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Anal Chem ; 93(27): 9541-9548, 2021 07 13.
Article in English | MEDLINE | ID: covidwho-1284670


Quantitative diagnostics that are rapid, inexpensive, sensitive, robust, and field-deployable are needed to contain the spread of infectious diseases and inform treatment strategies. While current gold-standard techniques are highly sensitive and quantitative, they are slow and require expensive equipment. Conversely, current rapid field-deployable assays available provide essentially binary information about the presence of the target analyte, not a quantitative measure of concentration. Here, we report the development of a molecular diagnostic test [quantitative recombinase polymerase amplification (qRPA)] that utilizes competitive amplification during a recombinase polymerase amplification (RPA) assay to provide semi-quantitative information on a target nucleic acid. We demonstrate that qRPA can quantify DNA, RNA, and viral titers in HIV and COVID-19 patient samples and that it is more robust to environmental perturbations than traditional RPA. These features make qRPA potentially useful for at-home testing to monitor the progress of viral infections or other diseases.

COVID-19 , Nucleic Acid Amplification Techniques , Humans , Molecular Diagnostic Techniques , Recombinases , SARS-CoV-2 , Sensitivity and Specificity
J Virol Methods ; 294: 114153, 2021 08.
Article in English | MEDLINE | ID: covidwho-1226314


The COVID-19 pandemic has resulted in an unparalleled need for viral testing capacity across the world and is a critical requirement for successful re-opening of economies. The logistical barriers to near-universal testing are considerable. We have designed an injection molded polypropylene anterior nares swab, the Rhinostic, with a screw cap integrated into the swab handle that is compatible with fully automated sample accessioning and processing. The ability to collect and release both human and viral material is comparable to that of several commonly used swabs on the market. SARS-CoV-2 is stable on dry Rhinostic swabs for at least 3 days, even at 42 °C, and elution can be achieved with small volumes. To test the performance of the Rhinostic in patients, 119 samples were collected with Rhinostic and the positive and negative determinations were 100 % concordant with samples collected using Clinical Laboratory Improvement Amendments (CLIA) use approved nasal swabs at a clinical lab. The Rhinostic swab and barcoded tube set can be produced, sterilized, and packaged cost effectively and is designed to be adopted by clinical laboratories using automation to increase throughput and dramatically reduce the cost of a standard SARS-CoV-2 detection pipeline.

COVID-19 Nucleic Acid Testing/instrumentation , Nasopharynx/virology , RNA, Viral/isolation & purification , SARS-CoV-2/isolation & purification , Specimen Handling/instrumentation , Specimen Handling/methods , Automation, Laboratory , COVID-19 Nucleic Acid Testing/methods , Humans , Nasopharynx/anatomy & histology , Polypropylenes
Nat Commun ; 11(1): 5920, 2020 11 20.
Article in English | MEDLINE | ID: covidwho-939437


Rapid, inexpensive, robust diagnostics are essential to control the spread of infectious diseases. Current state of the art diagnostics are highly sensitive and specific, but slow, and require expensive equipment. Here we report the development of a molecular diagnostic test for SARS-CoV-2 based on an enhanced recombinase polymerase amplification (eRPA) reaction. eRPA has a detection limit on patient samples down to 5 viral copies, requires minimal instrumentation, and is highly scalable and inexpensive. eRPA does not cross-react with other common coronaviruses, does not require RNA purification, and takes ~45 min from sample collection to results. eRPA represents a first step toward at-home SARS-CoV-2 detection and can be adapted to future viruses within days of genomic sequence availability.

Betacoronavirus/genetics , Betacoronavirus/isolation & purification , Nucleic Acid Amplification Techniques/methods , COVID-19 Testing , Clinical Laboratory Techniques , Coronavirus Infections/diagnosis , Humans , RNA/metabolism , RNA, Viral/genetics , RNA, Viral/isolation & purification , RNA-Directed DNA Polymerase/metabolism , Real-Time Polymerase Chain Reaction , Recombinases/metabolism , SARS-CoV-2 , Saliva/virology , Virion/genetics