WO2018016451A1 - Procédé à large spectre permettant de déterminer le type d'antigène flagellaire d'e. coli au moyen d'un procédé de pcr - Google Patents

Procédé à large spectre permettant de déterminer le type d'antigène flagellaire d'e. coli au moyen d'un procédé de pcr Download PDF

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WO2018016451A1
WO2018016451A1 PCT/JP2017/025817 JP2017025817W WO2018016451A1 WO 2018016451 A1 WO2018016451 A1 WO 2018016451A1 JP 2017025817 W JP2017025817 W JP 2017025817W WO 2018016451 A1 WO2018016451 A1 WO 2018016451A1
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coli
type
flic
seq
nucleic acid
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純 井口
将也 番上
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国立大学法人 宮崎大学
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids

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  • the present invention relates to a method for determining a broad flagellar antigen type of Escherichia coli using a PCR method. More specifically, the present invention relates to a primer that enables genetic classification according to the E. coli flagellar antigen type (H type) by PCR and a method for determining the broad flagellar antigen type of Escherichia coli using such a primer.
  • H type E. coli flagellar antigen type
  • O type classification is a technique for classifying sugar chain structures existing on the surface of Escherichia coli cells as antigens.
  • type H is a method of classifying flagella, which is a motor organ of E. coli, as an antigen.
  • O157 which is widely known in Japan as enterohemorrhagic Escherichia coli
  • E. coli having the O157 antigen It is known that multiple types of H-type are included, such as H39 and H45 in E. coli and H16 in non-pathogenic E. coli.
  • O103 which always ranks higher in the number of reports of enterohemorrhagic Escherichia coli in Japan, includes multiple types of H types such as H2, H11, and H25. From these facts, it is required that accurate type determination is performed not only for the O serum group but also for the H type.
  • Non-patent Document 3 the complete sequence information of the gene encoding the antigen has already been published (Non-patent Document 3).
  • the H-type determination method currently used is a determination method that uses an antigen as an identification target, and has the following problems, and is currently not functioning as a practical determination method. (1) In order to express flagella suitable for an antigen, it takes 2 to 3 days of pre-culture. (2) Judgment may be unclear due to low antigenicity (low aggregation). (3) It cannot be typed for strains that do not express flagella. (4) Type I immune serum sold by SSI is relatively expensive (about 50,000 yen per type).
  • an object of the present invention is to develop a practical determination method capable of quickly and easily determining an H type.
  • flagellin that constitutes flagella causes a difference in antigenicity due to the H type.
  • this flagellin is encoded by genes such as flkA and flmA, mainly fliC, and that these genes have a diversity region that determines H-type.
  • the present inventors have completed the invention relating to primers and the like, with the idea that H-type can be identified by performing nucleic acid amplification targeting the sex region.
  • the inventors found a sequence region useful for nucleic acid amplification in such a diversity region, and confirmed the usefulness of a primer set capable of amplifying this sequence region, thereby completing the invention related to the primer and the like. It has been made.
  • the inventors have also discovered a new problem in confirming the usefulness of the primer set. That is, the problem is that some types of H antigens are detected as different types of H antigens due to the nature of the gene.
  • the inventors have completed the invention of the H type determination method capable of specifying each H antigen by combining the primer sets and performing complementary detection in such a problem.
  • a first configuration of the present invention is a determination method according to H type of E. coli, wherein the determination according to H type is performed by performing nucleic acid amplification of a diversity region of a gene encoding flagellin constituting flagella of E. coli. It is.
  • an E. coli H type identification method according to the first configuration, wherein the gene is selected from one or more of fliC, flkA, fllA, and flmA.
  • the diversity region is selected from a sequence region obtained by subtracting 500 bases from 3 ′ and 300 bases from 3 ′ in each of the sequences represented by SEQ ID NOs: 1 to 53. It is the determination method according to E. coli H type according to the first feature characterized.
  • a fourth configuration of the present invention is the method according to E. coli H type according to the third configuration, wherein the diversity region includes a sequence region represented by SEQ ID NOs: 54 to 106.
