A thermophilic Cas13 enzyme for sensitive and specific one-pot detection of SARS-CoV-2

Abstract Robust, sensitive, and specific diagnostic platforms for early pathogen detection are essential for the identification of infected patients and management of current and future pandemics. CRISPR-Cas systems have been repurposed for SARS-CoV-2 detection in two-pot assays. Two-pot assays require extra steps and are prone to cross-contamination; however, the temperature range of current Cas enzymes limits the development of one-pot assays Here, we characterized TccCas13a, a thermophilic Cas13a enzyme with cis and trans activities from 37–70°C, and HheCas13a, which had a limited range and lower activity. We harnessed TccCas13a in a one-pot SARS-CoV-2 assay with two layers of amplification and TccCas13a-mediated collateral degradation of a single-stranded RNA reporter. This assay showed 95% sensitivity and 100% specificity compared with RT-qPCR on clinical samples. We also developed a mobile phone application to facilitate data reading, collection, and sharing. Our OPTIMA-dx detection module exhibits key features for point-of-care SARS-CoV-2 screening and pathogen detection in general.

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Innovations in Worksite Diagnosis of Urinary Tract Infections and the Occupational Health Nurse

Workplace Health & Safety ◽

10.1177/2165079919834310 ◽

2019 ◽

Vol 67 (6) ◽

pp. 268-274 ◽

Cited By ~ 2

Author(s):

Louise C. O’Keefe ◽

Paula Koelle ◽

Zac McGee ◽

L. Savannah Dewberry ◽

Carter Wright ◽

...

Keyword(s):

Urinary Tract ◽

Occupational Health ◽

Validation Study ◽

Pathogen Detection ◽

Point Of Care ◽

Health Clinic ◽

Clinical Samples ◽

Lost Work Time ◽

Tract Infections

Occupational health nurses play a key role in evaluating innovative technologies that can aid in providing safe and rapid care and reduce lost work time. A nurse-led employee health clinic participated in a validation study of a novel pathogen detection technique developed by GeneCapture, Inc. Their proposed portable urinary tract infection (UTI) in vitro diagnostic test was challenged with discarded, deidentified urine samples from patients presenting with typical UTI symptoms collected at two university clinics and two multiphysician practices. GeneCapture’s panel for this study was designed to rapidly identify the genetic signature of seven organisms: gram-negative Escherichia coli, Klebsiella pneumoniae, Proteus mirabilis, and Pseudomonas aeruginosa; gram-positive Enterococcus faecalis and Staphylococcus aureus; and fungal Candida species. The results from 40 clinical samples were in 95% agreement (90% specificity, 100% sensitivity) with traditional urine culture results from routine analysis. This successful occupational health nursing collaboration and validation study shows promise for point-of-care diagnoses and earlier treatment for workers with UTIs.

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Low-Cost Manually Assembled Open Source Reader for Isothermal Pathogen Detection from Saliva using RT-LAMP: SARS-CoV-2 Use Case

10.1101/2020.10.19.20215319 ◽

2020 ◽

Author(s):

John C. Bramley ◽

Jason E. Waligorski ◽

Colin L. Kremitzki ◽

Mariel J. Liebeskind ◽

Alex L. Yenkin ◽

...

Keyword(s):

Pathogen Detection ◽

Limit Of Detection ◽

Point Of Care ◽

Low Cost ◽

One Pot ◽

Home Testing ◽

Wide Range ◽

Platform Technology ◽

Crude Sample ◽

Analytical Limit

AbstractDistributed “Point-of-Care” or “at-Home” testing is an important component for a complete suite of testing solutions. This manuscript describes the construction and operation of a platform technology designed to meet this need. The ongoing COVID-19 pandemic will be used as the proof-of-concept for the efficacy and deployment of this platform. The technology outlined consists of a one-pot, reverse-transcription loop-mediated isothermal amplification (RT-LAMP) chemistry coupled with a low-cost and user-assembled reader using saliva as input. This platform is readily adapted to a wide range of pathogens due to the genetic basis of the reaction. A complete guide to the construction of the reader as well as the production of the reaction chemistry are provided here. Additionally, analytical limit of detection data and the results from saliva testing of SARS-CoV-2, are presented. The platform technology outlined here demonstrates a rapid, distributed, molecular point-of-care solution for pathogen detection using crude sample input.

