In-Gel Technology for PCR Genotyping and Pathogen Detection

Atrazhev, Alexey; Manage, Dammika P.; Stickel, Alexander J.; Crabtree, H. John; Pilarski, Linda M.; Acker, Jason P.

doi:10.1021/ac1013157

Cited by 29 publications

(37 citation statements)

References 33 publications

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“…In addition to the practical use of this assay for the qualitative analysis of field samples, the ability to perform real-time PCR for malaria directly from blood can be exploited in new technologies to bring molecular diagnostics for malaria to the point-of-care [18]. A number of technologies have been reported but they generally require an upstream module for DNA extraction [3,19,20].…”

Section: Discussionmentioning

confidence: 99%

Real-time PCR detection of Plasmodium directly from whole blood and filter paper samples

Taylor

Martin

Arango

et al. 2011

Malar J

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BackgroundReal-time PCR is a sensitive and specific method for the analysis of Plasmodium DNA. However, prior purification of genomic DNA from blood is necessary since PCR inhibitors and quenching of fluorophores from blood prevent efficient amplification and detection of PCR products.MethodsReagents designed to specifically overcome PCR inhibition and quenching of fluorescence were evaluated for real-time PCR amplification of Plasmodium DNA directly from blood. Whole blood from clinical samples and dried blood spots collected in the field in Colombia were tested.ResultsAmplification and fluorescence detection by real-time PCR were optimal with 40× SYBR® Green dye and 5% blood volume in the PCR reaction. Plasmodium DNA was detected directly from both whole blood and dried blood spots from clinical samples. The sensitivity and specificity ranged from 93-100% compared with PCR performed on purified Plasmodium DNA.ConclusionsThe methodology described facilitates high-throughput testing of blood samples collected in the field by fluorescence-based real-time PCR. This method can be applied to a broad range of clinical studies with the advantages of immediate sample testing, lower experimental costs and time-savings.

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Section: Discussionmentioning

confidence: 99%

Real-time PCR detection of Plasmodium directly from whole blood and filter paper samples

Taylor

Martin

Arango

et al. 2011

Malar J

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“…In previous studies “hydrogel” PCR technology was developed to package PCR reagents in a format that can be stored and is easy to use [23]. This included the creation of a customized field-ready real-time PCR machine, the ‘Gelcycler’.…”

Section: Resultsmentioning

confidence: 99%

“…Chips were stored in dark conditions to prevent photo-bleaching of SYBR green dye. Coverslips were treated with 3-(trimethoxysilyl)propyl methacrylate to promote adherence to the gel strip as described [23]. On desiccation, the gel shrinks away from the trench and stays attached to the coverslip, creating a channel underneath the strip where the sample can diffuse in.…”

Section: Methodsmentioning

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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“…Recent developments in PCR using CCD and in-gel technologies to analyze the results provide extended advantages to conventional PCR [36], yet detecting only DNA and RNA markers remains the major limitation of PCR-based techniques. The other advantage of LG-TIRF-MB when compared to PCR-based methods is that results are obtained in seconds, whereas PCR methods may require hours.…”

Section: Discussionmentioning

confidence: 99%

A Platform for Combined DNA and Protein Microarrays Based on Total Internal Reflection Fluorescence

Asanov¹,

Zepeda

Vaca

2012

Sensors

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We have developed a novel microarray technology based on total internal reflection fluorescence (TIRF) in combination with DNA and protein bioassays immobilized at the TIRF surface. Unlike conventional microarrays that exhibit reduced signal-to-background ratio, require several stages of incubation, rinsing and stringency control, and measure only end-point results, our TIRF microarray technology provides several orders of magnitude better signal-to-background ratio, performs analysis rapidly in one step, and measures the entire course of association and dissociation kinetics between target DNA and protein molecules and the bioassays. In many practical cases detection of only DNA or protein markers alone does not provide the necessary accuracy for diagnosing a disease or detecting a pathogen. Here we describe TIRF microarrays that detect DNA and protein markers simultaneously, which reduces the probabilities of false responses. Supersensitive and multiplexed TIRF DNA and protein microarray technology may provide a platform for accurate diagnosis or enhanced research studies. Our TIRF microarray system can be mounted on upright or inverted microscopes or interfaced directly with CCD cameras equipped with a single objective, facilitating the development of portable devices. As proof-of-concept we applied TIRF microarrays for detecting molecular markers from Bacillus anthracis, the pathogen responsible for anthrax.

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In-Gel Technology for PCR Genotyping and Pathogen Detection

Cited by 29 publications

References 33 publications

Real-time PCR detection of Plasmodium directly from whole blood and filter paper samples

Real-time PCR detection of Plasmodium directly from whole blood and filter paper samples

A lab-on-chip for malaria diagnosis and surveillance

A Platform for Combined DNA and Protein Microarrays Based on Total Internal Reflection Fluorescence

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