  • a fifth configuration of the present invention is the determination method according to Escherichia coli H type according to the first to fourth configurations, wherein the nucleic acid amplification of the diversity region is performed by a PCR method.
  • a sixth configuration of the present invention is the determination method according to E. coli H type according to the fifth configuration, wherein the nucleic acid amplification is performed using any one or a plurality of primer sets of primer sets 1 to 51. is there.
  • a primer that is used in the determination method according to E. coli H type described in the first to sixth aspects.
  • An eighth configuration of the present invention is a primer set that is used in the determination method according to E. coli H type according to the first to sixth configurations.
  • a ninth configuration of the present invention is a kit that includes the primer set described in the eighth configuration and is used in the determination method according to E. coli H type.
  • a primer set is defined as a minimum combination of primers necessary for nucleic acid amplification.
  • primer sets include forward primers and reverse primers when the nucleic acid amplification method is the PCR method, and FIP, F3 primer, BIP, and B3 primer when the LAMP method is used.
  • each of primer sets 1 to 51 is a primer used in the PCR method represented by SEQ ID NO: 107 to SEQ ID NO: 208, and one set each includes a forward primer and a reverse primer, as shown in FIGS. It is defined as including.
  • the present invention it is possible to provide a practical determination method capable of quickly and easily determining the H type. That is, according to the present invention, it has become possible to genetically quickly and accurately determine the H type of Escherichia coli, so that in the field of medical, public health and food hygiene, pathogenic E. coli infections including enterohemorrhagic Escherichia coli We can expect prevention and spread of infection more appropriately and quickly.
  • Diagram describing sequence information of H antigen, primer set, primer, etc. in the present invention (H1 to H19) Diagram describing sequence information of H antigen, primer set, primer, etc. in the present invention (H20 to H38) Diagram describing sequence information of H antigen, primer set, primer, etc.
  • the Escherichia coli H type discrimination determination method of the present invention is characterized in that determination by H type is performed by performing nucleic acid amplification of a diversity region of a gene encoding flagellin constituting flagella of E. coli.
  • fleliC, flkA, fllA, and flmA are exemplified as preferable target genes as genes encoding flagellin, and efficient primer design and nucleic acid amplification are enabled for diversity regions in these genes.
  • preferable target genes genes encoding flagellin
  • efficient primer design and nucleic acid amplification are enabled for diversity regions in these genes.
  • a specific sequence of the diversity region in such a gene for example, among the sequence regions shown in SEQ ID NOs: 1 to 53, 500 bases from the upstream 5 ′ end, 3 ′ downstream A sequence region obtained by subtracting 300 bases from the terminal side may be used.
  • SEQ ID NO: 1 to SEQ ID NO: 53 has SEQ ID NO: 1 as H1 (fliC, GenBank accession No.
  • SEQ ID NO: 2 as H2 (fliC, GenBank accession No. AIHA01000023), and SEQ ID NO: 3 as H3 (flkA, GenBank accession No. AB128916)
  • SEQ ID NO: 4 is H4 (fliC, GenBank accession No. AJ536600)
  • SEQ ID NO: 5 is H5 (fliC, GenBank accession No.
  • SEQ ID NO: 6 is H6 (fliC, GenBank accession No.AY249991)
  • sequence number 7 is H7 (fliC, GenBank accession No.AY337468)
  • sequence number 8 is H8 (fliC, GenBank accession No.AJ865465)
  • sequence number 9 is H9 (fliC, GenBank accession No.AY249994)
  • SEQ ID NO: 10 is H10 (fliC, GenBank accession No. AY249995)
  • SEQ ID NO: 11 is H11 (fliC, GenBank accession No. AY337465)
  • SEQ ID NO: 12 is H12 (fliC, GenBank accession No.
  • SEQ ID NO: 13 is H14 (fliC, GenBank accession No. AY249998)
  • SEQ ID NO: 14 is H15 (fliC, GenBank accession No. AY249999)
  • SEQ ID NO: 15 is H16 (FliC, GenBank accession No. AY337475)
  • SEQ ID NO: 16 is H17 (fliC, GenBank accession No. AJ515904)
  • SEQ ID NO: 17 is H18 (fliC, GenBank accession No. AY250001)
  • SEQ ID NO: 18 is H19 (fliC, GenBank accession No. AY250002)
  • SEQ ID NO: 19 is H20 (fliC, GenBank accession No.
  • SEQ ID NO: 20 is H21 (fliC, GenBank accession No. AIHL01000060)
  • SEQ ID NO: 21 is H23 (fliC, GenBank accession No. AB028476)
  • SEQ ID NO: 22 is H24 (fliC, GenBank accession No. AY250006)
  • SEQ ID NO: 23 is H25 (fliC, GenBank accession No. AGSG01000116)
  • SEQ ID NO: 24 is H26 (fliC, GenBank accession No. AY250008)
  • SEQ ID NO: 25 is H27 (FliC, GenBank accession No. AM231154)
  • SEQ ID NO: 26 is H28 (fliC, GenBank accession No.
  • SEQ ID NO: 27 is H29 (fliC, GenBank accession No. AY250012)
  • SEQ ID NO: 28 is H30 (fliC, GenBank accession No. AY250011)
  • SEQ ID NO: 29 is H31 (fliC, GenBank accession No.
  • SEQ ID NO: 30 is H32 (fliC, GenBank accession No.AY250014)
  • SEQ ID NO: 31 is H33 (fliC, GenBank accession No.AY250015)
  • SEQ ID NO: 32 is H34 (fliC, GenBank accession No.AY250016)
