JP2013220032A - Colon bacillus o-antigenic type inspection method using lamp method and inspection kit - Google Patents

Abstract

PROBLEM TO BE SOLVED: To develop a rapid and high accuracy type inspection method for EHEC O antigen instead of conventional serological O antigen classification.SOLUTION: A primer set with a specific sequence, a colon bacillus inspection method using the primer set, and an inspection kit are disclosed. They make it possible to attach a rapid and high accuracy type inspection method for O antigen classification instead of conventional serological O antigen classification. The detection and discrimination of E coli isolation can be performed by amplifying a colon bacillus O-antigen specific nucleic acid sequence by using the primer set.

Description

  The present invention relates to a test method and test kit for Escherichia coli O antigen type using the LAMP method.

  Infectious diseases caused by enterohemorrhagic Escherichia coli occur approximately 4000 cases annually in Japan. Some patients with infection may die from hemolytic uremic syndrome or encephalopathy, and enterohemorrhagic Escherichia coli sometimes has serious consequences. Therefore, enterohemorrhagic E. coli is one of the food poisoning bacteria that needs the most attention.

  To determine whether enterohemorrhagic Escherichia coli is a mass infection, to grasp the extent of contamination, to trace and identify the source and route of contamination, and to conduct epidemiological studies, the detailed classification of enterohemorrhagic Escherichia coli isolates and A comparison is required. As one of the methods, serological classification method (O antigen type classification) targeting cell antigens is carried out in almost all enterohemorrhagic Escherichia coli isolates.

  O antigen is a cell wall antigen in eubacteria. O antigen has a sugar chain structure, and it has the characteristic that the kind of saccharide | sugar which comprises O antigen, a coupling order, etc. differ with eubacteria. O antigen is used for detection, identification, and classification of eubacteria by utilizing the characteristic that this O antigen has a sugar chain structure that is different for each eubacteria. The same applies to E. coli, including enterohemorrhagic E. coli, and O antigen is used for detection, identification, and classification of E. coli isolates.

  About Escherichia coli Currently, 184 types of O antigen types have been determined by the Danish National Serological Institute (SSI), which is also the World Health Organization Cooperation Center for Reference and Research of Escherichia coli (Non-patent Document 1) ). In Japan, as a result of detailed examination of the O antigen type of about 13,000 strains of enterohemorrhagic Escherichia coli sent to the National Institute of Infectious Diseases from 2007 to 2011 by the National Institutes of Health and public health centers, The majority of the O antigenic strains that have been regarded as major, such as O26, O111, and O103, have occupied the majority. On the other hand, although the number of segregations was small, O antigen type strains with few (or no) reported cases in Japan and overseas have been found so far. When these confirmed O antigen type strains were combined, 86 types of O antigen types were confirmed as enterohemorrhagic Escherichia coli. In some cases, rare O antigenic strains have been isolated from critically ill patients such as bloody stool. In the future, these rare O antigenic strains will require careful attention.

Once enterohemorrhagic Escherichia coli infection occurs, medical laboratories and inspecting bacteria require rapid and accurate subclassification of enterohemorrhagic Escherichia coli to prevent the spread of infection. However, there are as many as 86 types of enterohemorrhagic Escherichia coli of O antigen type that have been confirmed at present, and all the enterohemorrhagic Escherichia coli are classified according to the current serological O antigen type classification. It is difficult to quickly classify isolates. In other words, only 50 types of O antigen immune sera are sold by domestic manufacturers, and only 33 types of the 86 types described above can be covered by domestic manufacturers. In addition, O antigen immune serum sold by SSI is expensive (about 50,000 yen per type), and it is virtually impossible for each laboratory (institution) to have all types.
Under these circumstances, it is desired to develop a rapid and highly accurate test method for enterohemorrhagic Escherichia coli O antigen type in place of the current serological O antigen classification.

International Publication No. 00/28082 Pamphlet

STATENS SERUM INSTUT, E.coli strains, Internet <URL: http://www.ssi.dk/English/SSI%20Diagnostica/Products%20from%20SSI%20Diagnostica/Bacterial%20strains/E%20coli.aspx> DedRoy et al. Animal Health Research Reviews 12 (2); 169-185

  Against the background of the above circumstances, an object of the present invention is to develop a rapid and highly accurate test method for enterohemorrhagic Escherichia coli O antigen type in place of the serological O antigen classification currently performed.

