Simultaneous Detection of Different Zika Virus Lineages via Molecular Computation in a Point-of-Care Assay

Bhadra, Sanchita; Saldaña, Miguel A.; Han, Hannah Grace; Hughes, Grant L.; Ellington, Andrew D.

doi:10.3390/v10120714

Cited by 15 publications

(14 citation statements)

References 73 publications

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“…Base-pairing to the toehold region is extremely sensitive to mismatches, ensuring specificity, and the programmability of both primers and probes makes possible rapid adaptation to new diseases or new disease variants. We have shown that higher order molecular information processing is also possible, such as integration of signals from multiple amplicons 17 . Overall, the use of sequence-specific probes allow construction of strand exchange computation circuits that act as ‘matter computers’, 5 – 8 something that is not generally possible within the context of a PCR reaction (which would of necessity melt the computational devices).…”

Section: Introductionmentioning

confidence: 99%

High-surety isothermal amplification and detection of SARS-CoV-2, including with crude enzymes

Bhadra

Lakhotia

et al. 2020

Preprint

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ABSTRACTIsothermal nucleic acid amplification tests (iNAT), such as loop-mediated isothermal amplification (LAMP), are good alternatives to polymerase chain reaction (PCR)-based amplification assays, especially for point-of-care and low resource use, in part because they can be carried out with relatively simple instrumentation. However, iNATs can generate spurious amplicons, especially in the absence of target sequences, resulting in false positive results. This is especially true if signals are based on non-sequence-specific probes, such as intercalating dyes or pH changes. In addition, pathogens often prove to be moving, evolving targets, and can accumulate mutations that will lead to inefficient primer binding and thus false negative results. Internally redundant assays targeting different regions of the target sequence can help to reduce such false negatives. Here we describe rapid conversion of three previously described SARS-CoV-2 LAMP assays that relied on non-sequence-specific readout into assays that can be visually read using sequence-specific fluorogenic oligonucleotide strand exchange (OSD) probes. We evaluate one-pot operation of both individual and multiplex LAMP-OSD assays and demonstrate detection of SARS-CoV-2 virions in crude human saliva.

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Section: Introductionmentioning

confidence: 99%

High-surety isothermal amplification and detection of SARS-CoV-2, including with crude enzymes

Bhadra

Lakhotia

et al. 2020

Preprint

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“…In this work, we developed a novel multiplex LAMP detection scheme to analyze multiple RNA targets simultaneously. Previous efforts to perform sequence-specific detection of LAMP products have relied on a branch migration-based strand displacement mechanism targeting the loop region of the LAMP dumbbell structure 28,29 . Our LAMP detection scheme also uses a strand displacement mechanism, but takes advantage of the LAMP reaction’s Bst polymerase to drive strand displacement using the 3’ end of the LAMP product as a primer.…”

Section: Discussionmentioning

confidence: 99%

Single-cell nucleic acid profiling in droplets (SNAPD) enables high-throughput analysis of heterogeneous cell populations

Hyman

Christopher

Romero

2020

Preprint

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Experimental methods that capture the individual properties of single cells are revealing the key role of cell-to-cell variability in countless biological processes. These single-cell methods are becoming increasingly important across the life sciences in fields such as immunology, regenerative medicine, and cancer biology. Existing single-cell analysis methods are often limited by their low analysis throughput, their inability to profile high-dimensional phenotypes, and complicated experimental workflows with slow turnaround times. In this work, we present Single-cell Nucleic Acid Profiling in Droplets (SNAPD) to analyze the transcriptional states of hundreds of thousands of single mammalian cells. Individual cells are encapsulated in aqueous droplets on a microfluidic chip and the content of each cell is profiled by amplifying a targeted panel of transcriptional markers. Molecular logic circuits then integrate this multi-dimensional information to categorize cells based on their transcriptional profile and produce a detectable fluorescence output. SNAPD analyzes over 100,000 cells per hour and can be used to quantify distinct cell types within populations, detect rare cells at frequencies down to 0.1%, and enrich specific cell types using microfluidic sorting. SNAPD provides a simple, rapid, low cost, and scalable approach to study complex phenotypes in heterogeneous cell populations.

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“…Other systems utilized fluorescently labeled oligonucleotide strand displacement probes that are incorporated into LAMP amplicons ( Bhadra et al, 2015 , 2018 ; Jiang et al, 2017 ; Priye et al, 2017 ). In an RT-LAMP assay for the multiplexed detection of Zika, dengue, and chikungunya viruses in urine samples, probes for each virus were labeled with a different dye to produce a color-coded fluorescent readout ( Yaren et al, 2017 ).…”

Section: Loop-mediated Isothermal Amplificationmentioning

confidence: 99%

Developments in integrating nucleic acid isothermal amplification and detection systems for point-of-care diagnostics

Pumford

Spaczai

et al. 2020

Biosensors and Bioelectronics

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Early disease detection through point-of-care (POC) testing is vital for quickly treating patients and preventing the spread of harmful pathogens. Disease diagnosis is generally accomplished using quantitative polymerase chain reaction (qPCR) to amplify nucleic acids in patient samples, permitting detection even at low target concentrations. However, qPCR requires expensive equipment, trained personnel, and significant time. These resources are not available in POC settings, driving researchers to instead utilize isothermal amplification, conducted at a single temperature, as an alternative. Common isothermal amplification methods include loop-mediated isothermal amplification, recombinase polymerase amplification, rolling circle amplification, nucleic acid sequence-based amplification, and helicase-dependent amplification. There has been a growing interest in combining such amplification methods with POC detection methods to enable the development of diagnostic tests that are well suited for resource-limited settings as well as developed countries performing mass screenings. Exciting developments have been made in the integration of these two research areas due to the significant impact that such approaches can have on healthcare. This review will primarily focus on advances made by North American research groups between 2015 and June 2020, and will emphasize integrated approaches that reduce user steps, reliance on expensive equipment, and the system's time-to-result.

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Simultaneous Detection of Different Zika Virus Lineages via Molecular Computation in a Point-of-Care Assay

Cited by 15 publications

References 73 publications

High-surety isothermal amplification and detection of SARS-CoV-2, including with crude enzymes

High-surety isothermal amplification and detection of SARS-CoV-2, including with crude enzymes

Single-cell nucleic acid profiling in droplets (SNAPD) enables high-throughput analysis of heterogeneous cell populations

Developments in integrating nucleic acid isothermal amplification and detection systems for point-of-care diagnostics

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