  • SEQ ID NO: 33 is H35 (flkA, GenBank accession No.EF392692)
  • SEQ ID NO: 34 is H36 (flkA, GenBank accession No. EF392693)
  • SEQ ID NO: 35 is H37 (fliC, GenBank accession No. AY250017)
  • SEQ ID NO: 36 is H38 (fliC, GenBank accession No.
  • SEQ ID NO: 37 is H39 (fliC, GenBank accession No. AY250019)
  • SEQ ID NO: 38 is H40 (fliC, GenBank accession No. AJ884568)
  • SEQ ID NO: 39 is H41 (fliC, GenBank accession No.
  • SEQ ID NO: 40 is H42 (fliC, GenBank accession No.AY250021), SEQ ID NO: 41 is H43 (fliC, GenBank accession No.AIGA01000038), SEQ ID NO: 42 is H44 (fllA, GenBank accession No.AB269770), SEQ ID NO: 43 is H45 (fliC, GenBank accession No.AY250023) , SEQ ID NO: 44 is H46 (fliC, GenBank accession No. AY250024), SEQ ID NO: 45 is H47 (flkA, GenBank accession No. EF39269 4), SEQ ID NO: 46 is H48 (fliC, GenBank accession No.
  • SEQ ID NO: 47 is H49 (fliC, GenBank accession No. AY250026)
  • SEQ ID NO: 48 is H51 (fliC, GenBank accession No. AY250027)
  • SEQ ID NO: 49 is H52 (fliC, GenBank accession No. AY250028)
  • SEQ ID NO: 50 is H53 (flkA, GenBank accession No. AB128917)
  • SEQ ID NO: 51 is H54 (flmA, GenBank accession No. AB128918)
  • SEQ ID NO: 52 is H55 (fllA , GenBank accession No. AB269771)
  • SEQ ID NO: 53 is based on each H antigen shown in H56 (fliC, GenBank accession No. AY250029).
  • sequence region shown in SEQ ID NOs: 1 to 53 the sequence region obtained by subtracting 500 bases from the upstream 5 ′ end and 300 bases from the 3 ′ end downstream” is further sequenced.
  • Nucleic acid amplification is preferably performed on a sequence including the sequence region represented by SEQ ID NO: 54 to SEQ ID NO: 106.
  • the sequence region represented by SEQ ID NO: 54 to SEQ ID NO: 106 was found by the inventors as a primer design region and a preferred sequence region in the amplification target. An effect is that amplification is possible.
  • region which is the object of these nucleic acid amplification naturally not only what was shown above but the nucleic acid sequence complementary to the sequence illustrated is included.
  • the method for performing nucleic acid amplification in the diversity region is not particularly limited as long as nucleic acid amplification is possible, and various nucleic acid amplification methods can be used.
  • nucleic acid amplification methods include the PCR method and the LAMP method.
  • reverse primers, forward primers, and primers for the diversity region can be designed and used as a primer set for nucleic acid amplification.
  • FIP, F3 primer, BIP, B3 primer for the diversity region these primers may be designed and used as a primer set for nucleic acid amplification.
  • a primer such as a loop primer may be used as a primer set.
  • the PCR method will be described as an example that is preferably used in the present invention.
  • specimen samples are required for E. coli testing by PCR.
  • 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.
  • the specimen sample for the E. coli test may be pretreated as usual and used as a sample for PCR. 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. .
  • an operation for nucleic acid amplification is performed using the primer set of the present invention.
  • an operation in this nucleic acid amplification for example, at least one primer set, a heat-resistant DNA polymerase, a substrate such as deoxynucleotide triphosphate is added to the reaction solution, heat denaturation, annealing, extension reaction is performed, and these cycles are performed. Nucleic acid amplification is performed by repeating the above.
  • the primer set of the present invention in the PCR method comprises a combination of a total of two primers consisting of a combination of forward primer (F) and reverse primer (R). What is necessary is just to use the nucleic acid sequence shown by the number 208 as a primer.
  • Each primer set allows nucleic acid amplification in the E. coli H antigen indicated by “+” in FIGS. 1 to 3 to 5 to 7.
  • these primer sets can be used alone, but it is preferable to perform complex H-type detection by combining a plurality of primer sets. That is, for some H antigens, multiple H antigens may be detected due to the nature of the gene. In such a case, the detection by combining a plurality of primer sets has the effect of making it possible to make a more accurate determination by E. coli H type.
  • the MP shown in FIG. 8 can be used as an example. That is, the MP shown in FIG. 8 covers all H antigens, and by combining these detections, it is possible to specify the H antigen of the target sample.
  • MP1 and MP2 are extremely useful because they combine 11 types of H-type related to enterohemorrhagic Escherichia coli that cause severe cases.
  • primer set of the present invention is not intended to be limited to the determination only for the target Escherichia coli H type, and may be used for the determination for other E. coli H types.
  • primer set 1 is used to detect E. coli H type other than H1 antigen.
  • thermostable DNA polymerase any commonly used thermostable DNA polymerase can be used.
