objective of this study is to clarify the details of the atypical O serogroup that is determined to be OUT, and to provide a classification technique that can classify it. [Means for solving the problem]

[0011] As a result of intensive research, the inventors have clarified the gene sequences of several atypical O serogroups that are judged to be OUT, and have completed an invention related to primers that enable the detection and classification of the O antigen-encoding genes of these atypical O serogroups by PCR. Furthermore, the inventors have completed an invention relating to a multiplex PCR kit that can specifically and comprehensively analyze multiple O serogroups of pathogenic E. coli, including such atypical O serogroups.

[0012] The present invention comprises the following configurations. [1] A multiplex PCR kit as set forth in any one of (1) to (4) below, characterized in that it comprehensively classifies the O serogroup of pathogenic Escherichia coli. (1) MP22 Primer set 9 (SEQ ID NO: 17, SEQ ID NO: 18), Primer set 12 (SEQ ID NO: 23, SEQ ID NO: 24), Primer set 21 (SEQ ID NO: 41, SEQ ID NO: 42), Primer set 23 (SEQ ID NO: 45, SEQ ID NO: 46), Primer set 26 (SEQ ID NO: 51, SEQ ID NO: 52), Primer set 27 (SEQ ID NO: 53, SEQ ID NO: 54), Primer set 31 (SEQ ID NO: 61, SEQ ID NO: 62), Primer set 32 ​​(SEQ ID NO: 63, SEQ ID NO: 64) (2) MP23 Primer set 1 (SEQ ID NO: 1, SEQ ID NO: 2), Primer set 10 (SEQ ID NO: 19, SEQ ID NO: 20), Primer set 17 (SEQ ID NO: 33, SEQ ID NO: 34), Primer set 18 (SEQ ID NO: 35, SEQ ID NO: 36), Primer set 20 (SEQ ID NO: 39, SEQ ID NO: 40), Primer set 25 (SEQ ID NO: 49, SEQ ID NO: 50), Primer set 33 (SEQ ID NO: 65, SEQ ID NO: 66) (3) MP24 Primer set 2 (SEQ ID NO: 3, SEQ ID NO: 4), Primer set 6 (SEQ ID NO: 11, SEQ ID NO: 12), Primer set 8 (SEQ ID NO: 15, SEQ ID NO: 16), Primer set 13 (SEQ ID NO: 25, SEQ ID NO: 26), Primer set 15 (SEQ ID NO: 29, SEQ ID NO: 30), Primer set 16 (SEQ ID NO: 31, SEQ ID NO: 32) (4) MP25 Primer set 3 (SEQ ID NO: 5, SEQ ID NO: 6), Primer set 4 (SEQ ID NO: 7, SEQ ID NO: 8), Primer set 5 (SEQ ID NO: 9, SEQ ID NO: 10), Primer set 7 (SEQ ID NO: 13, SEQ ID NO: 14), Primer set 11 (SEQ ID NO: 21, SEQ ID NO: 22), Primer set 14 (SEQ ID NO: 27, SEQ ID NO: 28)

[0013] [2] A primer set for identifying the O serogroup, which is included in the multiplex PCR kit described in [1], and is a primer set represented by any one of primer sets 1 to 15. [3] A primer for identifying the O serogroup, which is included in the primer set described in [2], and is a primer represented by any of SEQ ID NOs: 1 to 30. [4] A testing method for detecting or classifying the O serogroup by performing PCR using any one of the multiplex PCR kit described in [1], the primer set described in [2], and the primer described in [3]. [Effects of the Invention]

[0014] The present invention has clarified the details of the atypical O serogroup determined to be OUT, and has made it possible to provide a classification technique capable of classifying them. In other words, this invention makes it possible to rapidly and accurately detect and classify atypical O serogroup E. coli in a laboratory or research lab, which could not be classified using existing serological methods. This is expected to contribute to the prevention and spread of pathogenic E. coli infections, including enterohemorrhagic E. coli, in medical, public health, and food hygiene settings. [Brief explanation of the drawings]

[0015] [Figure 1] A diagram showing an overview of each O serogroup, including atypical O serogroups, and the primers used. [Figure 2] A diagram showing an overview of the multiplex PCR kit. [Figure 3]FIG. 1 shows the results of evaluating the specificity of a primer set. [Figure 4] FIG. 1 shows the results of an evaluation of the specificity of a multiplex PCR kit. DETAILED DESCRIPTION OF THE INVENTION

[0016] The present invention will now be described.

