Development and comparison of a novel multiple cross displacement amplification (MCDA) assay with other nucleic acid amplification methods for SARS-CoV-2 detection

Abstract

Objectives: To develop a novel multiple cross displacement amplification (MCDA) assay for COVID-19 and compare its speed and sensitivity to existing loop-mediated isothermal amplification (LAMP) and real-time PCR (RT-PCR) methods. Methods: Two MCDA assays targeting the SARS-CoV-2 N gene and ORF1ab was designed. The fastest time to detection and sensitivity of MCDA was compared to LAMP and RT-PCR using 7 DNA standards and transcribed RNA. Results: For the N gene, MCDA was consistently faster than LAMP and RT-PCR by 10 and 20 minutes, respectively with a fastest time to detection of 5.2 minutes. RT-PCR had the highest sensitivity with a limit of detection of 100 copies/reaction compared with MCDA (1000 copies/reaction) and LAMP (5000/reaction). For ORF1ab, MCDA and LAMP had similar speed with fastest time to detection at 9.7 and 8.4 minutes, respectively. LAMP was more sensitive for ORF1ab detection with 500 copies/reaction compared to MCDA (5000 copies/reaction). Conclusions: Different nucleic acid amplification methods provide different advantages. MCDA is the fastest nucleic acid amplification method for COVID-19 while RT-PCR is still the most sensitive. These advantages should be considered when determining the most suitable nucleic acid amplification methods for different applications.