  The inventors focused on two genes involved in O antigen synthesis in E. coli. That is, the O antigen sugar chain synthase gene (wzy) and the O antigen sugar chain transfer protein gene (wzx) present in the gene region responsible for O antigen synthesis on the Escherichia coli chromosome. These wzy and wzx are known to retain high nucleotide sequence specificity among O antigen types of E. coli.

  The inventor has clarified the nucleotide sequence of wzy or wzx for multiple types of E. coli strains whose nucleotide sequence information of the O antigen coding region is not known. The invention relating to the primer set for amplifying wzy or wzx was completed using the information on the nucleotide sequences of the 12 types of wzy or wzx and the known nucleotide sequence information. Furthermore, the inventors have completed an invention relating to a test method and test kit for E. coli O antigen type that specifically detects and discriminates the O antigen type of E. coli using these primer sets.

  The present invention has the following configuration.

  A first configuration of the present invention is a primer set for amplifying a base sequence specific to E. coli O antigen including any one or a plurality of primer sets described later, and each primer set includes primer set 1 SEQ ID NO: 1-4, primer set 2 is SEQ ID NO: 5-8, primer set 3 is SEQ ID NO: 9-12, primer set 4 is SEQ ID NO: 13-16, primer set 5 is SEQ ID NO: 17-20, primer set 6 is SEQ ID NO: 21-24, primer set 7 is SEQ ID NO: 25-28, primer set 8 is SEQ ID NO: 29-32, primer set 9 is SEQ ID NO: 33-36, primer set 10 is SEQ ID NO: 37-40, primer set 11 is SEQ ID NO: 41 to 44, primer set 12 is SEQ ID NO: 45 to 48, primer set 13 Is a primer set comprising the primers shown in SEQ ID NOs: 49-52 and primer set 14 shown in SEQ ID NOs: 53-56.

  In the second configuration of the present invention, the primer set 1 is the O41 antigen, the primer set 2 is the O48 antigen, the primer set 3 is the O69 antigen, the primer set 4 is the O74 antigen, the primer set 5 is the O76 antigen, Primer set 6 is O88 antigen, Primer set 7 is O113 antigen, Primer set 8 is O156 antigen, Primer set 9 is O170 antigen, Primer set 10 is O174 antigen, Primer set 11 is O175 antigen, Primer set The primer set according to the first configuration, wherein 12 is an O179 antigen, the primer set 13 is an O180 antigen, and the primer set 14 is a specific base sequence of an O181 antigen.

  The third configuration of the present invention is to detect and identify E. coli isolates by amplifying a base sequence specific to E. coli O antigen using the primer set described in the first or second configuration. This is a test method for E. coli O antigen.

  A fourth configuration of the present invention is the method for examining E. coli O antigen according to the third configuration, wherein the method for amplifying a base sequence specific to E. coli O antigen is the LAMP method.

  According to a fifth aspect of the present invention, there is provided a test kit comprising at least the primer set according to the first or second structure and a nucleic acid amplification reagent.

  The sixth configuration of the present invention is the test kit according to the fifth configuration, further comprising a fluorescent reagent.

  In the present invention, the sequences of the primers constituting the primer set are as follows.

Primer set 1
F3 (SEQ ID NO: 1): ACTTGTTTGATGGTTTTAGCT
B3 (SEQ ID NO: 2): ATCCCTTTTCTTGATATTTCCAT
FIP (SEQ ID NO: 3): CCATGCCTGCCTAAATAAATGAAAGTGCAATGATTATTCTATGCATAGG
BIP (SEQ ID NO: 4): TTGTGCAATTGGGATAAGCCGACATCAAATGACTCAATCCGG