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Direct detection of SARS-CoV-2 using CRISPR-Cas13a and a mobile phone

10.1101/2020.09.28.20201947 ◽

2020 ◽

Cited By ~ 7

Author(s):

Parinaz Fozouni ◽

Sungmin Son ◽

María Díaz de León Derby ◽

Gavin J Knott ◽

Carley N Gray ◽

...

Keyword(s):

Mobile Phone ◽

Nasal Swab ◽

Respiratory Virus ◽

Direct Detection ◽

Point Of Care ◽

Low Cost ◽

Clinical Samples ◽

Dna Targeting ◽

Global Pandemic ◽

Rna And Dna

The December 2019 outbreak of a novel respiratory virus, SARS-CoV-2, has become an ongoing global pandemic due in part to the challenge of identifying symptomatic, asymptomatic and pre- symptomatic carriers of the virus. CRISPR-based diagnostics that utilize RNA and DNA-targeting enzymes can augment gold-standard PCR-based testing if they can be made rapid, portable and accurate. Here we report the development of an amplification-free CRISPR-Cas13a-based mobile phone assay for direct detection of SARS-CoV-2 from nasal swab RNA extracts. The assay achieved ~100 copies/μL sensitivity in under 30 minutes and accurately detected a set of positive clinical samples in under 5 minutes. We combined crRNAs targeting SARS-CoV-2 RNA to improve sensitivity and specificity, and we directly quantified viral load using enzyme kinetics. Combined with mobile phone-based quantification, this assay can provide rapid, low-cost, point-of-care screening to aid in the control of SARS-CoV-2.

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Microfluidics-Enabled Digital Isothermal Cas13a Assay

10.1101/2021.08.18.21262201 ◽

2021 ◽

Author(s):

Frank X. Liu ◽

Johnson Q. Cui ◽

Hojeong Park ◽

Ka Wai Chan ◽

Tyler Leung ◽

...

Keyword(s):

Point Of Care ◽

Absolute Quantification ◽

Droplet Microfluidics ◽

Clinical Samples ◽

Quantitative Detection ◽

One Pot ◽

Viral Loads ◽

Assay Optimization ◽

Point Of Care Diagnostics ◽

Digital Format

AbstractThe isothermal molecular diagnosis with CRISPR has attracted particular interest for the sensitive, specific detection of nucleic acids. However, most of the assays with Cas enzymes were performed in bulk assays using multistep approaches and hard to realize quantitative detection. Herein, we report Microfluidics-Enabled Digital Isothermal Cas13a Assay (MEDICA), a digital format of SHERLOCK with enhanced robustness and sensitivity. We first address the macromolecular crowding problems when combining the recombinase polymerase amplification (RPA) and Cas13a detection into a one-pot SHERLOCK assay. After the assay optimization, the enhanced one-pot SHERLOCK (E-SHERLOCK) achieves high robustness and 200-fold increased sensitivity. Leveraging droplet microfluidics, we streamline the E-SHERLOCK to eliminate undesired input targets caused by pre-amplification before partition, enabling background-free absolute quantification. From the real-time monitoring, MEDICA enables qualitative detection within 10 min and absolute quantification within 25 min. For the proof of concept, we applied MEDICA to quantify HPV 16 and 18 viral loads in 44 clinical samples, indicating perfect accordance with qPCR results. MEDICA highlights the CRISPR-based isothermal assays are promising for the next generation of point-of-care diagnostics.

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Ultrasensitive and visual detection of SARS-CoV-2 using all-in-one dual CRISPR-Cas12a assay

Nature Communications ◽

10.1038/s41467-020-18575-6 ◽

2020 ◽

Vol 11 (1) ◽

Cited By ~ 13

Author(s):

Xiong Ding ◽

Kun Yin ◽

Ziyue Li ◽

Rajesh V. Lalla ◽

Enrique Ballesteros ◽

...

