Dataset Information

Integrated, Automated, Fast PCR System for Point-Of-Care Molecular Diagnosis of Bacterial Infection.

ABSTRACT: We developed an integrated PCR system that performs automated sample preparation and fast polymerase chain reaction (PCR) for application in point-of care (POC) testing. This system is assembled from inexpensive 3D-printing parts, off-the-shelf electronics and motors. Molecular detection requires a series of procedures including sample preparation, amplification, and fluorescence intensity analysis. The system can perform automated DNA sample preparation (extraction, separation and purification) in ?5 min. The variance of the automated sample preparation was clearly lower than that achieved using manual DNA extraction. Fast thermal ramp cycles were generated by a customized thermocycler designed to automatically transport samples between heating and cooling blocks. Despite the large sample volume (50 ?L), rapid two-step PCR amplification completed 40 cycles in ?13.8 min. Variations in fluorescence intensity were measured by analyzing fluorescence images. As proof of concept of this system, we demonstrated the rapid DNA detection of pathogenic bacteria. We also compared the sensitivity of this system with that of a commercial device during the automated extraction and fast PCR of Salmonella bacteria.

SUBMITTER: Lee D

PROVIDER: S-EPMC7827619 | biostudies-literature | 2021 Jan

REPOSITORIES: biostudies-literature

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Integrated, Automated, Fast PCR System for Point-Of-Care Molecular Diagnosis of Bacterial Infection.

Lee Dongkyu D Kim Deawook D Han Jounghyuk J Yun Jongsu J Lee Kang-Ho KH Kim Gyu Man GM Kwon Ohwon O Lee Jaejong J

Sensors (Basel, Switzerland) 20210107 2

We developed an integrated PCR system that performs automated sample preparation and fast polymerase chain reaction (PCR) for application in point-of care (POC) testing. This system is assembled from inexpensive 3D-printing parts, off-the-shelf electronics and motors. Molecular detection requires a series of procedures including sample preparation, amplification, and fluorescence intensity analysis. The system can perform automated DNA sample preparation (extraction, separation and purification) ...[more]

PMID: 33430443

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Project description:BackgroundIn this work, a platform was developed and tested to allow to detect a variety of candidate viral, bacterial and parasitic pathogens, for acute fever of unknown origin. The platform is based on a centrifugal microfluidic cartridge, the LabDisk ("FeverDisk" for the specific application), which integrates all necessary reagents for sample-to-answer analysis and is processed by a compact, point-of-care compatible device.Methodology/principal findingsA sample volume of 200 μL per FeverDisk was used. In situ extraction with pre-stored reagents was achieved by bind-wash-elute chemistry and magnetic particles. Enzymes for the loop-mediated isothermal amplification (LAMP) were pre-stored in lyopellet form providing stability and independence from the cold chain. The total time to result from sample inlet to read out was 2 h. The proof-of-principle was demonstrated in three small-scale feasibility studies: in Dakar, Senegal and Khartoum, Sudan we tested biobanked samples using 29 and 9 disks, respectively; in Reinfeld, Germany we tested spiked samples and analyzed the limit of detection using three bacteria simultaneously spiked in whole blood using 15 disks. Overall during the three studies, the FeverDisk detected dengue virus (different serotypes), chikungunya virus, Plasmodium falciparum, Salmonella enterica Typhi, Salmonella enterica Paratyphi A and Streptococcus pneumoniae.Conclusions/significanceThe FeverDisk proved to be universally applicable as it successfully detected all different types of pathogens as single or co-infections, while it also managed to define the serotype of un-serotyped dengue samples. Thirty-eight FeverDisks at the two African sites provided 59 assay results, out of which 51 (86.4%) were confirmed with reference assay results. The results provide a promising outlook for future implementation of the platform in larger prospective clinical studies for defining its clinical sensitivity and specificity. The technology aims to provide multi-target diagnosis of the origins of fever, which will help fight lethal diseases and the incessant rise of antimicrobial resistance.

