EP1409742A2 - Kit de test pour determiner la presence de souches escherichia coli enterohemorrhagiques (ehec) - Google Patents

Kit de test pour determiner la presence de souches escherichia coli enterohemorrhagiques (ehec)

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Publication number
EP1409742A2
EP1409742A2 EP02762153A EP02762153A EP1409742A2 EP 1409742 A2 EP1409742 A2 EP 1409742A2 EP 02762153 A EP02762153 A EP 02762153A EP 02762153 A EP02762153 A EP 02762153A EP 1409742 A2 EP1409742 A2 EP 1409742A2
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EP
European Patent Office
Prior art keywords
gene
seq
test kit
kit according
hybridizing
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP02762153A
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German (de)
English (en)
Inventor
Sabine Vollenhofer-Schrumpf
Manfred Schinkinger
Gert Fränzl
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Sy-Lab VGmbH
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Sy-Lab VGmbH
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Publication date
Application filed by Sy-Lab VGmbH filed Critical Sy-Lab VGmbH
Publication of EP1409742A2 publication Critical patent/EP1409742A2/fr
Withdrawn legal-status Critical Current

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    • 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
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
    • C12Q1/689—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for bacteria
    • 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
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/16—Primer sets for multiplex assays
    • 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
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/166—Oligonucleotides used as internal standards, controls or normalisation probes

