Rapid, simple, inexpensive, accurate, and sensitive point-of-care (POC) detection of viral pathogens in bodily fluids is a vital component of controlling the spread of infectious diseases. The predominant laboratory-based methods for sample processing and nucleic acid detection face limitations that prevent them from gaining wide adoption for POC applications in low-resource settings and self-testing scenarios. Here, we report the design and characterization of an integrated system for rapid sample-to-answer detection of a viral pathogen in a droplet of whole blood comprised of a 2-stage microfluidic cartridge for sample processing and nucleic acid amplification, and a clip-on detection instrument that interfaces with the image sensor of a smartphone. The cartridge is designed to release viral RNA from Zika virus in whole blood using chemical lysis, followed by mixing with the assay buffer for performing reverse-transcriptase loop-mediated isothermal amplification (RT-LAMP) reactions in six parallel microfluidic compartments. The battery-powered handheld detection instrument uniformly heats the compartments from below, and an array of LEDs illuminates from above, while the generation of fluorescent reporters in the compartments is kinetically monitored by collecting a series of smartphone images. We characterize the assay time and detection limits for detecting Zika RNA and gamma ray-deactivated Zika virus spiked into buffer and whole blood and compare the performance of the same assay when conducted in conventional PCR tubes. Our approach for kinetic monitoring of the fluorescence-generating process in the microfluidic compartments enables spatial analysis of early fluorescent “bloom” events for positive samples, in an approach called “Spatial LAMP” (S-LAMP). We show that S-LAMP image analysis reduces the time required to designate an assay as a positive test, compared to conventional analysis of the average fluorescent intensity of the entire compartment. S-LAMP enables the RT-LAMP process to be as short as 22 minutes, resulting in a total sample-to-answer time in the range of 17–32 minutes to distinguish positive from negative samples, while demonstrating a viral RNA detection as low as 2.70 × 10 2 copies per μl, and a gamma-irradiated virus of 10 3 virus particles in a single 12.5 μl droplet blood sample.
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Analytical methods for detection of Zika virus
Due to the recent outbreak of the Zika virus (ZIKV) in several regions, rapid, and accurate methods to diagnose Zika infection are in demand, particularly in regions that are on the frontline of a ZIKV outbreak. In this paper, three diagnostic methods for ZIKV are considered. Viral isolation is the gold standard for detection; this approach can involve incubation of cell cultures. Serological identification is based on the interactions between viral antigens and immunoglobulin G or immunoglobulin M antibodies; cross-reactivity with other types of flaviviruses can cause reduced specificity with this approach. Molecular confirmation, such as reverse transcription polymerase chain reaction (RT–PCR), involves reverse transcription of RNA and amplification of DNA. Quantitative analysis based on real-time RT–PCR can be undertaken by comparing fluorescence measurements against previously developed standards. A recently developed programmable paper-based detection approach can provide low-cost and rapid analysis. These viral identification and viral genetic analysis approaches play crucial roles in understanding the transmission of ZIKV.
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- Award ID(s):
- 1651359
- NSF-PAR ID:
- 10025583
- Date Published:
- Journal Name:
- MRS Communications
- ISSN:
- 2159-6859
- Page Range / eLocation ID:
- 1 to 10
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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- Free Publicly Accessible Full Text
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Accepted Manuscript1.0
- Journal Article:
- https://doi.org/10.1557/mrc.2017.20
