Specimens were cultured on MacConkey agar using the streak plate technique. From each plate, six individual colonies, if possible of different morphology, were picked und subcultured on sheep blood agar (Difco TM Columbia Blood Agar Base EH; Becton Dickinson AG, Allschwil, Switzerland). Isolates that were confirmed to possess stx (stx1 and/or stx2) by real-time PCR (LightCycler R 2.0 Instrument, Roche Diagnostics Corporation, Indianapolis, IN, USA) (EURL, 2013a) were selected for further analysis. From plates yielding more than one stx positive colony, one isolate was randomly chosen for subsequent characterization. Proportions of STEC in stool samples were defined as the numbers of stx positive colonies among six E. coli colonies.
Lightcycler r 2.0 instrument
Manufactured by Roche
Sourced in United States
The LightCycler R 2.0 Instrument is a real-time PCR system designed for sample analysis. It features a thermal cycler and optical detection unit to perform quantitative and qualitative nucleic acid amplification and analysis.
Automatically generated - may contain errors
4 protocols using lightcycler r 2.0 instrument
1
STEC Isolation and Characterization
2
Isolation and Characterization of Stx-Producing E. coli
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In the event of a stx-positive PCR result, one loopful each of the washed-off suspension was streaked onto on three to five STEC Chromagar plate (CHROMagar, Paris, France) and on three-to-five Brolacin agar plates (Bio-Rad, Hercules, CA, USA) to get single colonies. The plates were incubated overnight at 37 °C.
From each plate, 20–180 individual colonies were picked (mauve colonies on STEC Chromagar plates; yellow colonies on Brolacin agar plates) and suspended in 0.5 mL 0.85% NaCl. The suspensions were pooled in groups of ten colonies to simplify the screening process. The pooled suspensions were screened for stx1 and stx2 genes by real-time PCR (LightCycler R 2.0 Instrument, Roche Diagnostics Corporation, Indianapolis, IN, USA), using the QuantiFast Multiplex PCR Kit (Qiagen, Hombrechtikon, Switzerland) according to the guidelines of the European Union Reference Laboratory [25 ]. In the event of a positive PCR result for stx1 or stx2, the pool was taken apart and the ten colonies were tested individually. From plates yielding more than one stx positive colony, one presumptive STEC isolate was randomly chosen for subsequent characterization.
From each plate, 20–180 individual colonies were picked (mauve colonies on STEC Chromagar plates; yellow colonies on Brolacin agar plates) and suspended in 0.5 mL 0.85% NaCl. The suspensions were pooled in groups of ten colonies to simplify the screening process. The pooled suspensions were screened for stx1 and stx2 genes by real-time PCR (LightCycler R 2.0 Instrument, Roche Diagnostics Corporation, Indianapolis, IN, USA), using the QuantiFast Multiplex PCR Kit (Qiagen, Hombrechtikon, Switzerland) according to the guidelines of the European Union Reference Laboratory [25 ]. In the event of a positive PCR result for stx1 or stx2, the pool was taken apart and the ten colonies were tested individually. From plates yielding more than one stx positive colony, one presumptive STEC isolate was randomly chosen for subsequent characterization.
3
Enrichment and Detection of Shiga Toxin Genes
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Each fecal sample was enriched at a 1:10 ratio in Enterobacteriaceae enrichment (EE) broth (Becton, Dickinson, Heidelberg, Germany) for 24 h at 37 °C. One loopful of each of the enrichment cultures was cultured on sheep blood agar (Difco™ Columbia Blood Agar Base EH; Becton Dickinson AG, Allschwil, Switzerland) using the streak plate technique. The resulting colonies were washed off with 2 ml 0.85% NaCl and DNA was extracted by a standard lysis protocol. Screening for stx1 and stx2 genes was performed by real-time PCR (LightCycler R 2.0 Instrument, Roche Diagnostics Corporation, Indianapolis, IN, USA) using the QuantiFast Multiplex PCR Kit (Qiagen, Hombrechtikon, Switzerland) according to the guidelines of the European Union Reference Laboratory (EURL) [25 ].
4
Isolation and Characterization of STEC
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In the event of a stx positive PCR result, one loopful of suspension was streaked onto STEC Chromagar plates (CHROMagar, Paris, FR) and Brolacin agar plates (Bio-Rad, Hercules CA, USA) to get single colonies. The plates were incubated at 37 °C overnight. From each plate, 20–180 individual colonies were picked (mauve colonies on STEC Chromagar plates; yellow colonies on Brolacin agar plates) and suspended in 0.5 ml 0.85% NaCl. The suspensions were pooled in groups of material from ten colonies and screened for stx1 and stx2 genes by real-time PCR (LightCycler R 2.0 Instrument, Roche Diagnostics Corporation, Indianapolis, IN, USA) using the QuantiFast Multiplex PCR Kit (Qiagen, Hombrechtikon, Switzerland) according to the guidelines of the European Union Reference Laboratory (EURL)52 . In the event of a positive PCR result for stx1 or stx2, the pool was taken apart and the ten colonies were tested again individually. From plates yielding more than one stx1 and/or stx2 positive colony, one presumptive STEC isolate was randomly chosen for subsequent characterisation by whole genome sequencing (WGS) analysis. If the screening results indicated colonies with different stx types, the different corresponding colonies were included in the further analysis.
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