ABSTRACT
The COVID-19 pandemic, caused by severe acute respiratory coronavirus 2 (SARS-CoV-2), has become a public health emergency and widely spread around the world. Rapid, accurate and early diagnosis of COVID-19 infection plays a crucial role in breaking this pandemic. However, the detection accuracy is limited for current single-gene diagnosis of SARS-CoV-2. Herein, we develop an autonomous lab-on-paper platform for multiplex gene diagnosis of SARS-CoV-2 by combining reverse transcription recombinase polymerase amplification (RT-RPA) and CRISPR-Cas12a detection. The autonomous lab-on-paper is capable of simultaneously detecting nucleoprotein (N) gene and spike (S) gene of SARS-CoV-2 virus as well as human housekeeping RNAse P gene (an internal control) in a single clinical sample. With the developed platform, 102 copies viral RNA per test can be detected within one hour. Also, the lab-on-paper platform has been used to detect 21 swab clinical samples and obtains a comparable performance to the conventional RT-PCR method. Thus, the developed lab-on-paper platform holds great potential for rapid, sensitive, reliable, multiple molecular diagnostics of COVID-19 and other infectious diseases in resource-limited settings.
Subject(s)
COVID-19 , Pandemics , CRISPR-Cas Systems/genetics , Clustered Regularly Interspaced Short Palindromic Repeats , Humans , Nucleic Acid Amplification Techniques , RNA, Viral/genetics , SARS-CoV-2 , Sensitivity and SpecificityABSTRACT
Quantifying severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in clinical samples is crucial for early diagnosis and timely medical treatment of coronavirus disease 2019. Here, we describe a digital warm-start CRISPR (dWS-CRISPR) assay for sensitive quantitative detection of SARS-CoV-2 in clinical samples. The dWS-CRISPR assay is initiated at above 50 °C and overcomes undesired premature target amplification at room temperature, enabling accurate and reliable digital quantification of SARS-CoV-2. By targeting SARS-CoV-2's nucleoprotein gene, the dWS-CRISPR assay is able to detect down to 5 copies/µl SARS-CoV-2 RNA in the chip. It is clinically validated by quantitatively determining 32 clinical swab samples and three clinical saliva samples. Moreover, it has been demonstrated to directly detect SARS-CoV-2 in heat-treated saliva samples without RNA extraction. Thus, the dWS-CRISPR method, as a sensitive and reliable CRISPR assay, facilitates accurate SARS-CoV-2 detection toward digitized quantification.
Subject(s)
Biosensing Techniques , COVID-19 , CRISPR-Cas Systems/genetics , Clustered Regularly Interspaced Short Palindromic Repeats , Humans , Nucleic Acid Amplification Techniques , RNA, Viral , SARS-CoV-2ABSTRACT
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.
Subject(s)
Betacoronavirus/isolation & purification , Coronavirus Infections/virology , Pneumonia, Viral/virology , Betacoronavirus/genetics , COVID-19 , COVID-19 Testing , CRISPR-Cas Systems , Clinical Laboratory Techniques/methods , Coronavirus Infections/diagnosis , Genes, Viral , Humans , Molecular Diagnostic Techniques/methods , Nucleic Acid Amplification Techniques/methods , Pandemics , Pneumonia, Viral/diagnosis , Point-of-Care Systems , RNA, Viral/analysis , RNA, Viral/genetics , SARS-CoV-2 , Sensitivity and Specificity , Viral Proteins/analysis , Viral Proteins/geneticsABSTRACT
<p>Amid personal protective equipment shortage, clinicians, nurses, and other frontline workers across the world have faced threatening and/or firing for self-protection during this coronavirus disease 2019 (COVID-19) pandemic. This perspective describes the different challenges that the stressed and overworked frontline workers encounter when they raise concerns despite being right. It also highlights the importance of communication and appropriate execution upon hearing those concerns.</p>