Based on our previous published report, analytical sensitivity and specificity tests for N1 gene were 1 copy/µL RNA sensitive and 100% specific, respectively [8]. Out of 49 rRT-PCR positive samples, RT-LAMP detected 47 samples as positive and did not detect any of the rRT-PCR negative samples as positive. It was 95.9% sensitive (95% CI 86.0 to 99.5%) and 100% specific (95% CI 78.2–100%). Results showed that RT-LAMP did not detect two patients with low viral load (rRT-PCR CT value of 35.19 and 36.06). The RNA in these samples maybe degraded during storage or shipping. There is correlation between CT values of rRT-PCR and detection time of RT-LAMP. Figure 1 shows samples with high CT value took a longer time to amplify by RT-LAMP. According to Spearman’s rho test, there is strong correlation between CT values of rRT-PCR and detection time of RT-LAMP. Both are statistically significant, rho = 0.79, p < 0.001. When CT values of rRT-PCR increase, detection time of RT-LAMP also become longer. Nucleic acid extraction using chelating resin offers many advantages compared to conventional methods, such as spin/vacuum columns-based or paramagnetic beads-based extraction kits. These typical column- and paramagnetic beads-based methods are tedious as it involves multiple steps of processing and requires various consumables. A major drawback of these methods is the requirement of large quantities of biological samples for extraction [6]. However, using chelating resin for RNA extraction, only a minute volume of VTM samples (15 μL) was needed and the preparation of chelating resin was simple. The simple RNA preparation step improved diagnostic efficiency, cost and time. In addition, another benefit of using chelating resin for extraction was demonstrated by Walsh et al. [4] during their DNA extraction from forensic materials. They reported that DNA extraction from semen and blood stain samples using chelating resin was as sensitive if not more sensitive than proteinase K and phenol–chloroform extraction methods.

Fig. 1

Correlation between CT value of rRT-PCR and detection time of RT-LAMP. rRT-PCR, real time reverse transcription polymerase chain reaction; RT-LAMP, reverse transcriptase loop-mediated isothermal amplification; min, minutes; CT, cycle threshold

Coupled with the chelating resin extraction method, we achieved a promising result of 95.9% sensitivity for RT-LAMP. Flynn et al. [7] obtained 90% sensitivity during their optimization of RT-LAMP-based SARS-CoV-2 diagnostic protocol for saliva samples. Another study from Howson et al. [9] showed that overall sensitivity of direct RT-LAMP on saliva samples was approximately 83%.

Intriguingly, we managed to detect 10 samples with high CT values (rRT-PCR CT > 33.68–38.85) (Additional file 1: Table S1) in the current direct swab-to-RT-LAMP assay. This is a promising finding compared to other direct swab-to-RT-LAMP studies. Without further RNA extraction from nasopharyngeal swab samples, Lamb et al. [10] managed to detect 40% (N = 4/10) of SARS-CoV-2 (rRT-PCR CT < 24) samples by RT-LAMP. In another study by Wei et al. [11], out of 13 clinical swab positive samples (rRT-PCR CT < 31) tested, they only managed to detect 5 samples by RT-LAMP assay.

Various simplified preparation methods have been reported which can circumvent RNA extraction procedures due to the shortage of commercial kits in expanding diagnostic facilities of SARS-CoV-2. Fowler et al. [12] managed to obtain 67% sensitivity of RT-LAMP using VTM sample (1:20 dilution in water). Nie et al. [13] reported a direct RT-LAMP by heating nasopharyngeal swab samples at 95 °C for 30 s and cooling on ice for 2 min. In addition, Dao Thi et al. [14] managed to obtain 86% sensitivity RT-LAMP using swab samples either without any treatment or after heat treatment for 5 min at 95 °C during their development of colorimetric RT-LAMP assay. As for the high sensitivity of direct RT-LAMP, Yoshikawa et al. [15] also reported 1.43 × 103 copies of RNA by direct heating the swab samples at 95 °C for 10 min. However, we were not able to replicate these methods after several trials. The failure may be due to the presence of inhibitors, such as glucose in VTM.

Apart from real time turbidity detection of RT-LAMP results, the results can also be examined by observing the colour changes of the end product. We included HNB colorimetric dye in the LAMP assay during the preparation of the master mix. Carryover contamination can be eliminated as the dye was not added into the reaction tube upon completion of the amplification. Typical approach to confirm the LAMP results was performed by running the end products via gel electrophoresis. Unfortunately, running end products by gel electrophoresis may contribute to the issue of carryover contamination.

LoopAmp Real-Time Turbidimeter LA 500 used in this study can be upgraded with six control units, at which, 96 RT-LAMP tests can be performed in 1 h. For high-throughput testing, multiple instruments can be employed.

Even though SARS-CoV-2 antigen-detection tests that offer results in 15 min and as they become more widely available as at-home, self-administered and over-the-counter tests at pharmacy stores, our RT-LAMP assay still can be considered as an alternative for SARS-CoV-2 testing especially in resource limited areas. Most of these tests show lower sensitivity as compared to our previous published RT-LAMP assay (100% sensitivity) [16]. Meanwhile, Harmon et al. [17] reported 78.9% sensitivity using an at-home direct antigen rapid test kit. Another study reported by Shrestha et al. (2020) also revealed that low sensitivity of lateral flow antigen test kits for COVID-19 testing with 85% sensitivity [18]. Moreover, the developed RT-LAMP here costs only USD$1.95/reaction as compared to antigen detection test (USD$4.30).

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