Background
Corona Virus Disease 2019 (COVID-19) is a recently emerged life-threatening disease caused by SARS-CoV-2. It has the characteristics of rapid transmission, difficult treatment, rapid deterioration, and high mortality rate. Reliable and rapid diagnosis of SARS-CoV-2 infection is critical to treat patients and control transmission in time.
Methods
To detect SARS-CoV-2 nucleic acid faster and more convenient, RT-LAMP method (reverse transcription loop-mediated isothermal amplification) was developed. ORF1ab gene, E gene and N gene were selected as the target genes, because ORF1ab gene is the most specific and N gene is the most sensitive, while E gene has both sensitivity and specificity. Over thirty primer sets for them were designed, then the primer sets with rapid response and high specificity were screened out and their loop primers were designed and selected in order to improve the reaction speed further. Using these primers, RNA reverse transcription and nucleic acid amplification were performed in one step at 63 ℃ isothermal conditions, and the results can be judged by naked eyes through color change within 30 minutes. Finally, the optimal primer set for each gene was verified with over 208 clinical samples.
Results
ORF1ab gene, E gene and N gene were detected simultaneously by this method. ORF1ab gene has high specificity and the same sensitivity as RT-PCR currently used in clinic. Although N gene is less specific than ORF1ab, it is 80 times more sensitive than ORF1ab. The sensitivity and specificity of E gene are between them. Simultaneous detection of these three genes can neither cause false positives nor miss samples with low virus concentration, ensuring the sensitivity and specificity of SARS-CoV-2 detection. BLAST comparing results showed the primers for the three genes were highly specific for SARS-CoV-2. And the sequencing results of RT-LAMP products showed that the amplified fragments were unique to SARS-CoV-2. The accuracy of RT-LAMP assay was 99% as detecting 208 clinical specimens.
Conclusions
The RT-LAMP assay reported here has the advantages of rapid amplification, simple operation, and easy detection, which is useful for the rapid and reliable clinical diagnosis of SARS-CoV-2.