Sensitivity of RT-LAMP assay using in vitro synthesized RNA
We designed two SARS-CoV-2 specific LAMP primer sets that targeted conserved sequences in the ORF1b region (name: ORF1b-1 and ORF1b-2) (Table 1). To examine the sensitivity of the RT-LAMP assay for SARS-CoV-2, serial 10-fold dilutions of in vitro synthesized RNA possessing the target sequence of Wuhan-Hu-1 strain were used as template. All (6/6) or 83% (5/6) of the reactions with 50 copies of RNA yielded positive results with ORF1b-1 (RT-LAMP/ORF1b-1) or ORF 1b-2 primer sets (RT-LAMP/ORF1b-2), respectively (Table 2). Additionally, RT-LAMP/ORF1b-1 could detect samples containing 5 RNA copies once in six replicates. Moreover, both RT-LAMP/ORF1b-1 and RT-LAMP/ORF1b-2 could detect 500 RNA copies within 8 min and 50 RNA copies within 11.8 min (Table 2). These results indicate that this RT-LAMP assay can be used as a rapid and sensitive test for the detection of SARS-CoV-2 RNA.
Table 2  Sensitivity and detection time of RT-LAMP assay.
ORF1b-1
Copies/reaction  500  50  5  Water
Time (min)  7.5  9.0  -  -
7.3  8.8  10.5  -
8.0  11.8  -  -
6.8  9.3   -
6.8  9.0  -  -
7.3  8.5  -  -
Average  7.3  9.4  -  -
SD  0.4  1.1  -  -
Positive  6/6  6/6  1/6  0/6
ORF1b-2
Copies/ reaction  500  50  5  Water
Time (min)  8.0  11.8  -  -
7.8  -  -  -
7.3  11.5  -  -
7.5  9.0  -  -
7.3  9.5  -  -
6.8  10.3  -  -
Average  7.4  10.4  -  -
SD  0.4  1.1  -  -
Positive  6/6  5/6  0/6  0/6