[0017] The present invention was completed based on the following findings made by the inventors. (1) The inventors have clarified the specific sequences of four types (OgN32, OgN33, OgN34, and Og48va) of enterohemorrhagic Escherichia coli (atypical O serogroup) that were previously determined to be unclassifiable in Japan. (2) The inventors analyzed six types of atypical O serogroup (OgX18, OgX25, OgG5413, OgN-RKI3, OgN-RKI4, and OgS88) and found that these six types are homologous to previously reported sequences. (3) The inventors designed novel primers that enable the detection of 15 atypical O serogroups, including the 10 mentioned above. These novel primers were able to specifically detect the target O serogroups. (4) The inventors designed a multiplex PCR kit that combines the novel primers described in (3), primers designed for the O serogroup previously detected as atypical O serogroup EHEC, and primers designed for the Shigella O serogroup. This multiplex PCR kit was able to specifically detect the target O serogroup while maintaining the specificity of the primers. (5) Furthermore, the multiplex PCR kit enabled the detection of multiple O serogroups and the determination of their classification, as the O serogroups detected could be clearly distinguished based on the product length.

[0018] The multiplex PCR kit of the present invention (hereinafter abbreviated as "MP kit") is a multiplex PCR kit shown in any of (1) to (4) below, and is characterized by comprehensively classifying the O serogroup of pathogenic Escherichia coli. (1) MP22 Primer set 9 (SEQ ID NO: 17, SEQ ID NO: 18), Primer set 12 (SEQ ID NO: 23, SEQ ID NO: 24), Primer set 21 (SEQ ID NO: 41, SEQ ID NO: 42), Primer set 23 (SEQ ID NO: 45, SEQ ID NO: 46), Primer set 26 (SEQ ID NO: 51, SEQ ID NO: 52), Primer set 27 (SEQ ID NO: 53, SEQ ID NO: 54), Primer set 31 (SEQ ID NO: 61, SEQ ID NO: 62), Primer set 32 ​​(SEQ ID NO: 63, SEQ ID NO: 64) (2) MP23 Primer set 1 (SEQ ID NO: 1, SEQ ID NO: 2), Primer set 10 (SEQ ID NO: 19, SEQ ID NO: 20), Primer set 17 (SEQ ID NO: 33, SEQ ID NO: 34), Primer set 18 (SEQ ID NO: 35, SEQ ID NO: 36), Primer set 20 (SEQ ID NO: 39, SEQ ID NO: 40), Primer set 25 (SEQ ID NO: 49, SEQ ID NO: 50), Primer set 33 (SEQ ID NO: 65, SEQ ID NO: 66) (3) MP24 Primer set 2 (SEQ ID NO: 3, SEQ ID NO: 4), Primer set 6 (SEQ ID NO: 11, SEQ ID NO: 12), Primer set 8 (SEQ ID NO: 15, SEQ ID NO: 16), Primer set 13 (SEQ ID NO: 25, SEQ ID NO: 26), Primer set 15 (SEQ ID NO: 29, SEQ ID NO: 30), Primer set 16 (SEQ ID NO: 31, SEQ ID NO: 32) (4) MP25 Primer set 3 (SEQ ID NO: 5, SEQ ID NO: 6), Primer set 4 (SEQ ID NO: 7, SEQ ID NO: 8), Primer set 5 (SEQ ID NO: 9, SEQ ID NO: 10), Primer set 7 (SEQ ID NO: 13, SEQ ID NO: 14), Primer set 11 (SEQ ID NO: 21, SEQ ID NO: 22), Primer set 14 (SEQ ID NO: 27, SEQ ID NO: 28) The MP kit of the present invention makes it possible to efficiently and comprehensively classify multiple enterohemorrhagic Escherichia coli strains into O serogroups, including 15 strains that can be determined to belong to the atypical O serogroup.

