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PCRdrive: the largest qPCR assay archive to date and endless potential for lab workflow revitalization.

ABSTRACT: BACKGROUND:Primer design is a crucial step in establishing specific and sensitive qPCR assays. Even though numerous tools for primer design exist, the majority of resulting assays still requires extensive testing and optimisation or does not allow for high quality target amplification. We developed a workflow for designing qPCR assays. Unlike other tools, we compute a PCR assay including primer design, concentrations and the optimal PCR program. RESULTS:Gene expression assays were already generated in a total of 283,226 genes from three species and are continued for all genes of the major model species. The results are available online at https://pcrdrive.com/lab#/assay-database . The workflow involves filtering Primer3-generated primers by considering diverse parameters including specificity, single-nucleotide polymorphisms (SNPs), secondary structure as well as compatibility with standard qPCR assay conditions. The resulting assays consist of transcript-specific primer sequences, a reagents protocol as well as instrument settings which are provided in a web-based tool called PCRdrive. PCRdrive was designed to support PCR users in their PCR-related tasks and is equipped with handy functions, components of an electronic lab notebook (ELN) as well as teamworking opportunities. CONCLUSION:High quality ready to use qPCR assays for gene expression analysis are provided within the online platform PCRdrive. A built-in primer designer enables easy generation of assays which is not supported by any other tool. The wet lab optimisation of new assays can be transparently documented and shared within the team. PCRdrive also contains an archive of public PCRs which is updated regularly. Users may use the archive to publish their PCR to the community which makes it easy for other researchers worldwide to reproduce and validate the PCR. PCRdrive is a growing network of PCR users, simplifying and streamlining research through its useful existing features and continuous developments from the active development team.

SUBMITTER: Burger F

PROVIDER: S-EPMC6249872 | biostudies-other | 2018 Nov

REPOSITORIES: biostudies-other

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PCRdrive: the largest qPCR assay archive to date and endless potential for lab workflow revitalization.

Burger Florian F Angioni Michele M Russo Gianluca G Schad Martina M Kallarackal Jim J

BMC bioinformatics 20181122 1

<h4>Background</h4>Primer design is a crucial step in establishing specific and sensitive qPCR assays. Even though numerous tools for primer design exist, the majority of resulting assays still requires extensive testing and optimisation or does not allow for high quality target amplification. We developed a workflow for designing qPCR assays. Unlike other tools, we compute a PCR assay including primer design, concentrations and the optimal PCR program.<h4>Results</h4>Gene expression assays were ...[more]

PMID: 30466398

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Project description:BackgroundLike most hospitals, our hospital experienced COVID-19 pandemic-related supply chain shortages. Our additive manufacturing lab's capacity to offset these shortages was soon overwhelmed, leading to a need to improve the efficiency of our existing workflow. We undertook a work system analysis guided by the Systems Engineering Initiative for Patient Safety (SEIPS) construct which is based on human factors and quality improvement principles. Our objective was to understand the inefficiencies in project submission, review, and acceptance decisions, and make systematic improvements to optimize lab operations.MethodsContextual inquiry (interviews and workflow analysis) revealed suboptimal characteristics of the system, specifically, reliance on a single person to facilitate work and, at times, fractured communication with project sponsors, with root causes related to the project intake and evaluation process as identified through SEIPS tools. As interventions, the analysis led us to: 1) enhance an existing but underused project submission form, 2) design and implement an internal project scorecard to standardize evaluation of requests, and 3) distribute the responsibility of submission evaluation across lab members. We implemented these interventions in May 2021 for new projects and compare them to our baseline February 1, 2018 through - April 30, 2021 performance (1184 days).ResultsAll project requests were submitted using the enhanced project submission form and all received a standardized evaluation with the project scorecard. Prior to interventions, we completed 35/79 (44%) of projects, compared to 12/20 (60%) of projects after interventions were implemented. Time to review new submissions was reduced from an average of 58 days to 4 days. A more distributed team responsibility structure permitted improved workflow with no increase in staffing, allowing the Lab Manager to devote more time to engineering rather than administrative/decision tasks.ConclusionsBy optimizing our workflows utilizing a human factors approach, we improved the work system of our additive manufacturing lab to be responsive to the urgent needs of the pandemic. The current workflow provides insights for labs aiming to meet the growing demand for point-of-care manufacturing.

Project description:BackgroundMicroscopy and antigen detecting rapid diagnostic tests are the diagnostic tests of choice in management of clinical malaria. However, due to their limitations, the need to utilize more sensitive methods such as real-time PCR (qPCR) is evident as more studies are now utilizing molecular methods in detection of malaria. Some of the challenges that continue to limit the widespread utilization of qPCR include lack of assay standardization, assay variability, risk of contamination, and the need for cold-chain. Lyophilization of molecular assays can overcome some of these limitations and potentially enable widespread qPCR utilization.MethodsA recently published multiplex malaria qPCR assay was lyophilized by freezing drying into Sample-Ready™ format (MMSR). MMSR assay contained all the required reagents for qPCR including primers and probes, requiring only the addition of water and sample to perform qPCR. The performance of the MMSR assay was compared to the non-freeze dried, "wet" assay. Stability studies were done by maintaining the MMSR assays at four different ambient temperatures of 4°C, room temperature (RT), 37°C and 42°C over a period of 42 days, tested at seven-day intervals. Plasmodium falciparum and Plasmodium vivax DNAs were used for analysis of the MMSR assay either as single or mixed parasites, at two different concentrations. The CT values and the standard deviations (SD) were used in the analysis of the assay performance.ResultsThe limit of detection for the MMSR assay was 0.244 parasites/?L for Plasmodium spp. (PLU) and P. falciparum (FAL) assay targets compared to "wet" assay which was 0.39 and 3.13 parasites/?L for PLU and FAL assay targets, respectively. The MMSR assay performed with high efficiencies similar to those of the "wet" assay and was stable at 37°C for 42 days, with estimated shelf-life of 5 months. When used to analyse field clinical samples, MMSR assay performed with 100% sensitivity and specificity compared to the "wet" assay.ConclusionThe MMSR assay has the same robust performance characteristics as the "wet" assay and is highly stable. Availability of MMSR assay allows flexibility and provides an option in choosing assay for malaria diagnostics depending on the application, needs and budget.

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PCRdrive: the largest qPCR assay archive to date and endless potential for lab workflow revitalization.

Publications

PCRdrive: the largest qPCR assay archive to date and endless potential for lab workflow revitalization.

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