Single- and Two-Stage, Closed-Tube, Point-of-Care, Molecular Detection of SARS-CoV-2

<p>The 2019 novel coronavirus (COVID-19) is a newly emerged strain that has never been found in humans before. At present, the laboratory-based reverse transcription-polymerase chain reaction (RT-PCR) is the main method to confirm COVID-19 infection. The intensification of the COVID-19 epidemic overwhelms limited clinical resources in particular, but not only, in developing countries, resulting in many patients not being tested for the infection and in large queues of potentially infected individuals waiting to be tested while providing a breeding ground for the disease. We describe here a rapid, highly sensitive, point-of-care, molecular test amenable for use at home, in the clinic, and at points of entry by minimally trained individuals and with minimal instrumentation. Our test is based on loop mediated isothermal amplification (COVID-19 LAMP) and for higher sensitivity on nested nucleic acid, two stage isothermal amplification (COVID-19 Penn-RAMP). Both tests can be carried out in closed tubes with either fluorescence or colorimetric (e.g., leuco crystal violet LCV) detection. COVID-19 LAMP performs on par with COVID-19 RT-PCR. COVID-19 RAMP has 10 fold better sensitivity than COVID-19 LAMP and COVID-19 RT-PCR when testing purified targets and 100 times better sensitivity than COVID-19 LAMP and COVID-19 RT-PCR when testing rapidly prepared sample mimics. Due to fortunate scarcity of COVID-19 infections in the USA, we were not able to test our assays and methods with patient samples. We hope that such tests will be carried out by colleagues in impacted countries. Our Closed-Tube Penn-RAMP has the potential to significantly reduce false negatives while being amenable to use with minimal instrumentation and training. </p>

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A Single and Two-Stage, Closed-Tube, Molecular Test for the 2019 Novel Coronavirus (COVID-19) at Home, Clinic, and Points of Entry

10.26434/chemrxiv.11860137.v1 ◽

2020 ◽

Cited By ~ 28

Author(s):

Mohamed El-Tholotha ◽

Haim H. Bau ◽

Jinzhao Song

Keyword(s):

Point Of Care ◽

Isothermal Amplification ◽

Molecular Test ◽

Rt Pcr ◽

Two Stage ◽

Highly Sensitive ◽

Closed Tube ◽

The Usa ◽

Novel Coronavirus ◽

At Home

<p>The 2019 novel coronavirus (COVID-19) is a newly emerged strain that has never been found in humans before. At present, the laboratory-based reverse transcription-polymerase chain reaction (RT-PCR) is the main method to confirm COVID-19 infection. The intensification of the COVID-19 epidemic overwhelms limited clinical resources in particular, but not only, in developing countries, resulting in many patients not being tested for the infection and in large queues of potentially infected individuals waiting to be tested while providing a breeding ground for the disease. We describe here a rapid, highly sensitive, point-of-care, molecular test amenable for use at home, in the clinic, and at points of entry by minimally trained individuals and with minimal instrumentation. Our test is based on loop mediated isothermal amplification (COVID-19 LAMP) and for higher sensitivity on nested nucleic acid, two stage isothermal amplification (COVID-19 Penn-RAMP). Both tests can be carried out in closed tubes with either fluorescence or colorimetric (e.g., leuco crystal violet LCV) detection. COVID-19 LAMP performs on par with COVID-19 RT-PCR. COVID-19 RAMP has 10 fold better sensitivity than COVID-19 LAMP and COVID-19 RT-PCR when testing purified targets and 100 times better sensitivity than COVID-19 LAMP and COVID-19 RT-PCR when testing rapidly prepared sample mimics. Due to fortunate scarcity of COVID-19 infections in the USA, we were not able to test our assays and methods with patient samples. We hope that such tests will be carried out by colleagues in impacted countries. Our Closed-Tube Penn-RAMP has the potential to significantly reduce false negatives while being amenable to use with minimal instrumentation and training. </p>

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Aptamer Based Point of Care Diagnostic for the Detection of Food Allergens

10.21203/rs.3.rs-1060991/v1 ◽

2021 ◽

Author(s):

Sarah Stidham ◽

Valerie Villareal ◽

Vasant Chellappa ◽

Lucas Yoder ◽

Olivia Alley ◽

...

Keyword(s):

Molecular Detection ◽

Detection Method ◽

Point Of Care ◽

Protein Detection ◽

Peanut Protein ◽

The Novel ◽

Children And Families ◽

Peanut Allergens ◽

Consumer Device ◽

Detection Technologies

Abstract Aptamers, due to their small size, strong target affinity, and ease of chemical modification, are ideally suited for molecular detection technologies. Here, we describe successful use of aptamer technology in a consumer device for the detection of peanut antigen in food. The novel aptamer-based protein detection method is robust across a wide variety of food matrices and sensitive to peanut protein at concentrations as low as 12.5 ppm (37.5 µg peanut protein in the sample). Integration of the assay into a sensitive, stable, and consumer friendly portable device will empower users to easily and quickly assess the presence of peanut allergens in foods before eating. With most food reactions occurring outside the home, the type of technology described here has significant potential to improve lives for children and families.