  • examples of such an enzyme include pol I type DNA polymerase, ⁇ -type DNA polymerase, mixed DNA polymerase, and Hot ⁇ start DNA polymerase.
  • 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.
  • nucleic acid amplification products may be detected while performing a nucleic acid amplification reaction operation (RT-PCR).
  • RT-PCR nucleic acid amplification reaction operation
  • a detection method using a fluorescent dye such as an intercalator method or a hybridization method may be used.
  • the various reagents necessary for detecting nucleic acid amplification using the primer set of the present invention can be packaged in advance to form a test kit.
  • the test kit includes at least one primer set of the present invention and a nucleic acid amplification reagent.
  • nucleic acid amplification reagents include thermostable DNA polymerases, four types of dNTPs that serve as substrates for nucleic acid synthesis, buffers and salts that give suitable conditions for enzyme reactions, and protective agents that stabilize enzymes and templates. .
  • E. coli all H-type reference strains distributed from SSI were used. 2.
  • the strain DNA was purified using the Wizard Genomic DNA Purification Kit (Promega). Using this purified DNA, a nucleic acid amplification reaction was performed.
  • PCR reaction conditions and detection (1) According to Table 1, the reaction solution was mixed and reacted with a thermal cycler [94 ° C.-30 seconds, 58 ° C.-30 seconds, 72 ° C.-1 minute] ⁇ 25 cycles. (2) A portion of the reaction solution was electrophoresed on a 1.5% agarose gel, and after amplification with ethidium bromide, an amplification product (band) of the expected size was confirmed on a UV transilluminator.
  • FIG. 4 shows the result of electrophoresis.
  • a is the result of electrophoresis for the samples subjected to the amplification reaction for each H antigen from a to H19, b from H20 to H38, and c from H39 to H56.
  • a band appeared for H7 antigen which revealed that nucleic acid was being amplified.
  • other H antigens showed no bands and no nucleic acid amplification.
  • the amplification reaction of primer set 7 is “+” (with nucleic acid amplification) for H7, and “ ⁇ ” (nucleic acid amplification) for each other H antigen. None).
  • the results of detection of a plurality of non-target antigens in the target primer set are considered as follows.
  • the flagellar protein (flagellin) is encoded by the fliC gene, but H3 (flkA), H35 (flkA), H36 (flkA), H44 (fllA), H47 ( In flkA), H53 (flkA), H54 (flmA), and H55 (fllA), flagellin is encoded by a gene other than fliC (non-fliC).
  • Non-fliC-bearing strains have fliC on the chromosome, but the transcription of fliC is suppressed by the protein expressed from non-fliC. Therefore, the protein expressed by non-fliC, not the endogenous fliC (potential fliC), is a flagellin constituent protein (ie, H antigen).
  • the potential fliC of the H type reference strain used in this experiment is H3 (fliC_H16), H35 (fliC_H11), H36 (fliC_H11), H44 (fliC_H4 / H17), H47 (fliC_H21), H53 (fliC_H40) H54 (fliC_H21) and H55 (fliC_H38). Therefore, in the non-fliC type H reference strain, in addition to non-fliC, nucleic acid amplification of potential fliC was also confirmed, and as a result, multiple antigens are considered to have been detected.
  • the H-type can be specifically determined by prioritizing the non-fliC results. (5) In addition, it is possible to make a more accurate judgment by performing simultaneous detection with other primer sets.
  • Wild strains were obtained from joint research institutions. 2. The wild-type strains obtained were purified using the Wizard Genomic DNA Purification Kit (Promega). Using this purified DNA, a nucleic acid amplification reaction was performed in the same manner as in Experimental Example 2.
  • Table 2 shows the results of detection of wild-type E. coli strains having the target H antigen using each primer set.
  • Nucleic acid amplification was possible in all E. coli wild-type strains examined.
  • the Escherichia coli strains underlined in the table are Escherichia coli strains (EHEC) exhibiting enterohemorrhagic properties, and are the main EHEC. These EHECs could also be detected. 4). From these results, it was confirmed that the primer set of the present invention is useful for detection of E. coli wild-type strains.
  • each MP 10 types of MP shown in FIG. 8 were used.
  • each of these 10 types of MP includes a plurality of types of primer sets, and when all 10 types are combined, the combination covers all H antigens.
  • each MP is designed so that it can be clearly determined in consideration of its importance as H antigen and the number of bases of the amplified sequence.
  • FIG. 9 shows the electrophoresis results when standard antigen is detected using MP1.
  • MP1 was able to detect all H antigens corresponding to MP1.
  • the detected H antigens can be clearly distinguished by electrophoresis because the number of bases of the amplified sequences is different. 4).
  • MP1 and MP2 are combined with 11 types of H-type related to enterohemorrhagic Escherichia coli that cause severe cases, and are considered to be extremely useful.