Project description:IntroductionComa is a deep state of unconsciousness that can be caused by a variety of clinical conditions. Traditional tests for coma outcome prediction are based mainly on a set of clinical observations. Recently, certain event-related potentials (ERPs), which are transient electroencephalogram (EEG) responses to auditory, visual or tactile stimuli, have been introduced as useful predictors of a positive coma outcome (ie, emergence). However, such tests require the skills of clinical neurophysiologists, who are not commonly available in many clinical settings. Additionally, none of the current standard clinical approaches have sufficient predictive accuracies to provide definitive prognoses.ObjectiveThe objective of this study is to develop improved machine learning procedures based on EEG/ERP for determining emergence from coma.Methods and analysisData will be collected from 50 participants in coma. EEG/ERP data will be recorded for 24 consecutive hours at a maximum of five time points spanning 30 days from the date of recruitment to track participants' progression. The study employs paradigms designed to elicit brainstem potentials, middle-latency responses, N100, mismatch negativity, P300 and N400. In the case of patient emergence, data are recorded on that occasion to form an additional basis for comparison. A relevant data set will be developed from the testing of 20 healthy controls, each spanning a 15-hour recording period in order to formulate a baseline. Collected data will be used to develop an automated procedure for analysis and detection of various ERP components that are salient to prognosis. Salient features extracted from the ERP and resting-state EEG will be identified and combined to give an accurate indicator of prognosis.Ethics and disseminationThis study is approved by the Hamilton Integrated Research Ethics Board (project number 4840). Results will be disseminated through peer-reviewed journal articles and presentations at scientific conferences.Trial registration numberNCT03826407.

Project description:IntroductionEarly infant diagnosis (EID) and prompt linkage to care are critical to minimise the high morbidity and mortality associated with infant HIV infection. Attrition in the "EID cascade" is common; however, point-of-care (POC) EID assays with same-day result could facilitate prompt linkage of HIV-infected infant to treatment. Despite a number of POC EID assays in development, few have been independently evaluated and data on new technologies are urgently needed to inform policy.MethodsWe compared Alere q 1/2 Detect POC system laboratory test characteristics with the local standard of care (SOC), Roche CAP/CTM HIV-1 qualitative PCR in an independent laboratory-based evaluation in Cape Town, South Africa. Routinely EID samples collected between November 2013 and September 2014 were each tested by both SOC and POC systems. Repeat testing was done to troubleshoot any discrepancy between POC and SOC results.ResultsOverall, 1098 children with a median age of 47 days (IQR, 42-117) were included. Birth PCR (age <7 days) comprised of 8% (n = 92) tests while 56% (n = 620) of children tested as part of routine EID (ages 6-14 weeks). In the overall direct comparison, Alere q Detect achieved sensitivity of 95.5% (95% CI, 91.7-97.9%) and a specificity of 99.8% (95% CI, 99.1-100%). Following repeat testing of discordant samples and exclusion of any inconclusive results, the POC assay sensitivity and specificity were 96.9% (95% CI 93.4-98.9%) and 100% (lower 95% CI 98%) respectively. Among birth PCR tests the POC assay had slightly lower sensitivity (93.3% vs 96.5% in routine EID) and higher assay error rate (10% vs 5% in samples of older children, p = 0.04).ConclusionOur results indicate this POC assay performs well for EID in the laboratory. The high specificity and thus high positive predictive value would suggest a positive POC result may be adequate for immediate infant ART initiation. While POC testing for EID may have particular utility for birth testing at delivery facilities, the lower sensitivity and error rate requires further attention, as does field implementation of POC EID technologies in other clinical care settings.

Dataset Information

Integrated, Automated, Fast PCR System for Point-Of-Care Molecular Diagnosis of Bacterial Infection.

Publications

Integrated, Automated, Fast PCR System for Point-Of-Care Molecular Diagnosis of Bacterial Infection.

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