Definitions

  • EHEC enterohaemorrhagic Escherichia coli strains
  • the invention relates to a test kit for the detection of enterohaemorrhagic Escherichia coli strains (EHEC).
  • EHEC enterohaemorrhagic Escherichia coli strains
  • EHEC Escherichia coli
  • Enterohemorrhagic strains of Escherichia coli have been used as food pathogens in the past few years mainly in the USA, but also in some other countries, by causing sporadic, but often dramatic, and more and more frequent occurrences since 1993 Outbreaks of illnesses attracted attention. Infections occurred primarily through the consumption of meat that was not fully roasted (hamburgers), but also through the consumption of raw milk, yoghurt, sausage, apple cider, lettuce, vegetables or seedlings.
  • enterohaemorrhagic E. coli infection ranges from watery to bloody diarrhea to kidney damage such as hemolytic uraemic syndrome (HUS), which may lead to death (Griffin, PM, and Tauxe, RV (1991) : The Epidemiology of Infections Caused by Escherichia coli O157: H7, Other Enterohemorrhagic E. coli, and the Associated Hemolytic Uremic Syndrome. Epidemiol Rev 13: 60-98). Naturally, children, old people and people with immunodeficiency are particularly at risk.
  • HUS hemolytic uraemic syndrome
  • Enterohaemorrhagic E. coli is usually detected using classic microbiological, biochemical and serological methods (smear on plate, biochemical characterization, agglutination tests, enzyme-linked immunosorbent assay - ELISA) and cell culture methods (e.g. toxin test with Vero cells). Recently, however, detection using modern molecular biological or immunological methods has become increasingly important (Paton, AW, and Paton, JC (1998): Detection and Characterization of Shiga Toxigenic Escherichia coli by Using Multiplex PCR Assays for stxj, stx 2 , eaeA , Enterohemorrhagic E.
  • enterohaemorrhagic E. coli strains have some genetic peculiarities that distinguish them from non-pathogenic E. coli strains, they can be specifically detected using molecular biological methods that use these differences.
  • EHEC The most important genes typical for EHEC are the toxin genes SLT1 and SLT2 and their variants. Their gene products are largely responsible for the symptoms of an infection with EHEC. Another important gene for a pathogenicity factor of EHEC is the eaeA-Gcn. Since the vast majority of epidemics are caused by EHEC strains of the serotype O157: H7, the detection of this type is particularly important. Genes that are characteristic of this serotype are, for example, the genes rfl> E, a gene from the biosynthetic pathway of the O157 antigen, vm ⁇ fliC, the gene for the H7 antigen. At least the two genes for the toxins SLT1 and SLT2 and the 0157-specific gene rfbE are important for a test for routine food testing in order to clearly identify the dangerous pathogens as EHEC.
  • the object of the invention is to provide a test instrument with which the genes required for the detection of enterohaemorrhagic Escherichia coli strains or the serotype 0157 can be detected in a single reaction without major expenditure of equipment and time.
  • a control should be included in order to check the performance of the test.
  • the use of mutagenic or toxic substances should be avoided in this detection.
  • the aim is to provide a reliable tool for routine analysis in the quality control of food but also for the characterization of unknown E. coli strains isolated from any source.
  • the aim is to have a test instrument that enables even simply equipped laboratories to prove EHEC within a working day in the shortest possible time, with little hands-on time and without expensive equipment.
  • test kit which each has a pair of primers for amplifying at least a part of the SLT1 gene, the SLT2 gene, of the rfbE gene, the e ⁇ -4 gene and a control gene in a single reaction and each contain both an oligonucleotide as a capture probe and an oligonucleotide as a detector probe for hybridization of the amplificates obtained with the primers at room temperature, all capture probes on a common solid surface are immobilized and the detector probes are provided with a visually detectable marking.
  • the described molecular biological detection of EHEC by means of the test kit according to the invention offers significant advantages. Since there is neither a 100% reliable selection medium nor a clear biochemical test for EHEC, detection using microbiological, biochemical and serological methods is difficult. Other bacterial genera such as Escherichia hermannii or Hafnia spp. have, for example, a similar biochemical phenotype as O157: H7, or there are often cross-reactions of anti-O157 sera with other bacteria such as Citrobacter freundii or Yersinia enterocolitica.
  • Int J Food Microbiol 67: 71-80 is based on the fact that for the implementation of the detection reaction and for the Evaluation of the result requires no expensive special equipment such as microarray scanners and image processing systems or special software, hybridization cassettes or hybridization ovens, since the hybridization takes place at room temperature and the evaluation is carried out with the naked eye.
  • Another advantage is the use of capture probes in combination with directly labeled detection probes, so that there is a good specificity from the outset, which may only be guaranteed for microarrays by using at least two capture probes per amplificate (Chizhikov, V. et al, supra ).
  • a purification of amplificates or a removal of non-incorporated labels, as is essential when using fluorescence-labeled primers or nucleotides in microarrays, is in the the use of the test kit according to the invention is also not necessary, which reduces hands-on time and material consumption.
  • test kit is its exceptional sensitivity compared to other existing molecular biological detection methods for EHEC.
  • Fratamico et al. J Food Prot 63 (8): 1032-1037