Keyword(s):

Point Of Care ◽

Low Cost ◽

Disease Spread ◽

Clinical Samples ◽

Pcr Assay ◽

One Pot ◽

Nucleoprotein Gene ◽

Protospacer Adjacent Motif ◽

Recent Outbreak ◽

Novel Coronavirus

Abstract The recent outbreak of novel coronavirus (SARS-CoV-2) causing COVID-19 disease spreads rapidly in the world. Rapid and early detection of SARS-CoV-2 facilitates early intervention and prevents the disease spread. Here, we present an All-In-One Dual CRISPR-Cas12a (AIOD-CRISPR) assay for one-pot, ultrasensitive, and visual SARS-CoV-2 detection. By targeting SARS-CoV-2’s nucleoprotein gene, two CRISPR RNAs without protospacer adjacent motif (PAM) site limitation are introduced to develop the AIOD-CRISPR assay and detect the nucleic acids with a sensitivity of few copies. We validate the assay by using COVID-19 clinical swab samples and obtain consistent results with RT-PCR assay. Furthermore, a low-cost hand warmer (~$0.3) is used as an incubator of the AIOD-CRISPR assay to detect clinical samples within 20 min, enabling an instrument-free, visual SARS-CoV-2 detection at the point of care. Thus, our method has the significant potential to provide a rapid, sensitive, one-pot point-of-care assay for SARS-CoV-2.

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Development and evaluation of rapid novel isothermal amplification assays for important veterinary pathogens:Chlamydia psittaciandChlamydia pecorum

PeerJ ◽

10.7717/peerj.3799 ◽

2017 ◽

Vol 5 ◽

pp. e3799 ◽

Cited By ~ 26

Author(s):

Martina Jelocnik ◽

Md. Mominul Islam ◽

Danielle Madden ◽

Cheryl Jenkins ◽

James Branley ◽

...

Keyword(s):

Real Time ◽

Pathogen Detection ◽

Point Of Care ◽

Chlamydia Psittaci ◽

Isothermal Amplification ◽

Diagnostic Tools ◽

Clinical Samples ◽

Conserved Gene ◽

Thermal Cycler ◽

Species Specific

BackgroundChlamydia psittaciandChlamydia pecorumare important veterinary pathogens, with the former also being responsible for zoonoses, and the latter adversely affecting koala populations in Australia and livestock globally. The rapid detection of these organisms is still challenging, particularly at the point-of-care (POC). In the present study, we developed and evaluated rapid, sensitive and robustC. psittaci-specific andC. pecorum-specific Loop Mediated Isothermal Amplification (LAMP) assays for detection of these pathogens.Methods and MaterialsThe LAMP assays, performed in a Genie III real-time fluorometer, targeted a 263 bp region of theC. psittaci-specific Cps_0607 gene or a 209 bp region of aC. pecorum-specific conserved gene CpecG_0573, and were evaluated using a range of samples previously screened using species-specific quantitative PCRs (qPCRs). Species-specificity forC. psittaciandC. pecorumLAMP targets was tested against DNA samples from related chlamydial species and a range of other bacteria. In order to evaluate pathogen detection in clinical samples,C. psittaciLAMP was evaluated using a total of 26 DNA extracts from clinical samples from equine and avian hosts, while forC. pecorumLAMP, we tested a total of 63 DNA extracts from clinical samples from koala, sheep and cattle hosts. A subset of 36C. pecorumsamples was also tested in a thermal cycler (instead of a real-time fluorometer) using newly developed LAMP and results were determined as an end point detection. We also evaluated rapid swab processing (without DNA extraction) to assess the robustness of these assays.ResultsBoth LAMP assays were demonstrated to species-specific, highly reproducible and to be able to detect as little as 10 genome copy number/reaction, with a mean amplification time of 14 and 24 min forC. psittaciandC. pecorum, respectively. When testing clinical samples, the overall congruence between the newly developed LAMP assays and qPCR was 92.3% forC. psittaci(91.7% sensitivity and 92.9% specificity); and 84.1% forC. pecorum(90.6% sensitivity and 77.4% specificity). For a subset of 36C. pecorumsamples tested in a thermal cycler using newly developed LAMP, we observed 34/36 (94.4%) samples result being congruent between LAMP performed in fluorometer and in thermal cycler. Rapid swab processing method evaluated in this study also allows for chlamydial DNA detection using LAMP.DiscussionIn this study, we describe the development of novel, rapid and robustC. psittaci-specific andC. pecorum-specific LAMP assays that are able to detect these bacteria in clinical samples in either the laboratory or POC settings. With further development and a focus on the preparation of these assays at the POC, it is anticipated that both tests may fill an important niche in the repertoire of ancillary diagnostic tools available to clinicians.

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