Primer set 2
F3 (SEQ ID NO: 5): ATATTGCTGGGGGTTGGT
B3 (SEQ ID NO: 6): ATTGCAGTTGTTCCGAGT
FIP (SEQ ID NO: 7): CCAATCATGCCAGTTTCAATTAAGAAATGCAGTCGATGTTATTGGT
BIP (SEQ ID NO: 8): TGTGCATTTACCTCAAAGAGGATGGACATTGATGCTATCAAAAATGGA

Primer set 3
F3 (SEQ ID NO: 9): AATGTATTGTCTTGATGGCTG
B3 (SEQ ID NO: 10): CCTGAACTGTAAAAAGTAGGC
FIP (SEQ ID NO: 11): AGGGAAAAATGGTAGAACCCAATAATATTGTCTAGTGTTTATGGAGATG
BIP (SEQ ID NO: 12): ATTACGCAGAATCTAATCAAGCGATCAAGATATGAAGAGCAGCAA

Primer set 4
F3 (SEQ ID NO: 13): GATTATAGAGTATATGCATCAGGTA
B3 (SEQ ID NO: 14): CCGATTATATATGCAACCAACAA
FIP (SEQ ID NO: 15): CCAGACCATACCCAAATGGATTGAGATGAGCTATATGGTGCG
BIP (SEQ ID NO: 16): ATTCGATAACAGGCATTGACTTTCCGGCCAAGTTCAATCCCTAT

Primer set 5
F3 (SEQ ID NO: 17): CTTTTGTGGGTAGAATGAAGTT
B3 (SEQ ID NO: 18): GCTGACTAATGCAAAACCA
FIP (SEQ ID NO: 19): CGAACCCATCAATATCAGTTTCAGACATTCCTTATCTATTATTGCAGCAC
BIP (SEQ ID NO: 20): TGTCCGTTGGGGGGCTTATAATTATACCAAACTGATCCCAGTA

Primer set 6
F3 (SEQ ID NO: 21): GAGCCAAGTTTAAATGGCTAT
B3 (SEQ ID NO: 22): ACGGAATGAGCTGGTATG
FIP (SEQ ID NO: 23): CAAACGCCAAAATTAGTCTGTACTTTTATGGTTTAATGTTCTTGATGGTC
BIP (SEQ ID NO: 24): ATTTCAGGTGCGAAATTTTCATTCCAAAAGCCTTTCACCTTGAAG

Primer set 7
F3 (SEQ ID NO: 25): GCTGGGATTCTGGCATAT
B3 (SEQ ID NO: 26): GGAATATTATTCCTTAGAGCATTCA
FIP (SEQ ID NO: 27): ACTCCATATGTCAGAAACGCTCATATTCCATATGATACAATGGATTTAGC
BIP (SEQ ID NO: 28): TGCATGAAATGTTTAAATGCAGCGTAAACGTTGCTATAAATGGAAGC

Primer set 8
F3 (SEQ ID NO: 29): GGGGGACTATAAGTGTCATG
B3 (SEQ ID NO: 30): AGGGTCTTTGAAGCAGTAG
FIP (SEQ ID NO: 31): CCGCCTTTGTATATTGGAGTTACTAAACATAAATCAGAGATGTGAAAGTC
BIP (SEQ ID NO: 32): TGCAATAATATTTTGGTTGGCACTCTTGTTAATACTAAGGCTGGGAAT

Primer set 9
F3 (SEQ ID NO: 33): TGTTATAGTATTCGCTTTGTGTT
B3 (SEQ ID NO: 34): TTCACGAAGCATCACTACA
FIP (SEQ ID NO: 35): ACGTCCATTGGGATTTGTTATTGTAATTGGATTTATATATGCAGCAATCG
BIP (SEQ ID NO: 36): TTCTGGATTCATTAGACCGTCAGCAGCATATATAGAATGATAAGGCC

Primer set 10
F3 (SEQ ID NO: 37): ATTGGGATTACTATTAGGCTGG
B3 (SEQ ID NO: 38): CCTATCAGTATCTTTCATAAATGCT
FIP (SEQ ID NO: 39): ACCGCAGATATTAGCAATAGAAAGTGTTTTAAAAGGGAGTCAATTGAGA
BIP (SEQ ID NO: 40): ATTCTGATGGGGTGGAATTTAATCAAAATTAACCATGTAACAACAGTC