  • a multiplex PCR with 5 genes from EHEC with which a sensitivity of 1 cfu / g food (prepared) could be achieved, namely with an enrichment time of 12 hours, testing of a single strain and using agarose gel electrophoresis instead of hybridization.
  • hybridization not only increases the sensitivity of a molecular biological test by signal amplification during detection, but also the specificity.
  • the increased number of primers used can lead to unspecific reactions which lead to unspecific bands in gels and can severely impair test interpretation (Chizhikov, V., Rasooly, A., Chumakov, K., and Levy , DD (2001): Microarray analysis of microbial virulence factors. Appl. Environments. Microbiol. 67 (7): 3258-3263).
  • German standard DIN10134 "General process-specific requirements for the detection of microorganisms with the polymerase chain reaction (PCR) in food” stipulates, for example, that a PCR result must be verified by hybridization or sequencing. Occurring bands of the apparently correct size can Using agarose gel electrophoresis alone leads to false positive results, however, many molecular biological methods for the detection of EHEC do not meet the above-mentioned condition, but only use agarose gels to evaluate a test result (JP-A - 2001-095576; Hu, Y, Zhang, Q.
  • test kit The combination of pre-enrichment, DNA amplification and detection by the test kit according to the invention also allows routine laboratories equipped as standard to quickly and reliably detect EHEC. Only a thermal cycler and a shaker are required on additional equipment.
  • a preferred embodiment of the test kit is characterized in that the primer pair for the amplification of a part of the SLT1 gene has the nucleotide sequences SEQ ID NO: 1 and SEQ ID NO: 2.
  • the capture probe for hybridizing the SLT1 gene amplificate preferably has the nucleotide sequence SEQ ID NO: 3.
  • the nucleotide sequence of the detector probe is preferably SEQ ID NO: 4.
  • a further preferred embodiment of the test kit is characterized in that the primer pair for the amplification of a part of the SLT2 gene has the nucleotide sequences SEQ ID NO: 5 and SEQ ID NO: 6.
  • the capture probe for hybridizing the SLT2 gene amplificate preferably has the nucleotide sequence SEQ ID NO: 7.
  • the nucleotide sequence of the detector probe is preferably SEQ ID NO: 8.
  • test kit is characterized in that the primer pair for the amplification of part of the r ⁇ E gene has the nucleotide sequences SEQ ID NO: 9 and SEQ ID NO: 10.
  • the capture probe for hybridizing the rfbE gene amplificate preferably has the nucleotide sequence SEQ ID NO: 11.
  • the nucleotide sequence of the detector probe is preferably SEQ ID NO: 12.
  • test kit is characterized in that the primer pair for the amplification of a part of the eaeA gene has the nucleotide sequences SEQ ID NO: 13 and SEQ ID NO: 14.
  • the capture probe for hybridizing the ea ⁇ gene plicate preferably has the nucleotide sequence SEQ ID NO: 15.
  • the nucleotide sequence of the detector probe is preferably SEQ ID NO: 16.
  • Yet another preferred embodiment of the test kit is characterized in that the human KCNJ9 gene is provided as the control gene.
  • the pair of primers for amplifying part of the human KCNJ9-GG ⁇ .S preferably has the nucleotide sequences SEQ ID NO: 17 and SEQ ID NO: 18.
  • the capture probe for hybridizing the XCNJP gene amplificate preferably has the nucleotide sequence SEQ ID NO: 19.
  • the nucleotide sequence of the detector probe is preferably SEQ ID NO: 20.
  • test kit is characterized in that at least one oligonucleotide is provided for a hybridization negative control, which is immobilized on a solid surface.
  • the negative control can also consist, for example, of a mixture of several random oligonucleotides.
  • the oligonucleotides for the hybridization negative control preferably have the nucleotide sequences SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23 and SEQ ID NO: 24.
  • a coupling with alkaline phosphatase is preferably provided as the visually detectable marking of the detector probes, which coupling makes hybridization visible by means of enzymatic conversion of organic dyes.
  • the resulting double-stranded DNA fragments are then broken down into single strands (denatured), and one of the respective single strands is hybridized with oligonucleotides (capture probes) bound to any solid surface (membrane, microtiter plate, electrode, chip, polystyrene beads or similar) Nucleotide sequences are each complementary to the relevant single strands.
  • this complex is hybridized with further suitable oligonucleotides (detector probes) which bind to further complementary sites of the single DNA strands and which are provided with a label which can be detected directly or indirectly (see FIG. 1).
  • oligonucleotides detector probes
  • Another optical, autoradiographic or electrochemical detection would also be possible.
  • the individual preferred DNA sequences (primer pairs) for the amplification of four gene segments characteristic of EHEC and the internal PCR / hybridization positive control were especially designed to be used jointly in a single reaction (multiplex reaction).
  • the aim was to form amplification products that were as small as possible and similar in size in order to achieve the highest possible and also similar amplification efficiency for the individual gene segments and the shortest possible reaction time during amplification.
  • the preferred DNA sequences for the capture probes bound to the solid surface and for the labeled detector probes were further selected so that for all DNA sequences a specific hybridization and thus also a specific detection under the same conditions, namely at room temperature (20- 35 ° C), is made possible (multiplex hybridization).
  • DNA sequences according to claims 2 to 4 have proven to be particularly effective for the amplification and detection of part of the SLT1 gene from EHEC.
