Integrated Micropillar Polydimethylsiloxane Accurate CRISPR Detection (IMPACT) System for Rapid Viral DNA Sensing

ABSTRACTA fully Integrated Micropillar Polydimethylsiloxane Accurate CRISPR Detection (IMPACT) system is developed for viral DNA detection. This powerful system is patterned with high-aspect ratio micropillars to enhance reporter probe binding. After surface modification and probe immobilization, CRISPR Cas12a/crRNA complex is injected into the fully enclosed system. With the presence of double-stranded DNA target, the CRISPR enzyme is activated and non-specifically cleaves the ssDNA reporters initially immobilized on the micropillars. This collateral cleavage releases fluorescence dyes into the assay, and the intensity is linearly proportional to the target DNA concentration ranging from 0.1 to 10 nM. Importantly, this system does not rely on traditional dye-quencher labeled probe thus eliminating the fluorescence background presented in the assay. Furthermore, our one-step detection protocol is performed at isothermal conditions (37°C) without using complicated and time-consuming off-chip probe hybridization and denaturation. This miniaturized and fully packed IMPACT chip demonstrates rapid, sensitive, and simple nucleic acid detection and is an ideal candidate for the next generation molecular diagnostic platform for point-of-care (POC) applications, responding to emerging and deadly pathogen outbreaks.

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A fully integrated paperfluidic molecular diagnostic chip for the extraction, amplification, and detection of nucleic acids from clinical samples

Lab on a Chip ◽

10.1039/c5lc01392e ◽

2016 ◽

Vol 16 (4) ◽

pp. 753-763 ◽

Cited By ~ 124

Author(s):

Natalia M. Rodriguez ◽

Winnie S. Wong ◽

Lena Liu ◽

Rajan Dewar ◽

Catherine M. Klapperich

Keyword(s):

Nucleic Acids ◽

Point Of Care ◽

Low Cost ◽

Molecular Diagnostic ◽

Clinical Samples ◽

Fully Integrated

We present a low-cost, disposable, and fully-integrated paperfluidic molecular diagnostic chip for sample-to-result functionality at the point-of-care.

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Prototype Foamy Virus Integrase Displays Unique Biochemical Activities among Retroviral Integrases

Biomolecules ◽

10.3390/biom11121910 ◽

2021 ◽

Vol 11 (12) ◽

pp. 1910

Author(s):

Anthony J. Rabe ◽

Yow Yong Tan ◽

Ross C. Larue ◽

Kristine E. Yoder

Keyword(s):

Foamy Virus ◽

Inverse Correlation ◽

Divalent Metal ◽

Viral Dna ◽

Prototype Foamy Virus ◽

Dna Oligonucleotides ◽

Low Concentrations ◽

Target Dna ◽

One Step ◽

Hiv 1

Integrases of different retroviruses assemble as functional complexes with varying multimers of the protein. Retroviral integrases require a divalent metal cation to perform one-step transesterification catalysis. Tetrameric prototype foamy virus (PFV) intasomes assembled from purified integrase and viral DNA oligonucleotides were characterized for their activity in the presence of different cations. While most retroviral integrases are inactive in calcium, PFV intasomes appear to be uniquely capable of catalysis in calcium. The PFV intasomes also contrast with other retroviral integrases by displaying an inverse correlation of activity with increasing manganese beginning at relatively low concentrations. The intasomes were found to be significantly more active in the presence of chloride co-ions compared to acetate. While HIV-1 integrase appears to commit to a target DNA within 20 s, PFV intasomes do not commit to target DNA during their reaction lifetime. Together, these data highlight the unique biochemical activities of PFV integrase compared to other retroviral integrases.

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CRISPR-Cas12b-assisted nucleic acid detection platform

10.1101/362889 ◽

2018 ◽

Cited By ~ 6

Author(s):

Linxian Li ◽

Shiyuan Li ◽

Jin Wang

Keyword(s):

Nucleic Acid ◽

Molecular Diagnostic ◽

Nucleic Acid Detection ◽

Rna Detection ◽

Environmental Testing ◽

Cleavage Activity ◽

Target Nucleic Acid ◽

Reverse Transcription Step ◽

Diagnostic Technology ◽

One Step

AbstractRapid molecular diagnostic technology is very useful in many areas, including public health, environmental testing and criminal investigation. We recently showed that Cas12a had trans-cleavage activity upon collateral single-stranded DNA (ssDNA), with which the HOLMES platform (one-HOur Low-cost Multipurpose highly Efficient System) was developed. Here, we combine the thermophilic Cas12b, which also has the ssDNA trans-cleavage activity, with Loop-Mediated Isothermal Amplification (LAMP), and create HOLMESv2. In HOLMESv2, LAMP amplification and Cas12b trans-cleavage can be integrated into a one-step system with a constant temperature, which therefore brings much convenience in nucleic acid detection. Moreover, we also simplify the RNA detection procedures in HOLMESv2, using an RNA-dependent DNA polymerase for amplification and therefore omitting an extra reverse transcription step.One Sentence SummaryWe combine LAMP and Cas12b to develop HOLMESv2 for conveniently detecting target nucleic acid in a one-step approach.

