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Pathogen Detection with GeneGen®Test Systems

Easy-to-apply biomolecular detection of pathogenic microorganisms in foodstuffs! Our GeneGen®kits employ a novel DNA amplification and hybridization protocol using multiplex PCR followed by a shortened hybridization reaction (the GeneGen®technology) for the detection of pathogenic microorganisms from liquid cultures (e. g. enrichment cultures) or agar plates (single colonies).

User-friendly, fast, reliable!

  • No tedious pipetting!
    PCR tubes contain freeze-dried, ready-to-use master mix – only the extracted sample has to be added. Laborious pipetting of PCR reagents, pipetting errors and the danger of contamination are eliminated.
  • Convenient and time-saving!
    The special assay format enables a strong reduction of the standard protocol for DNA hybridizations and processing at ambient temperatures (20-30 ºC), thus providing a considerable saving of time and dispensing with the usual cumbersome incubation of hybridization reactions in water baths or hybridization ovens. To carry out hybridization, only an orbital shaker or a rocking platform is needed.
  • Reliable and sensitive!
    1-10 colony-forming units / 25g of foodstuff are detectable, in many cases after the pre-enrichment step, within 24 hours.
    Internal positive control is integrated!

The following test kits are available:

  • GeneGen®EHEC Detection Kit
    Detection and characterization of enterohemorrhagic Escherichia coli strains within 24 hours!
  • GeneGen®EHECplus Detection Kit
    Detection and characterization of enterohemorrhagic Escherichia coli strains and differential diagnosis of STEC 0104:H4 from food or fecal samples within 24 hours using 6 specific EHEC genes!

GeneGen® EHEC Detection Kit

DNA detection system based on a patented multiplex-PCR technique combined with a hybridisation step for the detection and characterization of enterohemorrhagic Escherichia coli strains from liquid cultures (e.g. enrichments) and from agar plates (single colonies).


Assay principle

The GeneGen® EHEC Detection Kit is intended for the detection and partial characterization of EHEC strains, based on molecular biology methods. Four typical genes that can be present in EHEC, the genes for Shiga-like toxins 1 and 2, an O157-specific gene, the intimin gene (eaeA), and an internal positive control are amplified in a single reaction (multiplex-PCR) and detected afterwards by sandwich hybridization and color development. The special assay format enables a strong reduction of the standard protocol for DNA hybridizations and processing at ambient temperature (20-30 ºC), thus providing a considerable saving of time and avoiding the usual cumbersome incubation of hybridizations in water baths or hybridization ovens.


Working steps:

step duration  
cultivation on plates or in liquidculture
(e.g. enrichment culture from minced meat)
 overnight
(16-18 h)
DNA extraction

 < 30 min
PCR

 approx. 2,5 h
hybridization

 30 min
colorimetric detection 60 min



In most cases, detection is possible within 24 hours without much effort and little hands-on time, the actual assay time (without cultivation) not exceeding 4 hours.

Beside O157, other EHEC serotypes (STEC = Shiga toxin producing Escherichia coli), enteropathogenic E. coli strains (EPEC) and other Shiga toxin producing bacterial genera, like Shigella dysenteriae, can be detected, depending on the gene(s) that is (are) present.

Samples:

  1. Escherichia coli, non-pathogenic

  2. enteropathogenic Escherichia coli strain (EPEC) with eaeA gene

  3. Escherichia coli, non-pathogenic

  4. EHEC (STEC): non-O157, with SLT2 gene and eaeA

  5. EHEC (STEC): non-O157, with SLT1 gene and eaeA

  6. Escherichia coli O157, no toxin genes, with eaeA

  7. Escherichia coli O157 (EHEC), with SLT2 gene and eaeA

  8. Escherichia coli O157 (EHEC), with SLT1 gene and eaeA

  9. Escherichia coli O157 (EHEC), with both toxin genes and eaeA

  10. Escherichia coli, strain from swine (non-O157, no eaeA gene, possesses a variant of the SLT2 gene)

  11. PCR negative control (water instead of sample)

  12. Invalid result (no signal for internal positive control)



GeneGen® EHECplus Detection Kit

DNA detection system based on a patented multiplex-PCR technique combined with a hybridisation step for the detection and characterization of enterohemorrhagic Escherichia coli strains from liquid cultures (e.g. enrichments) from agar plates (single colonies) or from fecal samples after enrichment.