Primer set 11
F3 (SEQ ID NO: 41): CTTGTTGCATATTATTATTCTAGCG
B3 (SEQ ID NO: 42): TATAATAAGAAGCAGGCCAATT
FIP (SEQ ID NO: 43): TCGATAATTTGACAGAACTGCTTCCTCTTCTGTTTAGACTTGATAAGGT
BIP (SEQ ID NO: 44): ACTTAGGAAAAGTGACTTCAACGGCAGACTAGTATAAACTGTTGTATCC

Primer set 12
F3 (SEQ ID NO: 45): AAATACTGCAGCGCTTTC
B3 (SEQ ID NO: 46): ACACTCAACAGTGATGTGAT
FIP (SEQ ID NO: 47): ATGCGATAATGCCAATAACCCAAGTAGCATTATTGTGTATGCTACTTTC
BIP (SEQ ID NO: 48): GTCTCAAACTGCAAGTGCTTTTATGTCTTTTTATGATGTCTTCGAATG

Primer set 13
F3 (SEQ ID NO: 49): GCTGAATATAGAATTATGGGACTT
B3 (SEQ ID NO: 50): GTGACATATAATAATGATAAGGCAC
FIP (SEQ ID NO: 51): GGTTAAGAAGCCAATATCACCAACTGTATATGATGATAGCATTCCCCTT
BIP (SEQ ID NO: 52): ATCAGAATAAACCACTTGTCGGACTAATGTTAACATAACTCGTGCA

Primer set 14
F3 (SEQ ID NO: 53): CCTATTGTTATCATTTTATTGCGGT
B3 (SEQ ID NO: 54): TGTAATGTTACTCGATCTGCC
FIP (SEQ ID NO: 55): GTTCGACGAAATATAAACCAGCAATGTTATACAAATTGTTTAACTCTCGG
BIP (SEQ ID NO: 56): GCATTCGCTGTTATAGGTGTGGCCATAATCCACTCGTATAGGTC

The primer set of the present invention is composed of F3, B3, FIP, and BIP primers. About each notation, it shows that it is a primer designed as follows.
[F3]
Outer primer designed to have an F3 region that is complementary to the F3c region.
[B3]
Outer primer designed to have a B3 region that is complementary to the B3c region.
[FIP]
Inner primer designed to have the F2 region complementary to the F2c region at the 3 'end and the same sequence as the F1c region at the 5' end.
[BIP]
Inner primer designed to have the B2 region, which is complementary to the B2c region, at the 3 'end and the same sequence as the B1c region at the 5' end.

According to the present invention, it has become possible to provide a rapid and highly accurate method for classifying O antigens in place of the serological O antigen classification currently performed. That is, by using the primer set in the present invention to amplify a specific nucleic acid sequence of E. coli O antigen, it was possible to detect and discriminate E. coli isolates quickly and with high accuracy.

Diagram showing wzy gene sequence of E. coli O41 antigen and 048 antigen Diagram showing wzy gene sequence of E. coli O69 antigen and 074 antigen Diagram showing wzy gene sequence of E. coli O76 antigen and 088 antigen Diagram showing wzy gene sequence of E. coli O156 antigen and 0170 antigen Diagram showing wzy gene sequence of E. coli O175 antigen and 0179 antigen Diagram showing wzy gene sequence of E. coli O180 antigen and 0181 antigen Diagram showing wzy gene sequence of E. coli O113 antigen and 0174 antigen Figure showing reaction solution composition Explanation of Pool template Diagram showing E. coli isolates contained in each pool in the Pool template The figure which showed the discrimination | determination result by the LAMP method of Examples 1-4 The figure which showed the discrimination | determination result by the LAMP method of Examples 5-8 The figure which showed the discrimination | determination result by the LAMP method of Examples 9-12 The figure which showed the discrimination | determination result by the LAMP method of Examples 13-14

  Hereinafter, the primer set of the present invention and the E. coli test method and test kit using the primer set will be described using the LAMP method as an example.

