ArticleJMIR research protocols2021
Multiplex Droplet Digital Polymerase Chain Reaction Assay for Rapid Molecular Detection of Pathogens in Patients With Sepsis: Protocol for an Assay Development Study.
Article in JMIR research protocols, 2021. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 3 papers.
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Who cites it
3 citing papers in PubMed, 7 citations in OpenAlex.
- Development of a multiplex ddPCR assay for simultaneous absolute quantification of bacterial, fungal, and human DNA.PloS one · 2026Article
- Clinical validation of a multiplex droplet digital PCR for diagnosing suspected bloodstream infections in ICU practice: a promising diagnostic tool.Critical care (London, England) · 2022Article
- REASSURED Multiplex Diagnostics: A Critical Review and Forecast.Biosensors · 2022Review
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Authors and funding
3 authors at 1 institution in 1 country.
Funding
No grant is acknowledged in the PubMed record.
Abstract
backgroundBlood cultures are the cornerstone of diagnosis for detecting the presence of bacteria or fungi in the blood, with an average detection time of 48 hours and failure to detect a pathogen occurring in approximately 50% of patients with sepsis. Rapid diagnosis would facilitate earlier treatment and/or an earlier switch to narrow-spectrum antibiotics.
objectiveThe aim of this study is to develop and implement a multiplex droplet digital polymerase chain reaction (ddPCR) assay as a routine diagnostic tool in the detection and identification of pathogens from whole blood and/or blood culture after 3 hours of incubation.
methodsThe study consists of three phases: (1) design of primer-probe pairs for accurate and reliable quantification of the most common sepsis-causing microorganisms using a multiplex reaction, (2) determination of the analytical sensitivity and specificity of the multiplex ddPCR assay, and (3) a clinical study in patients with sepsis using the assay. The QX200 Droplet Digital PCR System will be used for the detection of the following species-specific genes in blood from patients with sepsis: coa (staphylocoagulase) in Staphylococcus aureus, cpsA (capsular polysaccharide) in Streptococcus pneumoniae, uidA (beta-D-glucuronidase) in Escherichia coli, oprL (peptidoglycan-associated lipoprotein) in Pseudomonas aeruginosa, and the highly conserved regions of the 16S rRNA gene for Gram-positive and Gram-negative bacteria. All data will be analyzed using QuantaSoft Analysis Pro Software.
resultsIn phase 1, to determine the optimal annealing temperature for the designed primer-probe pairs, results from a gradient temperature experiment will be collected and the limit of detection (LOD) of the assay will be determined. In phase 2, results for the analytical sensitivity and specificity of the assay will be obtained after an optimization of the extraction and purification method in spiked blood. In phase 3, clinical sensitivity and specificity as compared to the standard blood culture technique will be determined using 301 clinical samples.
conclusionsSuccessful design of primer-probe pairs in the first phase and subsequent optimization and determination of the LOD will allow progression to phase 3 to compare the novel method with existing blood culture methods. INTERNATIONAL REGISTERED REPORT IDENTIFIER (IRRID): PRR1-10.2196/33746.
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