ABSTRACT
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is spreading all over the world and has caused millions of deaths. Several sample-to-answer platforms, including Cepheid Xpert Xpress SARS-CoV-2 (Xpert Xpress), have received emergency use authorization for SARS-CoV-2 nucleic acid detection as a point of care test in the United States. However, their application niche is unclear when compared with real-time RT-PCR assays cleared by the National Medical Products Administration in China. In this study, the clinical performance, sensitivity, and workflow of Xpert Xpress and two real-time RT-PCR kits (BioGerm kit and Sansure kit) were evaluated by the specimens from 86 symptomatic patients. The positive percent agreement of Xpert Xpress was 100% compared with 96.15% for the BioGerm kit and 90% for the Sansure kit. The negative percent agreement was 100% for all three assays. The limit of detection is 100 copies/mL for Xpert Xpress and 500 copies/mL for the BioGerm kit and Sansure kit. By serially diluting five positive specimens, the Xpert Xpress had better detection capability. In the workflow and throughput analysis, the turnaround time was 51 minutes for Xpert Xpress, 150 minutes for the BioGerm kit, and 210 minutes for the Sansure kit. This study provides some indication for diagnosis methods selection.
Subject(s)
COVID-19 Nucleic Acid Testing/standards , COVID-19/diagnosis , RNA, Viral/genetics , Reagent Kits, Diagnostic/standards , Real-Time Polymerase Chain Reaction/standards , SARS-CoV-2/genetics , Benchmarking , COVID-19/epidemiology , COVID-19 Nucleic Acid Testing/instrumentation , COVID-19 Nucleic Acid Testing/methods , China/epidemiology , Humans , Limit of Detection , Point-of-Care Testing , United States/epidemiologyABSTRACT
BACKGROUND: Real-time reverse transcription-PCR (rRT-PCR) has been the most effective and widely implemented diagnostic technology since the beginning of the COVID-19 pandemic. However, fuzzy rRT-PCR readouts with high Ct values are frequently encountered, resulting in uncertainty in diagnosis. METHODS: A Specific Enhancer for PCR-amplified Nucleic Acid (SENA) was developed based on the Cas12a trans-cleavage activity, which is specifically triggered by the rRT-PCR amplicons of the SARS-CoV-2 Orf1ab (O) and N fragments. SENA was first characterized to determine its sensitivity and specificity, using a systematic titration experiment with pure SARS-CoV-2 RNA standards, and was then verified in several hospitals, employing a couple of commercial rRT-PCR kits and testing various clinical specimens under different scenarios. FINDINGS: The ratio (10 min/5 min) of fluorescence change (FC) with mixed SENA reaction (mix-FCratio) was defined for quantitative analysis of target O and N genes, and the Limit of Detection (LoD) of mix-FCratio with 95% confidence interval was 1.2≤1.6≤2.1. Totally, 295 clinical specimens were analyzed, among which 21 uncertain rRT-PCR cases as well as 4 false negative and 2 false positive samples were characterized by SENA and further verified by next-generation sequencing (NGS). The cut-off values for mix-FCratio were determined as 1.145 for positive and 1.068 for negative. INTERPRETATION: SENA increases both the sensitivity and the specificity of rRT-PCR, solving the uncertainty problem in COVID-19 diagnosis and thus providing a simple and low-cost companion diagnosis for combating the pandemic. FUNDING: Detailed funding information is available at the end of the manuscript.
Subject(s)
Bacterial Proteins/metabolism , Betacoronavirus/genetics , CRISPR-Associated Proteins/metabolism , Clustered Regularly Interspaced Short Palindromic Repeats/genetics , Endodeoxyribonucleases/metabolism , RNA, Viral/metabolism , Real-Time Polymerase Chain Reaction/methods , Betacoronavirus/isolation & purification , COVID-19 , Coronavirus Infections/diagnosis , Coronavirus Infections/pathology , Coronavirus Infections/virology , Coronavirus Nucleocapsid Proteins , Humans , Limit of Detection , Nasal Cavity/virology , Nucleic Acid Amplification Techniques/methods , Nucleic Acid Amplification Techniques/standards , Nucleocapsid Proteins/genetics , Nucleocapsid Proteins/metabolism , Pandemics , Phosphoproteins , Pneumonia, Viral/diagnosis , Pneumonia, Viral/pathology , Pneumonia, Viral/virology , Polyproteins , RNA, Viral/genetics , Real-Time Polymerase Chain Reaction/standards , Reference Standards , SARS-CoV-2 , Viral Proteins/genetics , Viral Proteins/metabolismABSTRACT
The outbreak of SARS-CoV-2 is posing serious global public health problems. Facing the emergence of this pandemic, we established a portable microfluidic immunoassay system for easy-to-use, sensitive, rapid (<15 min), multiple, and on-site detection of IgG/IgM/Antigen of SARS-CoV-2 simultaneously. This integrated method was successfully applied for detecting SARS-CoV-2 IgM and IgG antibodies in clinical human serum as well as SARS-CoV-2 antigen in pharyngeal swabs from 26 patients with COVID-19 infection and 28 uninfected people. The assay demonstrated high sensitivity and specificity, which is promising for the diagnosis and monitoring as well as control of SARS-CoV-2 worldwide.