Comparative evaluation of 19 reverse transcription loop-mediated isothermal amplification assays for detection of SARS-CoV-2

Abstract

Coronavirus disease 2019 (COVID-19) caused by SARS-CoV-2 has caused a global pandemics. To facilitate the detection of SARS-CoV-2 infection, various RT-LAMP assays using 19 sets of primers had been developed, but never been compared. We performed comparative evaluation of the 19 sets of primers using 4 RNA standards and 29 clinical samples from COVID-19 patients. Six of 15 sets of primers were firstly identified to have faster amplification when tested with four RNA standards, and were further subjected to parallel comparison with the remaining four primer sets using 29 clinical samples. Among these 10 primer sets, Set-4 had the highest positive detection rate of SARS-CoV-2 (82.8%), followed by Set-10, Set-11, and Set-13 and Set-17 (75.9%). Set-14 showed the fastest amplification speed (Tt value < 8.5 min), followed by Set-17 (Tt value < 12.5 min). Based on the overall detection performance, Set-4, Set-10, Set-11, Set-13, Set-14 and Set-17 that target Nsp3, S, S, E, N and N gene regions of SARS-CoV-2, respectively, were determined to be better than the other primer sets. Two RT-LAMP assays with the Set-4 primers in combination with any one of four other primer sets (Set-14, Set-10, Set-11, and Set-13) were recommended to be used in the COVID-19 surveillance.

Figure 1

Genome location and evaluation strategy of 19 sets of SARS-CoV-2 RT-LAMP primers. The location of each primer set was detailed in Table 1.

Figure 2

Comparison of performance of 15 RT-LAMP assays using RNA standards. Two replicates were performed for each primer set. The replicates of the same primer set often generated completely overlapped amplification curves. A short bar was used to highlight the non-overlapping curves of the same primer. The curves of non-template control (NTC) are not shown. *The Tt values of the Set-8 were obtained by another repeated comparative experiments with Set-5and Set-9, both of which showed a consistent trend, but slightly lower Tt values than those shown here.

Figure 3

Comparison of performance of 10 selected RT-LAMP primer sets using 41 clinical RNA samples. (A) Positive rates and Tt values of 10 selected RT-LAMP assays. The 41 clinical samples included 29 SARS-CoV-2 positive and 12 negative clinical samples that were previously determined by RT-qPCR assay. The positive rate was calculated by dividing the number of positive sample by each primer set by total positive sample number of RT-qPCR assay (i.e. 29). (B) Paired comparison of Tt values of the primers Set-4, Set-10, Set-11, Set-13, Set-14 and Set-17. Because all RNA from clinical samples were fourfold diluted and some of them have very low viral load (high Ct values by RT-qPCR assay), some positive samples were not detected as positive by the RT-LAMP assay, which are defined as false-negative. We calculated the concordance rate by dividing the number of consistent results (true positive, true negative and false-negative) by any two primer sets by the total sample number (i.e. 41). SD standard deviation.

Figure 4

Sensitivity (A) and LOD (B) of the primer Set-4. NTC non-template control.