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Among retroviral integrases prototype foamy virus integrase displays unique biochemical activities

10.1101/2021.11.17.469013 ◽

2021 ◽

Author(s):

Anthony J Rabe ◽

Yow Yong Tan ◽

Ross C Larue ◽

Kristine E Yoder

Keyword(s):

Foamy Virus ◽

Inverse Correlation ◽

Divalent Metal ◽

Viral Dna ◽

Prototype Foamy Virus ◽

Dna Oligonucleotides ◽

Low Concentrations ◽

Target Dna ◽

One Step ◽

Hiv 1

Integrase enzymes of different retroviruses assemble as functional complexes with varying multimers of the protein. Retroviral integrases require a divalent metal cation to perform one-step transesterification catalysis. Tetrameric prototype foamy virus (PFV) intasomes assembled from purified integrase and viral DNA oligonucleotides were characterized for their activity in the presence of different cations. While most retroviral integrases are inactive in calcium, PFV intasomes appear to be uniquely capable of catalysis in calcium. The PFV intasomes also contrast other retroviral integrases by displaying an inverse correlation of activity with increasing manganese beginning at relatively low concentrations. The intasomes were found to be significantly more active in the presence of chloride co-ions compared to acetate. While HIV-1 integrase appears to commit to a target DNA within 20 seconds, PFV intasomes do not commit to target DNA during their reaction lifetime. Together these data highlight the unique biochemical activities of PFV integrase compared to other retroviral integrases.

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Molecular diagnostic of toxigenic Corynebacterium diphtheriae strain by DNA sensor potentially suitable for electrochemical point-of-care diagnostic

Talanta ◽

10.1016/j.talanta.2021.122161 ◽

2021 ◽

Vol 227 ◽

pp. 122161

Author(s):

Kasper Marchlewicz ◽

Iga Ostrowska ◽

Sławomir Oszwałdowski ◽

Aleksandra Zasada ◽

Robert Ziółkowski ◽

...

Keyword(s):

Point Of Care ◽

Corynebacterium Diphtheriae ◽

Molecular Diagnostic ◽

Dna Sensor

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Reply: One-step nucleic acid amplification assay also predicts axillary lymph node status in breast cancer patients: Further molecular diagnostic evidence

European Journal of Cancer ◽

10.1016/j.ejca.2013.09.009 ◽

2013 ◽

Vol 49 (18) ◽

pp. 3947-3948

Author(s):

Tomo Osako ◽

Takuji Iwase ◽

Futoshi Akiyama

Keyword(s):

Breast Cancer ◽

Nucleic Acid Amplification ◽

Molecular Diagnostic ◽

Lymph Node Status ◽

Axillary Lymph ◽

Axillary Lymph Node Status ◽

Breast Cancer Patients ◽

Nucleic Acid Amplification Assay ◽

Amplification Assay ◽

One Step

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Rapid Point-of-Care Testing for SARS-CoV-2 Virus Nucleic Acid Detection by an Isothermal and Nonenzymatic Signal Amplification System Coupled with a Lateral Flow Immunoassay Strip

Sensors and Actuators B Chemical ◽

10.1016/j.snb.2021.129899 ◽

2021 ◽

pp. 129899

Author(s):

Mingyuan Zou ◽

Feiya Su ◽

Rui Zhang ◽

Xinglu Jiang ◽

Han Xiao ◽

...

Keyword(s):

Nucleic Acid ◽

Signal Amplification ◽

Point Of Care ◽

Lateral Flow ◽

Lateral Flow Immunoassay ◽

Nucleic Acid Detection ◽

Point Of Care Testing ◽

Amplification System ◽

Signal Amplification System

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Fecal viral DNA shedding following clinical panleukopenia virus infection in shelter kittens: a prospective, observational study

Journal of Feline Medicine and Surgery ◽

10.1177/1098612x211023056 ◽

2021 ◽

pp. 1098612X2110230

Author(s):

Kyrsten J Janke ◽

Linda S Jacobson ◽

Jolene A Giacinti ◽

J Scott Weese

Keyword(s):

Small Sample Size ◽

Point Of Care ◽

Small Sample ◽

Enteric Pathogens ◽

Test Results ◽

Viral Dna ◽

Virus Shedding ◽

Feline Panleukopenia Virus ◽

Viral Shedding ◽

Point Of Care Test

Objectives The aims of this study were to determine the magnitude and duration of fecal viral DNA shedding after diagnosis of feline panleukopenia (FP) in a group of shelter cats using quantitative real-time PCR (qPCR); to assess the utility of a negative point-of-care test or the resolution of diarrhea and systemic signs as proxy measures for qPCR positivity; and to investigate patterns of additional enteric pathogens in relation to feline panleukopenia viral shedding duration. Methods Feline panleukopenia virus (FPV) infection in clinically affected shelter cats was confirmed by a commercial qPCR test. Observations were made on days 0, 3, 7, 14 and 21 post-diagnosis. Fecal flotation, FPV qPCR and the canine parvovirus IDEXX SNAP Parvo ELISA (SNAP) test were performed on fecal samples. Results Forty cats and kittens with confirmed panleukopenia were initially enrolled. Sixteen kittens were sampled until day 14, and 12 were followed to day 21. Median DNA viral copy numbers fell below the diagnostic cut-off by day 7, with 13/16, 6/16, 1/16 and 0/12 testing PCR-positive on days 3, 7, 14 and 21, respectively. The SNAP test was positive in 12/16 kittens on day 0 and only 3/16 on day 3. SNAP test results, diarrhea and systemic signs were inconsistent in relation to qPCR positivity post-diagnosis. Additional enteric pathogens were common. The presence of additional pathogen types was suggestive of a longer PCR shedding duration, but this was not tested statistically owing to the small sample size. Conclusions and relevance These findings suggest that cats should be isolated for at least 14 days after a diagnosis of FP, but that release from isolation after this point is reasonable, in association with a multifaceted infection control strategy. The study findings did not support using SNAP test results, diarrhea or systemic signs as proxy measures for virus shedding.

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