First, the LAMP method will be briefly explained.
The LAMP method (Loop-Mediated Isothermal Amplification) is a method of annealing the 3 'end of its own base sequence as a template and using it as a starting point for complementary strand synthesis. , Defined as a nucleic acid amplification method that enables an isothermal complementary strand synthesis reaction (WO 00/28082 pamphlet). Oligonucleotide primers used for nucleic acid amplification by the LAMP method are a total of 6 regions of the nucleotide sequence of the template nucleic acid, that is, the region of F3c, F2c, F1c from the 3 ′ end and the region of B3, B2, B1 from the 5 ′ end These are at least four types of primers that recognize the base sequences of the primers, and are called inner primers F and B and outer primers F and B, respectively. The complementary base sequences of F1c, F2c, and F3c are called F1, F2, and F3, and the complementary base sequences of B1, B2, and B3 are called B1c, B2c, and B3c, respectively.

Subsequently, a series of flow of the inspection method by the LAMP method will be described.
Specimen samples are required for E. coli testing by the LAMP method. As the specimen sample, any specimen specimen that is usually used can be used, and examples thereof include foods suspected of having E. coli infection and biological samples derived from humans and animals.
For the specimen sample for E. coli test, the pretreatment that is usually used may be performed and used as the sample for use in the LAMP method. For example, after degrading a tissue cell-derived protein with a proteolytic enzyme or the like, nucleic acid extraction or purification is performed using phenol or chloroform, or nucleic acid obtained using a commercially available extraction kit is extracted. .

  When the specimen sample is ready, an operation for nucleic acid amplification is performed using the primer set of the present invention. As an operation for this nucleic acid amplification, for example, in a reaction solution, at least one primer set, a template-dependent nucleic acid synthase, a substrate such as deoxynucleotide triphosphate, and the like are incubated at 60 to 65 ° C. for 15 minutes to 1 hour. do it.

  In the primer set of the present invention, primer set 1 (SEQ ID NO: 1 to 4) is mainly O41 antigen, primer set 2 (SEQ ID NO: 5 to 8) is mainly O48 antigen, and primer set 3 (SEQ ID NO: 9 to 12) is mainly O69. Antigen, primer set 4 (SEQ ID NO: 13 to 16) is mainly O74 antigen, primer set 5 (SEQ ID NO: 17 to 20) is mainly O76 antigen, primer set 6 (SEQ ID NO: 21 to 24) is mainly O88 antigen, primer set 7 (SEQ ID NO: 25 to 28) is mainly O113 antigen, primer set 8 (SEQ ID NO: 29 to 32) is mainly O156 antigen, primer set 9 (SEQ ID NO: 33 to 36) is mainly O170 antigen, primer set 10 (from SEQ ID NO: 37) 40) is mainly O174 antigen, primer set 11 (SEQ ID NOs: 41 to 44) Is mainly O175 antigen, primer set 12 (SEQ ID NO: 45 to 48) is mainly O179 antigen, primer set 13 (SEQ ID NO: 49 to 52) is mainly O180 antigen, and primer set 14 (SEQ ID NO: 53 to 56) is mainly O181. It is a primer set intended to amplify a specific base sequence of an antigen.

The primer set of the present invention can efficiently amplify the nucleic acid region of an E. coli isolate to be detected. Thereby, the E. coli isolate as a detection target can be detected at a high rate and quickly. In addition, it is possible to distinguish E. coli isolates by using a combination of primer sets.
In the present invention, these primer sets are not intended to be limited to the detection of only the main detection target type of E. coli isolates, and may be used to detect other types of E. coli isolates. For example, primer set 1 is used to detect E. coli isolates other than O41 antigen. When detecting and discriminating E. coli isolates, the target E. coli O antigen type is usually unknown. In such a case, it is possible to more accurately detect and discriminate E. coli isolates by using various primer sets in combination.

  As the template-dependent nucleic acid synthase, any conventionally used template-dependent nucleic acid synthase can be used. Examples of such an enzyme include Bst DNA polymerase, Bca (exo-) DNA polymerase, Klenow fragment of E. coli DNA polymerase I, and preferably Bst DNA polymerase.