  • the DNA sequences disclosed in claims 5 to 7 are directed to the amplification and detection of the SLT2 gene which also occurs in EHEC and most of its variants.
  • the subjects of claims 8 to 10 form DNA sequences which are suitable for the amplification and for the detection of a part of the rfbE gene from E. coli strains (in particular O157: H7) which have the O157 antigen.
  • the oligonucleotides described in claims 11 to 13 are used for the amplification and the detection of a part of the eaeA gene from E. coli strains.
  • Claims 15 to 17 describe oligonucleotides for the detection and detection of the human KCNJ9 gene as an internal positive control for the test.
  • oligonucleotides as a negative control for hybridization are set out in claim 19.
  • Bacterial strains Enterohaemorrhagic Escherichia co / z ' strains come from the American Type Culture Collection (ATCC). Table 1 shows a list of the used Strains and the genes that are present. The strains were kept on plate count agar plates, overnight cultures were prepared in liquid LB medium.
  • ATCC American Type Culture Collection
  • Target sequences The DNA sequences for SLT, rflE, eaeA and KCNJ9 genes, which were used for the design of suitable oligonucleotides, come from the GenBank database of the NIH (National Institutes of Health, www.ncbi.nlm .nih.gov) and were prepared and analyzed with the help of DNA analysis software such as GeneRunner (www, generunner.com) or PC / Gene (Intelligenetics Inc., Geneva, Switzerland). After multiple sequence comparisons (Higgins, D.G. and Sharp, P.M. (1988): CLUSTAL: a package for performing multiple sequence alignment on a microcomputer. Gene 73 (1): 237-244), highly conserved areas of the respective genes were selected.
  • amplification primers, capture probes and detector probes were then determined (Rychlik, W., Spencer, WJ, and Rhoads, RE: (1990): Optimization of the annealing temperature for DNA amplification in vitro. Nucl. Acids Res. 18 (21) : 6409-6412), taking care that the annealing temperatures of all primers for the multiplex amplification reaction do not differ too much from one another. Attempts were also made to find probes with a melting temperature that was as similar as possible. Thus, a simultaneous detection of all known variants of all relevant genes should be achieved under the same conditions.
  • Primer oligonucleotides, capture probes and detector probes The sequences of the primers and probes are given in Table 2 and in the attached sequence listing. In addition to the specific sequence, capture probes have 12 deoxy-thymidines at the 5 'end as "spacers" to the membrane. All oligonucleotides except labeled detector probes were obtained from VBC-GENOMICS Bioscience GmbH (Rennweg 95B, 1030 Vienna, Austria), detector probes were obtained from DNA Technology A / S (Science Park Aarhus, Gustav Wieds Vej 10A, DK-8000 Aarhus, Denmark) and in the described case were coupled with the enzyme alkaline phosphatase (Hermanson, G.
  • Meat samples Mashed mixed pork and beef were purchased from a butcher and kept refrigerated until used.
  • Bacterial count determination After calibration of the measurement method for all strains used by comparing the results with the results from the plate count method (number of colonies on plates), the bacterial count of the overnight cultures used for inoculation was determined by EHEC using a BacTrac 4100 analysis system (SY -LAB Automatic GmbH, Tullnerbach No 61-65, 3011 Neupurkersdorf, Austria) impedimetrically determined according to the manufacturer's instructions.
  • Genomic DNA from EHEC strains was obtained using commercially available spin column kits (NucleoSpin TM Tissue Kit from Macherey-Nagel GmbH & Co.KG, PO Box 10 13 52, D-52313 Düren, Federal Republic of Germany or High Pure PCR Template Preparation kit from Röche Diagnostics GmbH, Engelhomgasse 3, A-1211 Vienna, Austria) isolated from simple overnight cultures. DNA concentrations were determined in a SmartSpec3000 spectrophotometer (Bio-Rad Laboratories Ges.
  • the DNA was used for PCR 1 + 9 with water for molecular biology (Sigma,
  • thermostable DNA polymerase Saiki, RK, Gelfand, DH, Stoffel, S., Schare, SS, Higuchi, R., Hörn, GT, Mullis, KB, and Erlich, HA (1988): Primer-directed enzymatic amplification of DNA with a thermostable DNA polymerase. Science 239: 487-491).
  • Reaction conditions final concentrations: 1x amplification buffer, 2.5 mM MgCl 2 (both from Qiagen, Max-Volmer-Strasse, D-40724 Hilden, Federal Republic of Germany), nucleotide mix (each 200 ⁇ M dATP, dGTP, dCTP and dTTP or 200 ⁇ M each dATP, dGTP, dCTP and 600 ⁇ M dUTP), each 0.15 ⁇ M SLTl-fl and SLTl-rl, each 1 ⁇ M SLT2-fl, SLT2-r2, RFB-fl, RFB-rl, EAE-fl, EAE-rl , KCN-fl and KCN-rl, 0.7 ng / ⁇ l human DNA (Sigma, Hebbelplatz 7, A-1100 Vienna, Austria, Cat.
  • nucleotide mix each 200 ⁇ M dATP, dGTP, dCTP and dTTP or 200 ⁇ M each dATP,
  • Hybridization solution (final concentrations): 5xSSC, 0.1% N-lauroylsarcosine, 0.02% SDS, 1% blocking reagent (Röche), 70 pM SLT1-CO, 240 pM SLT2-CO1, 448 pM RFB-CO, 660 pM EAE -COl, 200 pM KCN-CO.
  • a mixture of the detection probes was heated at 45 ° C for about 10 minutes before addition to the hybridization solution.
  • the strip was then washed twice with at least 2 ml of washing solution (0.1X SSC, 0.1% SDS) at room temperature for at least 7 minutes.
  • washing solution 0.1X SSC, 0.1% SDS
  • the color reaction is stopped by swirling the strips in distilled water and evaluating the result immediately after the strips have dried.
  • Multiplex amplification / hybridization The amplification and hybridization conditions described in the material and methods provided very good results with all samples used. The signals for the individual genes were sufficiently strong and did not differ too much in their intensity. No cross-reactions were found between the different amplificates.
  • Random oligonucleotide Table 1 ATCC strains of Escherichia coli used
  • Fig. 2 Hybridization of artificially contaminated meat samples

