ABSTRACT
Rapid, sensitive and accurate diagnosis of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is of great need for effective quarantining and treatment. Real-time reverse-transcription polymerase chain reaction requiring thermocyling has been commonly used for diagnosis of SARS-CoV-2 though it may take two to 4 h before lengthy sample pretreatment process and require bulky apparatus and well-trained personnel. Since multiple reverse transcription loop-mediated isothermal amplification (multiple RT-LAMP) process without thermocycling is sensitive, specific and fast, an electromagnetically-driven microfluidic chip (EMC) was developed herein to lyse SARS-CoV-2 viruses, extract their RNAs, and perform qualitative analysis of three marker genes by on-chip multiple RT-LAMP in an automatic format within 82 min at a limit of detection of only â¼5000 copies per reaction (i.e. 200 virus/ µL). This compact EMC may be especially promising for SARS-CoV-2 diagnostics in resource-limited countries.
Subject(s)
COVID-19 , SARS-CoV-2 , COVID-19/diagnosis , COVID-19 Testing , Clinical Laboratory Techniques , Humans , Microfluidics , Molecular Diagnostic Techniques , Nucleic Acid Amplification Techniques , RNA, Viral/analysis , RNA, Viral/genetics , Reverse Transcription , SARS-CoV-2/genetics , Sensitivity and SpecificityABSTRACT
An integrated microfluidic platform (IMP) utilizing real-time reverse-transcription loop-mediated isothermal amplification (RT-LAMP) was developed here for detection and quantification of three genes of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2; i.e., coronavirus diseases 2019 (COVID-19)): RNA-dependent RNA polymerase, the envelope gene, and the nucleocapsid gene for molecular diagnosis. The IMP comprised a microfluidic chip, a temperature control module, a fluidic control module that collectively carried out viral lysis, RNA extraction, RT-LAMP, and the real-time detection within 90 min in an automatic format. A limit of detection of 5 × 103 copies/reaction for each gene was determined with three samples including synthesized RNAs, inactive viruses, and RNAs extracted from clinical samples; this compact platform could be a useful tool for COVID-19 diagnostics.