A novel method for sample delivery and testing of whole blood: gel strip PCR for point of care (POC) molecular diagnostics

Manage, Dammika P.; Lauzon, Jana; Atrazhev, Alexey; Pang, Xiaoli; Pilarski, Linda M.

doi:10.1039/c3lc50755f

Cited by 15 publications

(20 citation statements)

References 8 publications

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“…The hydrogel mastermix including all of the reagents necessary for real-time PCR from blood was added to each channel underneath the coverslip. The mastermix was polymerized and desiccated, yielding 12 self-contained hydrogel units intended for 12 blood samples to be tested in the PCR reaction [24,25]. During desiccation, the hydrogel shrinks away from the wax trough but stays attached to the coverslip, creating a route for sample delivery via the channel underneath each gel (Figure 1A).…”

Section: Resultsmentioning

confidence: 99%

A lab-on-chip for malaria diagnosis and surveillance

Taylor

Howell²,

Martin

et al. 2014

Malar J

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BackgroundAccess to timely and accurate diagnostic tests has a significant impact in the management of diseases of global concern such as malaria. While molecular diagnostics satisfy this need effectively in developed countries, barriers in technology, reagent storage, cost and expertise have hampered the introduction of these methods in developing countries. In this study a simple, lab-on-chip PCR diagnostic was created for malaria that overcomes these challenges.MethodsThe platform consists of a disposable plastic chip and a low-cost, portable, real-time PCR machine. The chip contains a desiccated hydrogel with reagents needed for Plasmodium specific PCR. Chips can be stored at room temperature and used on demand by rehydrating the gel with unprocessed blood, avoiding the need for sample preparation. These chips were run on a custom-built instrument containing a Peltier element for thermal cycling and a laser/camera setup for amplicon detection.ResultsThis diagnostic was capable of detecting all Plasmodium species with a limit of detection for Plasmodium falciparum of 2 parasites/μL of blood. This exceeds the sensitivity of microscopy, the current standard for diagnosis in the field, by ten to fifty-fold. In a blind panel of 188 patient samples from a hyper-endemic region of malaria transmission in Uganda, the diagnostic had high sensitivity (97.4%) and specificity (93.8%) versus conventional real-time PCR. The test also distinguished the two most prevalent malaria species in mixed infections, P. falciparum and Plasmodium vivax. A second blind panel of 38 patient samples was tested on a streamlined instrument with LED-based excitation, achieving a sensitivity of 96.7% and a specificity of 100%.ConclusionsThese results describe the development of a lab-on-chip PCR diagnostic from initial concept to ready-for-manufacture design. This platform will be useful in front-line malaria diagnosis, elimination programmes, and clinical trials. Furthermore, test chips can be adapted to detect other pathogens for a differential diagnosis in the field. The flexibility, reliability, and robustness of this technology hold much promise for its use as a novel molecular diagnostic platform in developing countries.

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Section: Resultsmentioning

confidence: 99%

A lab-on-chip for malaria diagnosis and surveillance

Taylor

Howell²,

Martin

et al. 2014

Malar J

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“…APPETITE’s data and specimen repository will be employed to conduct the next steps in PCR-based, point of care diagnostics and norovirus vaccine research. Academia and industry partners will work to develop: 1) an oral viral pathogen panel; and 2) simplified point-of-care diagnostic technology [ 2 , 3 , 56 ] to enable use in remote communities and in low and middle income countries to reduce AGE mortality [ 57 ].…”

Section: Discussionmentioning

confidence: 99%

Alberta Provincial Pediatric EnTeric Infection TEam (APPETITE): epidemiology, emerging organisms, and economics

