Rapid Molecular Detection of SARS-CoV-2 (COVID-19) Virus RNA Using Colorimetric LAMP

Yinhua Zhang; Nelson Odiwuor; Jin Xiong; Luo Sun; Raphael Ohuru Nyaruaba; Hongping Wei; Nathan A Tanner

We designed 5 full LAMP primers sets targeting SARS-CoV-2 RNA, with amplicon regions designed to the 5′ region of the ORF1a gene and Gene N. Each set was tested with synthetic DNA substrates and RNA transcribed from that DNA substrate before consideration for use clinically. To evaluate detection sensitivity, synthetic RNAs were serially diluted from ~120 million copies down to ~120 copies (per 25 μ L reaction) at 10fold intervals in LAMP reactions. All five primer sets showed similar detection sensitivity and could consistently detect as low as a few hundred copies, with sporadic detection of 120 copies (or 4.8 copies/μL; Fig. 1 ). The results from colorimetric detection were 100% in agreement with the real time detection. To estimate the relative efficiency using RNA or DNA templates, we compared synthetic RNA with similarly diluted gBlock dsDNA on real-time LAMP signal (Fig. 2) . For the 2 primer sets we compared, one showed slightly slower amplification and detection with RNA template while the other appeared slightly faster, confirming the RNA is efficiently converted to cDNA by the reverse transcriptase (WarmStart RTx) and subsequently amplified via LAMP by the DNA-dependent DNA polymerase (Bst 2.0 WarmStart).