Fully integrated multiplexed lab-on-a-card assay for enteric pathogens

Weigl, Bernhard H.; Gerdes, J.; Tarr, P.; Yager, Paul; Dillman, L.; Peck, Roger; Ramachandran, Sujatha; Lemba, M.; Kokoris, Mark S.; Nabavi, Mahmood; Battrell, F.; Hoekstra, Dirk; Klein, Eileen J.; Denno, Donna M.

doi:10.1117/12.644714

Cited by 19 publications

(16 citation statements)

References 12 publications

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“…This study was part of a project entitled 'Diarrhea Enteric Card (DEC)' aiming to develop a point-of-care microfluidic PCR-based multiplex assay for four common bacterial enteric pathogens (Yager et al, 2006;Weigl et al, 2006). The project was approved by the local and national ethical committees in Brazil (HIAS 80/06 and CONEPE 13523/2007, respectively).…”

Section: Methodsmentioning

confidence: 99%

Campylobacter jejuni infection and virulence-associated genes in children with moderate to severe diarrhoea admitted to emergency rooms in northeastern Brazil

Quetz

Lima

Havt

et al. 2012

Journal of Medical Microbiology

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Campylobacter is an important cause of foodborne gastroenteritis. We determined the occurrence of Campylobacter jejuni and Campylobacter coli, using culture-based methods and PCRs targeting virulence-associated genes (VAGs) among children aged ¡14 years who were treated for diarrhoea at emergency rooms in northeastern Brazil. Genomic DNA was extracted directly from stool samples collected from 366 children. A questionnaire was also applied to qualify the clinical conditions presented by each child at the time of admission. C. jejuni and C. coli were detected in 16.4 % (60/366) and 1.4 % (5/366) of the diarrhoeal samples, respectively, by PCR, a much higher proportion than that detected by conventional methods. C. jejuni VAGs were detected in the following proportions of hipO-positive samples: ciaB, 95 % (57/60); dnaJ, 86.7 % (52/60); racR, 98.3 % (59/60); flaA, 80 % (48/60); pldA, 45 % (27/60); cdtABC, 95 % (57/60); and pVir 0 % (0/ 60). Particular symptoms, such as blood in faeces, vomiting, fever, and/or abdominal pain, were not associated with detection of C. jejuni nor were they associated with any particular VAG or combination of VAGs (P.0.05). C. jejuni and its VAGs were detected in a substantial proportion of the children admitted. Further efforts shall be directed towards elucidating whether these genetic factors or their expressed proteins play a role in Campylobacter pathogenesis. INTRODUCTIONThe genus Campylobacter, a group of thermotolerant, microaerophilic, Gram-negative bacteria, includes a number of pathogens that primarily cause gastrointestinal disease in humans, particularly Campylobacter jejuni and Campylobacter coli. Campylobacter-associated gastroenteritis is thought to occur through zoonotic transmission, being acquired from exposure to tainted food and/or contaminated drinking water (Sherman et al., 2010). Campylobacter infection frequently presents as self-limiting acute enteritis with diarrhoea, malaise, fever and abdominal pain, sometimes with vomiting and the presence of blood in faeces (Allos, 2001); disruption of epithelial cells and inflammation of the intestinal mucosa are hallmark features of severe cases (Beltinger et al., 2008). C. jejuni and C. coli cause significant morbidity worldwide, especially in children (Amieva, 2005;Tam et al., 2003;Wang et al., 2008; Fernández et al., 2008).Adherence and colonization are crucial steps in the pathogenesis of C. jejuni. Flagella have a major role in invasion; markedly reduced internalization in vitro has been reported with flaA 2 C. jejuni mutants (Wassenaar, 1997). The genes racR and dnaJ are determinants for C. jejuni colonization and are presumably expressed in response to conditions encountered in the intestinal microenviroment, such as differences in temperature between environmental reservoirs and human intestines (Konkel et al., 1998; Brás et al., 1999 antigen B protein, which confers invasive properties, as shown by C. jejuni ciaB null mutants which display a significant reduction in internalization (Konkel et al., 1999). Also, ...

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Section: Methodsmentioning

confidence: 99%

Campylobacter jejuni infection and virulence-associated genes in children with moderate to severe diarrhoea admitted to emergency rooms in northeastern Brazil

Quetz

Lima

Havt

et al. 2012

Journal of Medical Microbiology

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“…100 Work developing a multiplexed PCR system for bacteria was announced in 2006, with promising early results. 101 In 2010, Ramalingam and colleagues presented the simultaneous detection of four waterborne bacteria in a PDMS PCR array, which utilized capillary flow for sample loading. 102 In 2012, Agrawal and coworkers showed simultaneous detection of E. coli and S. tyhimurium on a circular microfluidics design manufactured in PDMS using magnetic nanoparticles to control and concentrate samples.…”

Section: Waterborne Pathogensmentioning

confidence: 99%

Miniaturized Detection Systems

Bridle¹

2014

Waterborne Pathogens

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“…The amplification step is usually achieved through one of a number of nucleic acid amplification methods (e.g., PCR, NASBA, TMA, LAMP, etc) all of which require that the reaction vessel be heated at a carefully maintained temperature(s). Despite several interesting attempts to develop small, portable NA assays, [1,2] the vast majority remain lab-based and require complex sample preparation as well as an instrument for heating (and/or heatcycling) and detection. Development of a NA assay platform that requires absolutely no instrument, power, or external reagents now appears possible.…”

Section: Malaria and Other Organisms With Dna Genomesmentioning

confidence: 99%

Non-instrumented nucleic acid amplification assay

et al. 2008

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We have developed components of a diagnostic disposable platform that has the dual purpose of providing molecular diagnostics at the point of care (POC) as well as stabilizing specimens for further analysis via a centralized surveillance system. This diagnostic is targeted for use in low-resource settings by minimally trained health workers. The disposable device does not require any additional instrumentation and will be almost as rapid and simple to use as a lateral flow strip test-yet will offer the sensitivity and specificity of nucleic acid amplification tests (NAATs). The low-cost integrated device is composed of three functional components: (1) a sample-processing subunit that generates clean and stabilized DNA from raw samples containing nucleic acids, (2) a NA amplification subunit, and (3) visual amplicon detection sub-unit. The device integrates chemical exothermic heating, temperature stabilization using phase-change materials, and isothermal nucleic acid amplification. The aim of developing this system is to provide pathogen detection with NAAT-level sensitivity in low-resource settings where there is no access to instrumentation. If a disease occurs, patients would be tested with the disposable in the field. A nucleic acid sample would be preserved within the spent disposable which could be sent to a central laboratory facility for further analysis if needed.

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Fully integrated multiplexed lab-on-a-card assay for enteric pathogens

Cited by 19 publications

References 12 publications

Campylobacter jejuni infection and virulence-associated genes in children with moderate to severe diarrhoea admitted to emergency rooms in northeastern Brazil

Campylobacter jejuni infection and virulence-associated genes in children with moderate to severe diarrhoea admitted to emergency rooms in northeastern Brazil

Miniaturized Detection Systems

Non-instrumented nucleic acid amplification assay

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