Development and comparison of novel multiple cross displacement amplification (MCDA) assays with other nucleic acid...

The development of alternative isothermal amplification assays including multiple cross displacement amplification (MCDA) may address speed and portability limitations of real-time PCR (rt-PCR) methods for SARS-CoV-2 detection. We developed a novel SARS-CoV-2 MCDA assay and compared its speed and sensitivity to loop-mediated isothermal amplification (LAMP) and rt-PCR. Two MCDA assays targeting SARS-CoV-2 N gene and ORF1ab were designed. The fastest time to detection and sensitivity of MCDA was compared to LAMP and rt-PCR using DNA standards and transcribed RNA. For the N gene, MCDA was faster than LAMP and rt-PCR by 10 and 20 min, respectively with fastest time to detection at 5.2 min. rt-PCR had the highest sensitivity with the limit of detection at 10 copies/µl compared with MCDA (100 copies/µl) and LAMP (500 copies/µl). For ORF1ab, MCDA and LAMP had similar speed with fastest time to detection at 9.7 and 8.4 min, respectively. LAMP was more sensitive for ORF1ab detection with 50 copies/µl compared to MCDA (500 copies/µl). In conclusion, different nucleic acid amplification methods provide different advantages. MCDA is the fastest nucleic acid amplification method for SARS-CoV-2 while rt-PCR is the most sensitive. These advantages should be considered when determining the most suitable nucleic acid amplification methods for different applications.