[0019] In another embodiment, the present invention is configured as a PCR primer set. In other words, it is composed of a primer set designated as any one of primer sets 1 to 15, which can detect and classify 15 types of O serogroups that can be determined to be atypical O serogroups.

[0020] In another embodiment, the present invention is configured as a PCR primer. That is, it is composed of primers designated as primers 1 to 30, which can detect and classify 15 types of O serogroups that can be determined to be atypical O serogroups.

[0021] The present invention uses the MP kit, primer set, and primers (hereinafter collectively referred to as "primers, etc.") to perform nucleic acid amplification by PCR, and detects pathogenic E. coli or classifies it into the O serogroup.

[0022] A sample is required for E. coli testing using the PCR method. Any commonly used sample can be used as this sample, such as food suspected of being infected with E. coli, or biological samples from humans or animals.

[0023] Samples for E. coli testing can be prepared for PCR by conventional pretreatment, such as decomposing tissue cell-derived proteins with protease, followed by nucleic acid extraction and purification using phenol and chloroform, or by extracting the resulting nucleic acid using a commercially available extraction kit. Once the specimen sample is prepared, a procedure for nucleic acid amplification is carried out using the primers of the present invention, etc. In this nucleic acid amplification procedure, for example, at least one set of primer set, a heat-resistant DNA polymerase, and a substrate such as deoxynucleotide triphosphate are added to a reaction solution, and thermal denaturation, annealing, and extension reactions are carried out, and nucleic acid amplification is carried out by repeating these cycles.

[0024] The primer set in the PCR method includes a combination of a forward primer (F) and a reverse primer (R), totaling two primers, and the nucleic acid sequences shown in sequence numbers 1 to 66 in Figure 1 can be used as primers. In the present invention, although these primer sets can be used individually, it is preferable to use an MP kit that combines multiple primer sets to detect and classify O serogroups, which has the effect of enabling comprehensive O serogroup detection and classification tests with a small number of tests, and enabling efficient testing in a short time. Such a multiplex kit can be designed to clearly distinguish the lengths of the nucleic acid amplification products to be detected by the primer sets, while taking into consideration the absence of side reactions due to the primers used. MP kits that can be used are preferably MP21, MP22, MP23, MP24, and MP25, and more preferably MP22, MP23, MP24, and MP25.

[0025] The thermostable DNA polymerase may be any commonly used thermostable DNA polymerase, such as pol I type DNA polymerase, α type DNA polymerase, mixed type DNA polymerase, hot start DNA polymerase, etc.

[0026] Once the nucleic acid amplification reaction operation is completed, detection of the nucleic acid amplification product is performed. For the detection of the nucleic acid amplification product, any commonly used detection method can be used. For example, detection using a labeled oligonucleotide that specifically recognizes the amplified base sequence, the fluorescent intercalator method, detection by agarose gel electrophoresis, etc. Also, detection of the nucleic acid amplification product may be performed while conducting the nucleic acid amplification reaction operation (RT-PCR). In this case, a detection method using a fluorescent dye, such as the intercalator method or the hybridization method, may be used.

[0027] When performing detection of nucleic acid amplification using the primer set of the present invention, various reagents necessary therefor can be packaged in advance and made into a kit to obtain a test kit. In this case, the test kit includes at least one set of the primer set of the present invention and reagents for nucleic acid amplification. Examples of reagents for nucleic acid amplification include a heat-resistant DNA polymerase, four types of dNTPs that serve as substrates for nucleic acid synthesis, buffers and salts that provide suitable conditions for the enzyme reaction, and protective agents that stabilize the enzyme and template.

Examples

[0028] The present invention will be described in detail with reference to examples.

[0029] <<I. Analysis of base sequence information>> 1. Genetic analysis was performed on non-typeable O serogroup EHEC (about 40 strains) isolated from infected patients in the country, and the full base sequence information was determined. Furthermore, based on the determined base sequence information, their O antigen-encoding gene groups were compared and analyzed with the previously reported O antigen-encoding gene information.

