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1.
Science ; 372(6545): 914-915, 2021 05 28.
Article in English | MEDLINE | ID: covidwho-1501517
3.
Nat Biomed Eng ; 5(7): 643-656, 2021 07.
Article in English | MEDLINE | ID: covidwho-1324420

ABSTRACT

The accurate and timely diagnosis of disease is a prerequisite for efficient therapeutic intervention and epidemiological surveillance. Diagnostics based on the detection of nucleic acids are among the most sensitive and specific, yet most such assays require costly equipment and trained personnel. Recent developments in diagnostic technologies, in particular those leveraging clustered regularly interspaced short palindromic repeats (CRISPR), aim to enable accurate testing at home, at the point of care and in the field. In this Review, we provide a rundown of the rapidly expanding toolbox for CRISPR-based diagnostics, in particular the various assays, preamplification strategies and readouts, and highlight their main applications in the sensing of a wide range of molecular targets relevant to human health.


Subject(s)
CRISPR-Cas Systems/genetics , Communicable Diseases/diagnosis , Nucleic Acid Amplification Techniques/methods , Nucleic Acids/analysis , Communicable Diseases/microbiology , Communicable Diseases/virology , Genetic Diseases, Inborn/diagnosis , Humans , Nucleic Acid Amplification Techniques/economics , Nucleic Acids/metabolism , Point-of-Care Systems , Polymorphism, Single Nucleotide , Sequence Analysis, DNA
4.
PLoS One ; 15(11): e0238612, 2020.
Article in English | MEDLINE | ID: covidwho-902011

ABSTRACT

BACKGROUND: Rapid and extensive testing of large parts of the population and specific subgroups is crucial for proper management of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infections and decision-making in times of a pandemic outbreak. However, point-of-care (POC) testing in places such as emergency units, outpatient clinics, airport security points or the entrance of any public building is a major challenge. The need for thermal cycling and nucleic acid isolation hampers the use of standard PCR-based methods for this purpose. METHODS: To avoid these obstacles, we tested PCR-independent methods for the detection of SARS-CoV-2 RNA from primary material (nasopharyngeal swabs) including reverse transcription loop-mediated isothermal amplification (RT-LAMP) and specific high-sensitivity enzymatic reporter unlocking (SHERLOCK). RESULTS: Whilst specificity of standard RT-LAMP assays appears to be satisfactory, sensitivity does not reach the current gold-standard quantitative real-time polymerase chain reaction (qPCR) assays yet. We describe a novel multiplexed RT-LAMP approach and validate its sensitivity on primary samples. This approach allows for fast and reliable identification of infected individuals. Primer optimization and multiplexing helps to increase sensitivity significantly. In addition, we directly compare and combine our novel RT-LAMP assays with SHERLOCK. CONCLUSION: In summary, this approach reveals one-step multiplexed RT-LAMP assays as a prime-option for the development of easy and cheap POC test kits.


Subject(s)
Betacoronavirus/genetics , Coronavirus Infections/diagnosis , Nucleic Acid Amplification Techniques/methods , Pneumonia, Viral/diagnosis , RNA, Viral/metabolism , Betacoronavirus/isolation & purification , COVID-19 , COVID-19 Testing , Clinical Laboratory Techniques , Coronavirus Infections/virology , Humans , Nasopharynx/virology , Pandemics , Pneumonia, Viral/virology , RNA, Viral/genetics , Real-Time Polymerase Chain Reaction , SARS-CoV-2 , Sensitivity and Specificity
5.
Nat Biomed Eng ; 4(12): 1140-1149, 2020 12.
Article in English | MEDLINE | ID: covidwho-733522

ABSTRACT

Nucleic acid detection by isothermal amplification and the collateral cleavage of reporter molecules by CRISPR-associated enzymes is a promising alternative to quantitative PCR. Here, we report the clinical validation of the specific high-sensitivity enzymatic reporter unlocking (SHERLOCK) assay using the enzyme Cas13a from Leptotrichia wadei for the detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-the virus that causes coronavirus disease 2019 (COVID-19)-in 154 nasopharyngeal and throat swab samples collected at Siriraj Hospital, Thailand. Within a detection limit of 42 RNA copies per reaction, SHERLOCK was 100% specific and 100% sensitive with a fluorescence readout, and 100% specific and 97% sensitive with a lateral-flow readout. For the full range of viral load in the clinical samples, the fluorescence readout was 100% specific and 96% sensitive. For 380 SARS-CoV-2-negative pre-operative samples from patients undergoing surgery, SHERLOCK was in 100% agreement with quantitative PCR with reverse transcription. The assay, which we show is amenable to multiplexed detection in a single lateral-flow strip incorporating an internal control for ribonuclease contamination, should facilitate SARS-CoV-2 detection in settings with limited resources.


Subject(s)
COVID-19/diagnosis , CRISPR-Associated Proteins/genetics , Molecular Diagnostic Techniques/methods , Nucleic Acid Amplification Techniques/methods , RNA, Viral/genetics , SARS-CoV-2/genetics , COVID-19/virology , Humans , Leptotrichia/enzymology , Pandemics/prevention & control
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