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Molecular detection and point-of-care testing in Ebola virus disease and other threats: a new global public health framework to stop outbreaks

Expert Review of Molecular Diagnostics ◽

10.1586/14737159.2015.1079776 ◽

2015 ◽

Vol 15 (10) ◽

pp. 1245-1259 ◽

Cited By ~ 16

Author(s):

Gerald J Kost ◽

William Ferguson ◽

Anh-Thu Truong ◽

Jackie Hoe ◽

Daisy Prom ◽

...

Keyword(s):

Public Health ◽

Ebola Virus ◽

Molecular Detection ◽

Virus Disease ◽

Point Of Care ◽

Ebola Virus Disease ◽

Global Public Health ◽

Point Of Care Testing

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Molecular beacon assay development for Severe Acute Respiratory Syndrome Coronavirus 2 detection

10.22541/au.161652048.85831942/v1 ◽

2021 ◽

Author(s):

Josué Carvalho ◽

Jéssica Lopes Nunes ◽

Joana Figueiredo ◽

Tiago Santos ◽

André Miranda ◽

...

Keyword(s):

Molecular Beacon ◽

Point Of Care ◽

Total Duration ◽

Fluorescence Analysis ◽

Population Level ◽

Clinical Samples ◽

Test Duration ◽

Upper Respiratory Tract ◽

Testing Strategy ◽

Two Stage

The fast spread of SARS-CoV-2 has led to a global pandemic, calling for fast and accurate assays to allow infection diagnosis and prevention of transmission. We aimed to develop a molecular beacon (MB)-based detection assay for SARS-CoV-2, designed to, detect the ORF1ab and S genes, proposing a two-stage COVID-19 testing strategy, using MBs to detect the presence of target amplicons by fluorescence analysis. Two MBs were designed, optimized in terms of concentration, fluorescence plateaus of hybridization, reaction temperature and best real-time results. A total of 450 nasopharyngel and throat swab samples (418 positive and 32 negative) were tested with the MB assay and the fluorescence levels compared with the cycle threshold (Ct) values obtained from a commercial RT-PCR test in terms of test duration, sensitivity and specificity. Our results show that the samples with higher fluorescence levels correspond to those with low Ct values, suggesting a correlation between viral load and increased MB fluorescence. The proposed assay represents a fast (total duration of 2 h 20 min including amplification and fluorescence reading stages) and simple way of detecting SARS-CoV-2 in clinical samples from the upper respiratory tract. Our two-stage testing strategy is suitable for further development into a point-of-care assay and potentially scalable to population level.

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Microfluidic Chip with Two-Stage Isothermal Amplification Method for Highly Sensitive Parallel Detection of SARS-CoV-2 and Measles Virus

Micromachines ◽

10.3390/mi12121582 ◽

2021 ◽

Vol 12 (12) ◽

pp. 1582

Author(s):

Qin Huang ◽

Xiaohui Shan ◽

Ranran Cao ◽

Xiangyu Jin ◽

Xue Lin ◽

...

Keyword(s):

Microfluidic Chip ◽

Measles Virus ◽

Point Of Care ◽

Isothermal Amplification ◽

Molecular Diagnostic ◽

Nasopharyngeal Swab ◽

Two Stage ◽

Parallel Detection ◽

Amplification Method ◽

Specificity And Sensitivity

A two-stage isothermal amplification method, which consists of a first-stage basic recombinase polymerase amplification (RPA) and a second-stage fluorescence loop-mediated isothermal amplification (LAMP), as well as a microfluidic-chip-based portable system, were developed in this study; these enabled parallel detection of multiplex targets in real time in around one hour, with high sensitivity and specificity, without cross-contamination. The consumption of the sample and the reagent was 2.1 μL and 10.6 μL per reaction for RPA and LAMP, respectively. The lowest detection limit (LOD) was about 10 copies. The clinical amplification of about 40 nasopharyngeal swab samples, containing 17 SARS-CoV-2 (severe acute respiratory syndrome coronavirus) and 23 measles viruses (MV), were parallel tested by using the microfluidic chip. Both clinical specificity and sensitivity were 100% for MV, and the clinical specificity and sensitivity were 94.12% and 95.83% for SARS-CoV-2, respectively. This two-stage isothermal amplification method based on the microfluidic chip format offers a convenient, clinically parallel molecular diagnostic method, which can identify different nucleic acid samples simultaneously and in a timely manner, and with a low cost of the reaction reagent. It is especially suitable for resource-limited areas and point-of-care testing (POCT).

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