[0030] 2. A part of the results of the comparison and analysis is shown in Table 1. (1) Among the untypeable O serogroups of EHEC, four types (OgN32, OgN33, OgN34, Og48va) had novel O antigen-encoding gene clusters. That is, the sequences clarified by the inventors were novel sequences, and each was registered in GenBank as LC550084 for OgN32, LC550085 for OgN33, LC550086 for OgN34, and LC550087 for Og48va. (2) Also, six types (OgX18, OgX25, OgG5413, OgN-RKI3, OgN-RKI4, OgS88) were found to be homologous to previously reported sequences derived from untypeable O serogroup Escherichia coli.

[0031] 3. In addition to the above 10 types of untypeable O serogroups, five types (OgN2, OgN4, OgN13, OgN15, OgN17) derived from untypeable O serogroups determined and published in the inventors' previous research were added to design primers. 4. By targeting the above 15 types of O serogroups for detection, primers or primer sets were designed to cover most of the untypeable O serogroup EHEC at present, and subsequent examinations were carried out.

Table 1

[0032] <<II. Design of PCR Primers>> 1. PCR primers capable of specifically determining 15 types of O antigen-encoding gene clusters were designed targeting each wzx or wzy. As design conditions, the primer length was 19 - 21 bases, the G + C content was 50 - 55%, and the product length obtained was designed within the range of 130 - 890 bp. 2. Fig. 1 shows an overview of each O antigen-encoding genotype and detection primers. In Fig. 1, the PCR primers included in the multiplex PCR kit described later are shown together for reference. (1) In Fig. 1, as described above, the 15 newly designed primers are indicated with underlines. (2) Thereafter, design and examination were carried out using the 33 types of primer sets shown in FIG. 1.

[0033] <<III. Design of Multiplex PCR Kit>> 1. In addition to the above 15 types of novel primers, primers targeting the O antigen coding gene groups of Escherichia coli or Shigella dysenteriae, which were mainly designed by the inventors in the past, were used to design a multiplex PCR kit. That is, in addition to pathogenic Escherichia coli including atypical O serogroup Escherichia coli, a multiplex PCR kit was designed that can comprehensively detect the O serogroups of Shigella dysenteriae, which may be found in conjunction with the detection of enterohemorrhagic Escherichia coli and has a high homology with Escherichia coli. 2. FIG. 2 shows the general outline of the multiplex PCR kit. (1) As multiplex PCR kits (hereinafter referred to as "MP"), MP-21, MP-22, MP-23, MP-24, and MP-25 were designed. (2) Note that MP-22 contains 2 types of novel primers, MP-23 contains 2 types, MP-24 contains 5 types, and MP-25 contains 6 types.

[0034] <<IV. Experimental Method>> 1. For the PCR reaction, DreamTaq Green PCR master mix (2×) (Thermo Fisher Scientific) was used. 2. Regarding the reaction conditions, in the case of single primer PCR, 94°C - 30 seconds, 58°C - 30 seconds, 72°C - 1 minute were used as one cycle, and 25 cycles were performed. 3. In the case of multiplex PCR, after reacting at 94°C for 1 minute, 94°C - 30 seconds, 58°C - 30 seconds, 72°C - 1 minute were used as one cycle, and 25 cycles were performed, and finally, it was reacted at 72°C for 2 minutes. 4. 2 μL of the reaction solution was electrophoresed on a 1% agarose gel, stained with ethidium bromide, and the amplification products were confirmed on a UV transilluminator.

[0035] <<V. Experimental Results>> 1. For each primer set, amplification was performed using only the target reference strains from the total O serogroups of Escherichia coli (O1 - O188), previously reported untypable O serogroup genotypes (OgN1, OgN8, OgN9, OgN10, OgN12, OgN31, OgN3, OgN5), and 15 reference strains (one strain each) of untypable O serogroups used in this development, and the specificity was evaluated. 2. The results are shown in Figure 3. (1) Each primer set showed bands only at the target product length, and no other bands were confirmed. (2) From these results, it was confirmed that for all the primer sets shown in Figure 1, each target O antigen could be specifically detected.