When the nucleic acid amplification reaction operation is completed, the nucleic acid amplification product is detected. Any commonly used detection method can be used to detect the nucleic acid amplification product. For example, detection using a labeled oligonucleotide that specifically recognizes the amplified base sequence, detection by a fluorescent intercalator method, agarose gel electrophoresis, and the like. In the agarose gel electrophoresis method, a large number of bands with different base lengths are detected in a ladder form in the LAMP amplification product.
In addition, the LAMP method may be capable of visual detection and is preferred when rapid detection of E. coli isolates is required. That is, in the LAMP method, a large amount of substrate is consumed by nucleic acid synthesis, and pyrophosphate, a by-product, reacts with coexisting magnesium to become magnesium pyrophosphate, and the reaction solution becomes cloudy to the extent that it can be visually confirmed. Isolate detection is possible. In this case, it is also possible to confirm the detection of E. coli isolates using a measuring instrument such as a spectrophotometer that can optically observe the turbidity increase during the reaction and the turbidity after the completion of the reaction.

Various reagents necessary for detection of nucleic acid amplification using the primer set of the present invention can be packaged in advance and used as a test kit. In this case, the test kit includes at least one primer set of the present invention and a nucleic acid amplification reagent. Examples of nucleic acid amplification reagents include four types of dNTPs that serve as substrates for nucleic acid synthesis, template-dependent nucleic acid synthases with strand displacement activity, enzymes with reverse transcription activity, buffers and salts that give conditions suitable for enzymatic reactions , Protecting agents that stabilize enzymes and templates.
The test kit in the present invention can further contain a fluorescence detection reagent. Thus, the presence or absence of nucleic acid amplification can be confirmed by visual observation with UV irradiation or the like, so that the convenience can be improved. An example of this fluorescence detection reagent is calcein.

  Hereinafter, the present invention will be described in detail on the basis of examples. However, as a matter of course, the present invention is not limited to the following contents.

<Experiment method>
1. Targeted E. coli O antigen types In the examples, 14 types of E. coli O antigen types (O41, O48, O69, O74, O76, O88, O113, O156, O170, O174, O175, O179, O180, O181) were targeted. did.

2. Acquisition of base sequence information
(1) For 12 E. coli O antigen types (O41, O48, O69, O74, O76, O88, O156, O170, O175, O179, O180, O181) Since it was not known, the nucleotide sequence was determined using an E. coli O antigen type standard strain. DNA is purified from each E. coli O antigen type standard strain, amplified using PCR with primers designed on both sides of the O antigen coding region using them as template DNA (amplified product is 15-25 kb), and the amplified product is shot It was determined by the cancer sequence method. From the obtained sequence information, annotation was made based on the NCBI integrated database, and a gene expected to be wzy or wzx was identified (FIGS. 1 to 6, SEQ ID NOs: 57 to 68).
(2) For two types of E. coli O antigen types (O113, O174), the nucleotide sequences already known (Paton and Paton. Infec and Immun 67 (11); 5930-5937, Beutin et al. J Clin Microbiol43 (10); 169-185) was obtained from GenBank (FIG. 7, SEQ ID NOs: 69, 70).

3. Design of primers Four primers (FIP, BIP, F3, B3) were designed based on the obtained wzy or wzx nucleotide sequences.

4). Nucleic acid amplification operation and detection operation by LAMP method
(1) A 96-well PCR reaction plate was used for the reaction.
(2) The reaction solution was mixed with the composition shown in Fig. 8, and reacted at 63 ° C for 59 minutes and at 80 ° C for 2 minutes using a thermal cycler (ABI3100).
(3) The PCR reaction lid was opened and turned upside down, placed on a UV transilluminator, irradiated with 312 nm UV, and the presence or absence of color was confirmed with a gel imaging device.
(4) Since the reaction solution contains a reagent (Calcein) that fluoresces when DNA is amplified, only the E. coli O antigen type can be detected by UV irradiation after the amplification reaction. At this time, since the migration confirmation process can be omitted, more rapid and simple detection is possible.

