Novel reverse transcription-multiple inner primer loop-mediated isothermal amplification (RT-MIPLAMP) for visual and sensitive detection of SARS-CoV-2

Abstract

Since the end of 2019, outbreaks of COVID-19 pandemics have continued in different areas worldwide, which exacerbates the need for rapid, sensitive and simple methods for diagnosis. Currently, COVID-19 diagnosis mainly relies on reverse transcription-polymerase chain reaction (RT-PCR), which requires sophisticated instruments. Reverse transcription-loop mediated isothermal amplification (RT-LAMP), due to its isothermal nature and high specificity, can be used as an alternative. In this paper, a novel visual reverse transcription-multiple inner primer loop-mediated isothermal amplification (RT-MIPLAMP) method is established based on RT-LAMP by adding a pair of inner primers. The RT-MIPLAMP method has a higher sensitivity and shorter reaction time compared with conventional RT-LAMP. By using RT-MIPLAMP, as low as 6 × 10³ copies per mL in vitro transcribed (IVT) N gene can be detected within 55 min. Meanwhile, as low as 6 × 10⁴ copies per mL IVT N gene is detectable with conventional RT-LAMP within 80 min. The feasibility of visual RT-MIPLAMP is also validated by detecting the N gene spiked into one healthy volunteer's saliva and the full-length RNA in pseudoviruses, indicating the great potential of visual RT-MIPLAMP for SARS-CoV-2 identification.