Isothermal gene amplification coupled MALDI-TOF MS for SARS-CoV-2 detection

Area of Science:

• Molecular Biology
• Biotechnology
• Infectious Diseases

Background:

• Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic necessitates accurate and rapid diagnostic tools.
• Current RT-qPCR methods for SARS-CoV-2 detection are time-consuming, require complex temperature control, and risk false negatives.
• Effective pandemic control relies on large-scale screening and early diagnosis of infected individuals.

Purpose of the Study:

• To develop a novel, rapid, and accurate assay for SARS-CoV-2 detection.
• To overcome the limitations of existing RT-qPCR based diagnostic methods.
• To provide a tool for efficient large-scale screening and COVID-19 pandemic control.

Main Methods:

• A three-way junction induced exponential rolling circle amplification (3WJ-eRCA) combined with MALDI-TOF MS assay was developed.
• The assay targets both nucleocapsid (N) and open reading frame 1ab (orf1ab) genes of SARS-CoV-2.
• Detection was performed using an isothermal process at 55°C, with results obtained within 30 minutes.

Main Results:

• The 3WJ-eRCA combined MALDI-TOF MS assay demonstrated high specificity for SARS-CoV-2, distinguishing it from other coronaviruses (SARS-CoV, MERS-CoV, bat-SL-CoVZC45).
• The assay successfully detected SARS-CoV-2 pseudovirus in various matrices including water, saliva, and urine.
• Simultaneous detection of N and orf1ab genes was achieved in a single test.

Conclusions:

• The developed 3WJ-eRCA combined MALDI-TOF MS assay is a promising tool for rapid and accurate SARS-CoV-2 detection.
• This isothermal method offers a significant advantage over traditional RT-qPCR, reducing processing time and complexity.
• The assay's potential for large-scale screening supports its role in effective COVID-19 pandemic control efforts.