5. Pool
Detection and discrimination of Escherichia coli O antigen type by template and single template
(1) The LAMP method with each primer set was performed using 184 standard strains covering all E. coli O antigen types.
(2) As the first step, a comprehensive detection operation was performed using Pool template, and as the second step, a detection operation was performed to determine O antigen using Single template.
(3) Use any number of pools (37 from 1 to 37 in this example) for the Pool template, and any number of types (5 in this example) for each pool. , E. coli O antigen type standard strain DNA is included (FIGS. 9 and 10. Pool Template No. in FIG. 10 indicates that the E. coli O antigen type is included in the pool of that number.) . Thereby, when fluorescence is confirmed by Pool template, it means that nucleic acid amplification was performed with respect to one or more of the five types of E. coli O antigen standard strains in the pool where fluorescence was present.
(4) Each single template contains one E. coli O antigen type standard strain DNA in each hole. As a result, when fluorescence was confirmed by the single template, it means that the nucleic acid amplification of the E. coli O antigen standard strain in the hole where the fluorescence was present was performed.

<Experimental result>
The results are shown in FIGS.

1. In Example 1, primer set 1 (SEQ ID NOs: 1 to 4) was used. Fluorescence was confirmed at No. 9 of the Pool template, and it was confirmed that nucleic acid amplification was performed on any one or a plurality of E. coli O antigen standard strains O41, O42, O43, O44, and O45. Next, when a single template was used for confirmation, fluorescence at O41 was confirmed (FIG. 11). Therefore, it was confirmed that the primer set 1 of Example 1 specifically detected O41.
2. In Example 2, primer set 2 (SEQ ID NOs: 5 to 8) was used. Fluorescence was confirmed with Pool template No. 10, and when confirmed with Single template, fluorescence at O48 was confirmed (FIG. 11). Therefore, it was confirmed that the primer set 2 of Example 2 was specifically detected by O48.
3. In Example 3, primer set 3 (SEQ ID NOs: 9 to 12) was used. Fluorescence was confirmed at No. 14 of the Pool template and confirmed by Single template, and fluorescence at O69 was confirmed (FIG. 11). Therefore, it was confirmed that the primer set 3 of Example 3 was specifically detected by O69.
4). In Example 4, primer set 4 (SEQ ID NOs: 13 to 16) was used. Fluorescence was confirmed at No. 15 of the Pool template and confirmed by Single template, and fluorescence at O74 was confirmed (FIG. 11). Therefore, it was confirmed that the primer set 4 of Example 4 was specifically detected by O74.

5. In Example 5, primer set 5 (SEQ ID NOs: 17 to 20) was used. Fluorescence was confirmed at No. 15 of the Pool template and confirmed by Single template, and fluorescence at O76 was confirmed (FIG. 12). Therefore, it was confirmed that the primer set 5 of Example 5 was specifically detected by O76.
6). In Example 6, primer set 6 (SEQ ID NOs: 21 to 24) was used. Fluorescence was confirmed at No. 18 of the Pool template and confirmed by Single template, and fluorescence at O88 was confirmed (FIG. 12). Therefore, it was confirmed that the primer set 6 of Example 6 was specifically detected by O88.
7. In Example 7, primer set 7 (SEQ ID NOs: 25 to 28) was used. Fluorescence was confirmed at Pool template No. 22 and confirmed by Single template, and fluorescence at O113 was confirmed (FIG. 12). Therefore, it was confirmed that the primer set 7 of Example 7 specifically detected O113.
8). In Example 8, primer set 8 (SEQ ID NOs: 29 to 32) was used. Fluorescence was confirmed at Pool template No. 31 and confirmed at Single template, and fluorescence at O156 was confirmed (FIG. 12). Therefore, it was confirmed that the primer set 8 of Example 8 specifically detected O156.