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Abstract

Le problème décrit par la présente invention est de fournir un procédé de détermination pratique permettant d'effectuer rapidement et facilement la détermination du type H. La solution de l'invention porte sur un procédé de détermination du typage H d'Escherichia coli caractérisé en ce qu'il consiste à déterminer un typage H en effectuant une amplification d'acide nucléique d'une région polymorphe d'un gène qui code pour la flagelline constituant les flagelles d'E. coli. Plus précisément, grâce à la présente invention, le type H d'E. coli peut être déterminé de manière rapide et précise, et il est donc possible de prévenir de manière plus appropriée et plus rapide une infection par E. coli entéropathogène ou la propagation infectieuse d'E. coli entéropathogène, y compris E. coli entérohémorragique dans des environnements médicaux ou de santé publique/d'hygiène alimentaire.
PCT/JP2017/025817 2016-07-22 2017-07-14 Procédé à large spectre permettant de déterminer le type d'antigène flagellaire d'e. coli au moyen d'un procédé de pcr WO2018016451A1 (fr)

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CZ308046B6 (cs) * 2018-06-29 2019-11-20 FakultnĂ­ nemocnice Brno Způsob typizace bakteriálních kmenů Escherichia coli a oligonukleotidy pro použití při tomto způsobu

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1999061458A1 (fr) * 1998-05-21 1999-12-02 The University Of Sydney Antigenes et detection de ces antigenes
JP2013220032A (ja) * 2012-04-12 2013-10-28 Univ Of Miyazaki Ｌａｍｐ法を用いた大腸菌ｏ抗原型の検査方法および検査キット

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1999061458A1 (fr) * 1998-05-21 1999-12-02 The University Of Sydney Antigenes et detection de ces antigenes
JP2013220032A (ja) * 2012-04-12 2013-10-28 Univ Of Miyazaki Ｌａｍｐ法を用いた大腸菌ｏ抗原型の検査方法および検査キット

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
IGUCHI ET AL: "A complete view of the genetic diversity of the Escherichia coli O-antigen biosynthesis gene cluster", DNA RES., vol. 22, no. l, 2015, pages 101 - 107, XP055452935 *
MASAYA BANJO ET AL.: "Chokan Shukketsusei Daichokin ni Kanren suru Benmo Kogengata (H- gata) o Hantei suru PCR-ho no Kaihatsu", DAI 37 KAI JAPANESE SOCIETY OF FOOD MICROBIOLOGY GAKUJUTSU SOKAI KOEN YOSHISHU, 29 August 2016 (2016-08-29), pages 62 *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CZ308046B6 (cs) * 2018-06-29 2019-11-20 FakultnĂ­ nemocnice Brno Způsob typizace bakteriálních kmenů Escherichia coli a oligonukleotidy pro použití při tomto způsobu

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