Detection of C. difficile toxin as a model assay for performing fully automated high-throughput RT-PCR on clinical stool samples

Eigner, Ulrich; Nörz, Dominik; Reucher, Svenja; Furrer, Jan; Sun, Jing‐Tao; Chu, Kristina T.; Kolb, Melissa; Hefner, Nadine; Pfefferle, Susanne; Lütgehetmann, Marc

doi:10.1016/j.mimet.2020.105882

Cited by 6 publications

(5 citation statements)

References 35 publications

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“…Implementation on a fully-automated high-throughput sample-to-result platform like the cobas6800 further improves speed and scalability (sample-to-result time of approx. 3.5 h and throughput of 386 samples per 8 h [30]). Depending on local needs, users may be able to use existing SNP-assays to assemble customized multiplex-panels for circulating lineages, or use the design principles outlined above to implement additional assays when new Spike gene SNPs become relevant in the future.…”

Section: Discussionmentioning

confidence: 99%

Rapid Automated Screening for SARS-CoV-2 B.1.617 Lineage Variants (Delta/Kappa) through a Versatile Toolset of qPCR-Based SNP Detection

et al. 2021

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Background: The recent emergence of distinct and highly successful SARS-CoV-2 lineages has substantial implications for individual patients and public health measures. While next-generation-sequencing is routinely performed for surveillance purposes, RT-qPCR can be used to rapidly rule-in or rule-out relevant variants, e.g., in outbreak scenarios. The objective of this study was to create an adaptable and comprehensive toolset for multiplexed Spike-gene SNP detection, which was applied to screen for SARS-CoV-2 B.1.617 lineage variants. Methods: We created a broad set of single nucleotide polymorphism (SNP)-assays including del-Y144/145, E484K, E484Q, P681H, P681R, L452R, and V1176F based on a highly specific multi-LNA (locked nucleic acid)-probe design to maximize mismatch discrimination. As proof-of-concept, a multiplex-test was compiled and validated (SCOV2-617VOC-UCT) including SNP-detection for L452R, P681R, E484K, and E484Q to provide rapid screening capabilities for the novel B.1.617 lineages. Results: For the multiplex-test (SCOV2-617VOC-UCT), the analytic lower limit of detection was determined as 182 IU/mL for L452R, 144 IU/mL for P681R, and 79 IU/mL for E484Q. A total of 233 clinical samples were tested with the assay, including various on-target and off-target sequences. All SNPs (179/179 positive) were correctly identified as determined by SARS-CoV-2 whole genome sequencing. Conclusion: The recurrence of SNP locations and flexibility of methodology presented in this study allows for rapid adaptation to current and future variants. Furthermore, the ability to multiplex various SNP-assays into screening panels improves speed and efficiency for variant testing. We show 100% concordance with whole genome sequencing for a B.1.617.2 screening assay on the cobas6800 high-throughput system.

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Section: Discussionmentioning

confidence: 99%

Rapid Automated Screening for SARS-CoV-2 B.1.617 Lineage Variants (Delta/Kappa) through a Versatile Toolset of qPCR-Based SNP Detection

et al. 2021

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“…However, Roche has since received “Emergency use authorization” by the FDA for their own SARS-CoV-2 IVD assay and have made the test available commercially [ 12 ]. This provides a common ground across different testing sites for quantification of RT-PCR data, especially when taking the high inter-run consistency of the instrument into account, as previously demonstrated for other clinical targets [ 13 ].…”

Section: Introductionmentioning

confidence: 99%

Pushing beyond specifications: Evaluation of linearity and clinical performance of the cobas 6800/8800 SARS-CoV-2 RT-PCR assay for reliable quantification in blood and other materials outside recommendations

Nörz

Frontzek²,

Eigner³

et al. 2020

Journal of Clinical Virology

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Highlights Effective reduction of SARS-CoV-2 infectivity by chemical inactivation without affecting assay performance. SARS-CoV-2 IVD for the cobas 6800/8800 is linear over up to six log steps in different materials including human plasma. Minimal variance of CT values between testing sites indicates high comparability of quantification results.

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“…As a comparison, EIA against GDH or EIA against toxin A/B can reach 10 3 CFU/g (feces) [58] . The LOD of PCR assays varies from 10 1 to 10 4 CFU/mL (CFU/g) [59–61] . Overall, RFD‐CD2 based assays displayed very comparable LODs with these two detection methods.…”

Section: Resultsmentioning

confidence: 96%

“…[58] The LOD of PCR assays varies from 10 1 to 10 4 CFU/mL (CFU/g). [59][60][61] Overall, RFD-CD2 based assays displayed very comparable LODs with these two detection methods. The culturing methods, however, can reach an LOD of 10 1 to 10 2 CFU/mL depending on the recovery method.…”

Section: Detection Sensitivity Of Rfd-cd2 As a Fluorescent Sensormentioning

confidence: 99%

In Vitro Selection and Characterization of a DNAzyme Probe for Diverse Pathogenic Strains of Clostridium difficile

Qian

Chang

et al. 2023

Chemistry A European J

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Clostridium difficile frequently causes an infectious disease known as Clostridium difficile infection (CDI), and there is an urgent need for the development of more effective rapid diagnostic tests for CDI. Previously we have developed an RNA-cleaving fluorogenic DNAzyme (RFD) probe, named RFD-CD1, that is capable of detecting a specific strain of C. difficile but is too specific to recognize other pathogenic C. difficile strains. To overcome this issue, herein we report RFD-CD2, another RFD that is not only highly specific to C. difficile but also capable of recognizing diverse pathogenic C. difficile strains. Extensive sequence and structure characterization establishes a pseudoknot structure and a significantly minimized sequence for RFD-CD2. As a fluorescent sensor, RFD-CD2 can detect C. difficile at a concentration as low as 100 CFU/mL, thus making this DNAzyme an attractive molecular probe for rapid diagnosis of CDI caused by diverse strains of C. difficile.

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Detection of C. difficile toxin as a model assay for performing fully automated high-throughput RT-PCR on clinical stool samples

Cited by 6 publications

References 35 publications

Rapid Automated Screening for SARS-CoV-2 B.1.617 Lineage Variants (Delta/Kappa) through a Versatile Toolset of qPCR-Based SNP Detection

Rapid Automated Screening for SARS-CoV-2 B.1.617 Lineage Variants (Delta/Kappa) through a Versatile Toolset of qPCR-Based SNP Detection

Pushing beyond specifications: Evaluation of linearity and clinical performance of the cobas 6800/8800 SARS-CoV-2 RT-PCR assay for reliable quantification in blood and other materials outside recommendations

In Vitro Selection and Characterization of a DNAzyme Probe for Diverse Pathogenic Strains of Clostridium difficile

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