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Abstract

L'invention concerne un kit de test pour déterminer la présence de souches Escherichia coli entérohémorrhagiques (EHEC), ce kit comprenant deux amorces pour l'amplification d'au moins une partie du gène SLT1, du gène SLT2, du gène rfbE , du gène eaeA et d'un gène de contrôle lors d'une seule réaction. Il contient également une oligonucléotide en tant que sonde de capture et une oligonucléotide en tant que sonde de détection pour l'hybridation des produits d'amplification obtenus avec les amorces à température ambiante, toutes les sondes de capture étant immobilisées sur une surface solide commune et les sondes de détection étant dotées d'un marquage visible. Ce kit de test permet de déterminer la présence de souches Escherichia coli entérohémorrhagiques de manière fiable et simple lors d'une seule réaction.
EP02762153A 2001-07-26 2002-07-26 Kit de test pour determiner la presence de souches escherichia coli enterohemorrhagiques (ehec) Withdrawn EP1409742A2 (fr)

Applications Claiming Priority (3)

Application Number Priority Date Filing Date Title
AT0117201A AT411832B (de) 2001-07-26 2001-07-26 Test-kit für den nachweis der bakteriellen gene slt1, slt2 und rfbe
AT11722001 2001-07-26
PCT/AT2002/000222 WO2003010332A2 (fr) 2001-07-26 2002-07-26 Kit de test pour determiner la presence de souches escherichia coli enterohemorrhagiques (ehec)

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EP1409742A2 true EP1409742A2 (fr) 2004-04-21

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WO2005005659A1 (fr) * 2003-07-14 2005-01-20 Statens Serum Institut Diagnostic d'escherichia coli (dec) et de shigella spp diarrheogenes
CN100441696C (zh) * 2004-10-22 2008-12-10 中国疾病预防控制中心传染病预防控制所 肠出血性大肠杆菌o157：h7菌株的检测方法

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US5654417A (en) * 1995-04-14 1997-08-05 Children's Hospital And Medical Center Nucleic acid probes for detecting E. coli O157:H7
DE19814828A1 (de) * 1998-04-02 1999-10-07 Roche Diagnostics Gmbh Spezifisches und sensitives Nukleinsäurenachweisverfahren
FR2777907B1 (fr) * 1998-04-28 2002-08-30 Pasteur Sanofi Diagnostics Sequences nucleotidiques pour la detection des escherichia coli enterohemorragiques (ehec)
DE19946296A1 (de) * 1999-09-28 2001-03-29 Roche Diagnostics Gmbh Multiplex-PCR zum Nachweis von EHEC-Infektionen

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ATA11722001A (de) 2003-11-15
WO2003010332A3 (fr) 2003-11-06
AT411832B (de) 2004-06-25

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