[0036] 3. For each MP, the same specificity evaluation as for the primer sets was performed. 4. The results are shown in Figure 4. (1) In each MP, bands were shown only at the respective target product lengths, and no other bands were confirmed. (2) From these results, it was confirmed that even when detection was performed using MPs combined with each primer, the O antigen - coding gene of the target O serogroup could be specifically detected.

[0037] <<VI. Evaluation of Practicality>> 5. Multiplex PCR of MP - 1 to MP - 25 was performed using enterotoxigenic Escherichia coli (149 strains) isolated from patients. (1) For 146 strains tested, it was possible to classify them into any type (Og type). (2) Among the 146 classified strains, OgN13, OgN15, and OgN5413 were each detected in one strain.

[0038] 6. Multiplex PCR was performed using enterohemorrhagic Escherichia coli (366 strains) isolated from cattle. (1) Among the 366 strains tested, for 365 strains excluding one strain, it was possible to classify them into any type (Og type). (2) Of the 365 strains classified, OgN4 (10 strains), OgN13 (1 strain), OgN32 (17 strains), OgN-RKI4 (2 strains), OgG5413 (1 strain), OgX18 (12 strains), and OgX25 (6 strains) were detected.

[0039] 7. Atypical O-serogroup EHEC isolated from two HUS cases in 2017 and 2018 and from four patients with bloody stools between 2010 and 2019 were classified as OgX18 by PCR. This suggests that EHEC OgX18 may be a highly pathogenic emerging strain that requires close monitoring.

Claims

[Claim 1] A multiplex PCR kit comprising any one of the primers (1) to (4) below, which is characterized by comprehensively classifying the O serogroup of pathogenic Escherichia coli. (1) MP22 containing all primer sets shown below Primer set 9 (SEQ ID NO: 17, SEQ ID NO: 18), Primer set 12 (SEQ ID NO: 23, SEQ ID NO: 24), Primer set 21 (SEQ ID NO: 41, SEQ ID NO: 42), Primer set 23 (SEQ ID NO: 45, SEQ ID NO: 46), Primer set 26 (SEQ ID NO: 51, SEQ ID NO: 52), Primer set 27 (SEQ ID NO: 53, SEQ ID NO: 54), Primer set 31 (SEQ ID NO: 61, SEQ ID NO: 62), Primer set 32 ​​(SEQ ID NO: 63, SEQ ID NO: 64) (2) MP23 containing all primer sets shown below Primer set 1 (SEQ ID NO: 1, SEQ ID NO: 2), Primer set 10 (SEQ ID NO: 19, SEQ ID NO: 20), Primer set 17 (SEQ ID NO: 33, SEQ ID NO: 34), Primer set 18 (SEQ ID NO: 35, SEQ ID NO: 36), Primer set 20 (SEQ ID NO: 39, SEQ ID NO: 40), Primer set 25 (SEQ ID NO: 49, SEQ ID NO: 50), Primer set 33 (SEQ ID NO: 65, SEQ ID NO: 66) (3) MP24 containing all primer sets shown below Primer set 2 (SEQ ID NO: 3, SEQ ID NO: 4), Primer set 6 (SEQ ID NO: 11, SEQ ID NO: 12), Primer set 8 (SEQ ID NO: 15, SEQ ID NO: 16), Primer set 13 (SEQ ID NO: 25, SEQ ID NO: 26), Primer set 15 (SEQ ID NO: 29, SEQ ID NO: 30), Primer set 16 (SEQ ID NO: 31, SEQ ID NO: 32) (4) MP25 containing all primer sets shown below Primer set 3 (SEQ ID NO: 5, SEQ ID NO: 6), Primer set 4 (SEQ ID NO: 7, SEQ ID NO: 8), Primer set 5 (SEQ ID NO: 9, SEQ ID NO: 10), Primer set 7 (SEQ ID NO: 13, SEQ ID NO: 14), Primer set 11 (SEQ ID NO: 21, SEQ ID NO: 22), Primer set 14 (SEQ ID NO: 27, SEQ ID NO: 28)

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