et al. 2015

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BackgroundEach year in Canada there are 5 million episodes of acute gastroenteritis (AGE) with up to 70 % attributed to an unidentified pathogen. Moreover, 90 % of individuals with AGE do not seek care when ill, thus, burden of disease estimates are limited by under-diagnosing and under-reporting. Further, little is known about the pathogens causing AGE as the majority of episodes are attributed to an “unidentified” etiology. Our team has two main objectives: 1) to improve health through enhanced enteric pathogen identification; 2) to develop economic models incorporating pathogen burden and societal preferences to inform enteric vaccine decision making.Methods/DesignThis project involves multiple stages: 1) Molecular microbiology experts will participate in a modified Delphi process designed to define criteria to aid in interpreting positive molecular enteric pathogen test results. 2) Clinical data and specimens will be collected from children aged 0–18 years, with vomiting and/or diarrhea who seek medical care in emergency departments, primary care clinics and from those who contact a provincial medical advice line but who do not seek care. Samples to be collected will include stool, rectal swabs (N = 2), and an oral swab. Specimens will be tested employing 1) stool culture; 2) in-house multiplex (N = 5) viral polymerase chain reaction (PCR) panel; and 3) multi-target (N = 15) PCR commercially available array. All participants will have follow-up data collected 14 days later to enable calculation of a Modified Vesikari Scale score and a Burden of Disease Index. Specimens will also be collected from asymptomatic children during their well child vaccination visits to a provincial public health clinic. Following the completion of the initial phases, discrete choice experiments will be conducted to enable a better understanding of societal preferences for diagnostic testing and vaccine policy. All of the results obtained will be integrated into economic models.DiscussionThis study is collecting novel samples (e.g., oral swabs) from previously untested groups of children (e.g., those not seeking medical care) which are then undergoing extensive molecular testing to shed a new perspective on the epidemiology of AGE. The knowledge gained will provide the broadest understanding of the epidemiology of vomiting and diarrhea of children to date.

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“…The industry needs a fast, sensitive and easy to use molecular method to definitively identify pathogens in food. To facilitate rapid and frequent monitoring of food processing to screen for pathogens, here, we have employed our cassette PCR technology [ 12 – 16 ] on an improved and reliable miniaturized testing platform and on an improved cassette.…”

Section: Introductionmentioning

confidence: 99%

Monitoring food pathogens: Novel instrumentation for cassette PCR testing

Hunt¹,

Figley²,

Manage

et al. 2018

PLoS ONE

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In this manuscript, we report the design and development of a fast, reliable instrument to run gel-based cassette polymerase chain reactions (PCR). Here termed the GelCycler Mark II, our instrument is a miniaturized molecular testing system that is fast, low cost and sensitive. Cassette PCR utilizes capillary reaction units that carry all reagents needed for PCR, including primers and Taq polymerase, except the sample, which is loaded at the time of testing. Cassette PCR carries out real time quantitative PCR followed by melt curve analysis (MCA) to verify amplicon identity at the expected melt temperature (Tm). The cassette PCR technology is well developed, particularly for detecting pathogens, and has been rigorously validated for detecting pathogenic Escherichia coli in meat samples. However, the work has been hindered by the lack of a robust and stable instrument to carry out the PCR, which requires fast and accurate temperature regulation, improved light delivery and fluorescent recording, and faster PCR reactions that maintain a high sensitivity of detection. Here, we report design and testing of a new instrument to address these shortcomings and to enable standardized testing by cassette PCR and commercial manufacture of a robust and accurate instrument that can be mass produced to deliver consistent performance. As a corollary to our new instrument development, we also report the use of an improved design approach using a machined aluminum cassette to meet the new instrument standards, prevent any light bleed across different trenches in each cassette, and allow testing of a larger number of samples for more targets in a single run. The GelCycler Mark II can detect and report E. coli contamination in 41 minutes. Sample positives are defined in as having a melt curve comparable to the internal positive control, with peak height exceeding that of the internal negative control. In a fractional analysis, as little as 1 bacterium per capillary reaction unit is directly detectable, with no enrichment step, in 35 cycles of PCR/MCA, in a total time of 53 minutes, making this instrument and technology among the very best for speed and sensitivity in screening food for pathogenic contamination.

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A novel method for sample delivery and testing of whole blood: gel strip PCR for point of care (POC) molecular diagnostics

Abstract: Testing of whole blood in miniaturized PCR is compromised by the opaque nature of whole blood that leads to physical masking of a fluorescent signal. We demonstrate a method to perform real-time PCR with whole blood that avoids interference from the opacity of whole blood.

Cited by 15 publications

References 8 publications

A lab-on-chip for malaria diagnosis and surveillance

A lab-on-chip for malaria diagnosis and surveillance

Alberta Provincial Pediatric EnTeric Infection TEam (APPETITE): epidemiology, emerging organisms, and economics

Monitoring food pathogens: Novel instrumentation for cassette PCR testing

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