An ultrasensitive amplification technology enabling the analysis of minimal amounts of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA is crucial for the early diagnosis of coronavirus disease 2019 (COVID-19). However, conventional reverse transcription-polymerase chain reaction (RT-PCR) techniques frequently result in false-negatives because of insufficient sensitivity for less than 100 target RNA molecules in a reaction solution. In this study, we succeeded in establishing ultrasensitive amplification of SARS-CoV-2 RNA by immobilizing DNA polymerase into highly ordered nanopores of mesoporous silica. The optimized silica pores enabled highly selective and efficient RT-PCR amplification from targeted single-copy RNA, even in the presence of nontarget background DNA. Additionally, in the presence of high concentrations of PCR inhibitors, the DNA polymerase-mesoporous silica composite showed a markedly improved resistance to inhibition. Furthermore, the composite exhibited extremely robust characteristics, maintaining amplification activity stably over a long period of time, despite storage in conditions completely free of bovine serum albumin (BSA) and 50% glycerol, which are considered essential in the storage of conventional PCR enzymes. Mesoporous silica holds promise as a sensitive and reliable platform for the analysis or diagnostics of single-copy RNA.
Matsuura et al. (Thu,) studied this question.