Stx1 and Stx2 in the recovered samples were quantified by using an ELISA kit, RIDASCREEN® Verotoxin (R-Biopharm AG, Darmstadt, Germany) in accordance with the manufacturer’s instructions. The concentration of Stx1 and Stx2 was quantified by measuring absorbance at 450 nm relative to respective Stx1 and Stx2 standards.
Ridascreen verotoxin
Manufactured by R-Biopharm
Sourced in Germany
The RIDASCREEN® Verotoxin is a laboratory equipment product used for the detection of Verotoxin (Shiga toxin) produced by E. coli. It is an enzyme immunoassay that allows for the qualitative determination of Verotoxin in stool samples.
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Lab products found in correlation
Onestepplus real time pcr system, by Thermo Fisher Scientific (1 mentions)
High capacity cdna reverse transcription kit, by Thermo Fisher Scientific (1 mentions)
Faststart universal sybr green master, by Roche (1 mentions)
Dnase 1, by Roche (1 mentions)
Sv total rna isolation system, by Promega (1 mentions)
5 protocols using ridascreen verotoxin
1
Quantifying Shiga Toxins via ELISA
2
Quantification of Shiga Toxin Production
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Stx production was semi-quantified by using an enzyme immunoassay (EIA, Ridascreen® Verotoxin, R-Biopharm, Germany). Supernatants of cultures at 18 h were obtained by centrifugation at 12,000 ×g for 10 min, diluted 1/10 in LB and then analyzed according to manufacturer instructions. Regarding supernatants from cultures treated with acids, they were neutralized before performing the ELISA.
The results were spectrophotometrically measured at 450 nm and classified as weak positive (1+) if the extinction was >0.1–0.5 above the negative control, moderate (2+) (>0.5–1.0) and strongly positive 3+ (>1.0–2.0) to 4+ (>2.0). The assays were done twice.
The results were spectrophotometrically measured at 450 nm and classified as weak positive (1+) if the extinction was >0.1–0.5 above the negative control, moderate (2+) (>0.5–1.0) and strongly positive 3+ (>1.0–2.0) to 4+ (>2.0). The assays were done twice.
3
Detecting Stx1 and Stx2 in Bacterial Cultures
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We used the RIDASCREEN® Verotoxin (R5701, R-Biopharm Latinoamérica S.A., City of Buenos Aires, Argentina) test to detect Stx1 and Stx2. Following the manufacturer’s specifications, we assayed the supernatants of the bacterial cultures treated 18 h with CBHG (6 mg/ml) to determine extracellular Stx. Measurements were taken photometrically with a wavelength of 450 nm. The average was calculated and compared with untreated bacterial cultures.
4
Quantifying EHEC Toxin Production
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Cultures of strain ZAP1620, a phage-type (PT) 21/28 EHEC isolate, were cultured overnight in LB medium. After dilution 1/100, cultures were grown in the presence or absence of AHU3 (50 μM) to exponential phase (OD600 = 0.3–0.4) before phage lysis was induced by the addition of mitomycin C (5 μg/ml). Lysis was allowed to proceed for 1.5 h after which cultures were filtered (0.22 μm filter). Supernatant samples containing the toxin were applied directly to a RIDASCREEN® verotoxin (R-Biopharm) 96-well microtitre plate and assayed for toxin levels by ELISA according to manufacturer guidelines.
5
Quantifying Shiga Toxin Expression in E. coli
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The expression of stx 2 was evaluated 90 min after mitomycin C addition. Total RNA was purified from 500 μl aliquots (SV total RNA isolation system; Promega) and treated with DNase I (Roche Diagnostics GmbH) at 37 °C for 1 h. cDNA was synthesized from 1 μg total RNA with the High Capacity cDNA Reverse Transcription Kit (Applied Biosystems) and diluted 1/10 for qPCR reactions. Two qPCRs were carried out for each sample on a OneStep Plus Real-Time PCR System (Applied Biosystems). Primers 2SF and 2R (de Sablet et al., 2008) were used to amplify the stx 2a gene, and TufAqR plus TufAqF for the tufA gene (de Sablet et al., 2008) . Each reaction was performed in a 20 μl reaction mix containing 10 μl 2× SYBR Green mix (FastStart Universal SYBR Green Master, Roche) and either primers 2SF and 2R at 400 nM each, or TufAqR and TufAqF at 300 nM each. Expression levels of stx 2a were normalized against tufA.
In order to evaluate extracellular Stx2a production, supernatants from cultures at 18 h were obtained by centrifugation at 12,000 ×g for 10 min, diluted 1/10 in LB and analyzed with an enzyme immunoassay (EIA, Ridascreen Verotoxin, R-Biopharm) according to the manufacturer instructions.
In order to evaluate extracellular Stx2a production, supernatants from cultures at 18 h were obtained by centrifugation at 12,000 ×g for 10 min, diluted 1/10 in LB and analyzed with an enzyme immunoassay (EIA, Ridascreen Verotoxin, R-Biopharm) according to the manufacturer instructions.
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