9. In Example 9, primer set 9 (SEQ ID NOs: 33 to 36) was used. Fluorescence was confirmed at No. 34 of the Pool template, and when confirmed by Single template, fluorescence at O170 was confirmed (FIG. 13). Therefore, it was confirmed that the primer set 9 of Example 9 specifically detected O170.
10. In Example 10, primer set 10 (SEQ ID NOs: 37 to 40) was used. Fluorescence was confirmed at Pool template No. 35 and confirmed by Single template, and fluorescence at O174 was confirmed (FIG. 13). Therefore, it was confirmed that the primer set 10 of Example 10 specifically detected O174.
11. In Example 11, primer set 11 (SEQ ID NOs: 41 to 44) was used. Fluorescence was confirmed at Pool template No. 35 and confirmed by Single template, and fluorescence at O175 was confirmed (FIG. 13). Therefore, it was confirmed that the primer set 11 of Example 11 specifically detected O175.
12 In Example 12, primer set 12 (SEQ ID NOs: 45 to 48) was used. Fluorescence was confirmed at Pool template No. 36, and when confirmed by Single template, fluorescence at O179 was confirmed (FIG. 13). Therefore, it was confirmed that the primer set 12 of Example 12 specifically detected O179.

13. In Example 13, primer set 13 (SEQ ID NOs: 49 to 52) was used. Fluorescence was confirmed at Pool template No. 36 and confirmed by Single template, and fluorescence at O181 was confirmed (FIG. 14). Therefore, it was confirmed that the primer set 13 of Example 13 specifically detected O181.
14 In Example 14, primer set 14 (SEQ ID NOs: 53 to 56) was used. Fluorescence was confirmed at Pool template # 36 and confirmed at Single template, and fluorescence at O180 was confirmed (FIG. 14). Therefore, it was confirmed that the primer set 14 of Example 14 was specifically detected by O180.

  It was confirmed that the O antigen type of Escherichia coli can be genetically rapidly and accurately discriminated by each primer set of this example. In particular, 12 types of O antigen types except O170 and O175 have been confirmed as O antigen types of enterohemorrhagic Escherichia coli so far. Therefore, it can be expected that the results of the present invention will be effective in the future in the field of medical care, public health and food hygiene to prevent pathogenic E. coli infections including intestinal hemorrhagic E. coli and prevent the spread of infection.

The present invention can be used in a technique for classifying and identifying infectious disease-causing Escherichia coli in the medical field, the public health field, and the food hygiene field.

Claims (6)

A primer set for amplifying a base sequence specific to Escherichia coli O antigen containing one or more of the following primer sets, each primer set,
Primer set 1 is SEQ ID NO: 1-4
Primer set 2 is SEQ ID NO: 5-8,
Primer set 3 is SEQ ID NO: 9-12,
Primer set 4 is SEQ ID NO: 13-16,
Primer set 5 is SEQ ID NO: 17-20,
Primer set 6 is SEQ ID NO: 21-24,
Primer set 7 is SEQ ID NO: 25-28,
Primer set 8 is SEQ ID NO: 29-32,
Primer set 9 is SEQ ID NO: 33-36,
Primer set 10 is SEQ ID NO: 37-40,
Primer set 11 is SEQ ID NO: 41-44,
Primer set 12 is SEQ ID NO: 45-48,
Primer set 13 is SEQ ID NO: 49-52,
Primer set 14 is SEQ ID NO: 53-56,
A primer set comprising:
The primer set 1 is O41 antigen,
The primer set 2 is O48 antigen,
The primer set 3 is O69 antigen,
The primer set 4 is O74 antigen,
The primer set 5 is an O76 antigen,
The primer set 6 is an O88 antigen,
The primer set 7 is an O113 antigen,
The primer set 8 is O156 antigen,
The primer set 9 is O170 antigen,
The primer set 10 is an O174 antigen,
The primer set 11 is O175 antigen,
The primer set 12 is an O179 antigen,
The primer set 13 is O180 antigen,
The primer set 14 is O181 antigen,
The primer set according to claim 1, wherein the specific base sequence is amplified.
A method for testing E. coli O antigen, comprising amplifying a base sequence specific to E. coli O antigen using the primer set according to claim 1 to detect and identify an E. coli isolate.
4. The method for examining E. coli O antigen according to claim 3, wherein the method for amplifying a base sequence specific to E. coli O antigen is the LAMP method.
A test kit comprising at least the primer set according to claim 1 and a reagent for nucleic acid amplification.
The test kit according to claim 5, further comprising a fluorescent reagent.