Assay principle

The GeneGen® EHEC Detection Kit is intended for the detection and partial characterization of EHEC strains, based on molecular biology methods. Six typical genes that can be present in EHEC, the genes for Shiga-like toxins 1 and 2, an O157-specific gene, the intimin gene (eaeA), the flagelling gene of (flicH4) and the gen for iron uptake (iha) of the serotype O104:H4 as well as an internal positive control are amplified in a single reaction (multiplex-PCR) and detected afterwards by sandwich hybridization and color development. The special assay format enables a strong reduction of the standard protocol for DNA hybridizations and processing at ambient temperature (20-30 ºC), thus providing a considerable saving of time and avoiding the usual cumbersome incubation of hybridizations in water baths or hybridization ovens.


Working steps:

step duration

cultivation on plates or in liquidculture
(e.g. enrichment culture from foods or fecal samples) and  DNA extraction

overnight
(16-18 h)

PCR

approx. 2,5 h

hybridization

30 min

colorimetric detection

60 min




Detection is possible within 24 hours without much effort and little hands-on time, the actual assay time (without cultivation) not exceeding 4 hours.
Beside O157, other EHEC serotypes (STEC = Shiga toxin producing Escherichia coli), enteropathogenic E. coli strains (EPEC), STEC 0104:H4 and other Shiga toxin producing bacterial genera, like Shigella dysenteriae, can be detected, depending on the gene(s) that is (are) present.




Samples:

  1. positive control-DNA GeneGen® EHECplus Kit (all targets+)

  2. EHEC (SLT 1+)

  3. EHEC (SLT 2+, eae+, iha+)

  4. German Outbreak isolate STEC O104:H4 (SLT 2+, iha+, fliC H4+)

  5. EHEC (SLT 1+, eae+, iha+)

  6. E. coli, serotype O157, non- toxinogen (O157+,eae+, iha+)

  7. EHEC, serotype O157 (SLT 2+, O157+, eae+, iha+)

  8. EHEC, serotype O157 (SLT 1+, O157+, eae+, iha+)

  9. EHEC EDL933, serotype O157 (SLT 1+, SLT 2+, O157+, eae+, iha+)

  10. Escherichia coli, isolate from pig (SLT 2+, iha+)

  11. enteropathogenic E. coli (eae+)

  12. Escherichia coli (non pathogen)

  13. Reagent control (water)

  14. invalid result (negative for internal positive control)




Quick reference procedure

  1. Perform enrichment or plate culture for 16-18 hours.
  2. For single colonies: Resuspend a single colony in 100µl of molecular biology grade water and proceed with step 6. For liquid cultures: Remove 100µl of culture and transfer to a tube.
  3. Pellet bacteria by centrifugation.
  4. Discard supernatant, wash bacterial pellet with 100µl of molecular biology grade water and centrifuge as above.
  5. Discard supernatant and resuspend bacteria in 100µl of molecular biology grade water.
  6. Heat sample to 95-100°C, let cool down and centrifuge.
  7. For fecal samples only: Extract the DNA using the GeneGen extraction reagent provided with the GeneGen Fecal kit using a sonication bath.
  8. Apply 25µl of supernatant in PCR.
  9. Prepare test strips, denature PCR products and prepare hybridization mix.
  10. Hybridize for 30 min.
  11. Wash 2x10 min and prepare developing solution.
  12. Equilibrate strips for 1 min.
  13. Color development (up to 1 hr. max).


Download GeneGen Catalogue
in *pdf Format (Acrobat Reader)