A Novel Reverse Transcription Loop-Mediated Isothermal Amplification Method for Rapid Detection of SARS-CoV-2

Abstract

COVID-19 has become a major global public health burden, currently causing a rapidly growing number of infections and significant morbidity and mortality around the world. Early detection with fast and sensitive assays and timely intervention are crucial for interrupting the spread of the COVID-19 virus (SARS-CoV-2). Using a mismatch-tolerant amplification technique, we developed a simple, rapid, sensitive and visual reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay for SARS-CoV-2 detection based on its N gene. The assay has a high specificity and sensitivity, and robust reproducibility, and its results can be monitored using a real-time PCR machine or visualized via colorimetric change from red to yellow. The limit of detection (LOD) of the assay is 118.6 copies of SARS-CoV-2 RNA per 25 µL reaction. The reaction can be completed within 30 min for real-time fluorescence monitoring, or 40 min for visual detection when the template input is more than 200 copies per 25 µL reaction. To evaluate the viability of the assay, a comparison between the RT-LAMP and a commercial RT-qPCR assay was made using 56 clinical samples. The SARS-CoV-2 RT-LAMP assay showed perfect agreement in detection with the RT-qPCR assay. The newly-developed SARS-CoV-2 RT-LAMP assay is a simple and rapid method for COVID-19 surveillance.

One-Step Rapid Quantification of SARS-CoV-2 Virus Particles via Low-Cost Nanoplasmonic Sensors in Generic Microplate Reader and Point-of-Care Device

“…It is evident that the infection may be effectively reduced and eventually controlled with swift and large-scale testing for early diagnostics [3][4][5][6] . Current testing methods include PCR nucleic acid detection with either thermal cycling 7,8 or isothermal amplification [9][10][11] , serological IgM/IgG antibody testing 12 as well as viral protein detections 13 in nasopharyngeal swap. The PCR based nucleic acid testing is very sensitive and accurate in early infection diagnostics but it generally requires multiple and lengthy processes including virus lysis, RNA extraction, reverse transcription and amplification and is prone to sample contamination 14,15 .…”

A comparative study of isothermal nucleic acid amplification methods for SARS-CoV-2 detection at point-of-care

“…Different virological and serological approaches for rapidly detecting SARS-CoV-2 at POC have been introduced. Virological diagnosis directly detects the viral nucleic acids via isothermal nucleic acid amplification tests (iNAATs) [9][10][11][12] and CRISPR Cas12 based method [13]. Serological tests detect the rising titers of antibody between acute and convalescent stages of infection or detect IgM in primary infection [14][15][16][17].…”

A Novel Two-Step, Direct-to-PCR Method for Virus Detection off Swabs using Human Coronavirus 229E

“…The traditional protocol for preparing a sample for PCR based detection often involves procedures of swabbing a patient, processing the sample to lyse the virus, extract, and purify its nucleotides, and then amplify the puri ed genetic material via PCR for detection of a gene product needed to con rm the patient's suspected diagnosis [3]. In the face of the COVID-19 pandemic, attempts have been made to perform PCR based diagnostics for SARS-CoV-2 with reduced time and cost, especially as the plastics and reagents needed for traditional viral nucleotide extractions have become scarce in the face of an exponentially increased global demand [4,5,6,7].…”

“…Though some success has been seen with thermal and enzymatic digestion attempts on viral samples to expose the genetic material for extraction-less PCR detection, no method has shown high viral lysis in under one minute when dealing with clinical concentrations of virus on swabs [5,6,8].…”