Establishment of the PNA RT-LAMP assay. Two (2) sets of primer and PNA probes targeting two (2) specific regions in ORF1ab and N gene of SARS-CoV-2 were designed for amplification and detection of SARS-CoV-2. The sequences of the oligonucleotides were aligned against publicly available 56303 SARS-CoV-2 sequences downloaded from NCBI database by February 2021 which contain full genomic information on CLC Main Workbench. All alignments showed 100% of identity against the queries showing that in silico analysis predicted that the assay can amplify and detect all SARS-CoV-2 isolates analyzed in this study. Results are summarized in Table 1.
The assay targets one (1) specific region in each ORF1ab and N gene of the SARS-CoV-2 in two separate tubes with FAM fluorescence channel. Each tube contains primer and probe targeting human RNase P gene in HEX fluorescence channel as an internal control for parallel evaluation of sample quality/quantity and the test performance.
Both reverse transcription and LAMP reactions take place at 60°C using the M-MLV Reverse Transcriptase and Bst DNA polymerase. During the amplification, dual labeled PNA probes can be incorporated into the amplification products. Upon the incorporation, fluorescence is generated and can be monitored by the fluorescence reader on the real-time PCR detection platforms in a real time fashion.
The analytical specificity of the assay. The analytical specificity of the test was evaluated using 35 microorganisms shown in Table 2 which are frequently found in the human respiratory tract spiked in clinical negative nasopharyngeal (NP) swab specimen at concentrations of 106 CFU/mL or higher for bacteria and 105 pfu/mL or higher for viruses. In addition, RNA isolate from the SARS-CoV-2 negative human nasal wash was tested for specificity against the human normal nasal microflora. No detectable amplification curve was observed in FAM detection channel for SARS-CoV-2 ORF1ab and N genes, whereas the internal control RNase P in HEX detection channel did show 100% detection rate as expected in all three (3) test replicates for all organisms as well as for the nasal wash. Those results showed that the exclusivity of the assay against the microorganisms tested in this study is 100% (0% of false positivity) for both ORF1ab and N gene amplicon sets. Results are summarized in Table 2.
Clinical performance of the assay against commercial Real-time PCR test. A clinical evaluation of the PNA RT-LAMP assay was performed that evaluating a total of 270 blinded clinical NP swab specimens including 70 SARS-CoV-2 positive and 200 negative individual, leftover, de-identified specimens collected in the Chungnam National University Hospital which were previously tested using commercially available FDA EUA authorized real-time PCR test targeting SARS-CoV-2 specific RdRp and E genes (PowerCheck 2019-nCoV real-time PCR kit, Kogene Biotech).
Both clinical sensitivity and specificity of the PNA RT-LAMP assay against the real-time PCR test result were confirmed to be 100% (Sensitivity 95% CI: 94.80%-100.00%; Specificity 95% CI: 98.10%-100.00%). This result showed that the clinical performance of the PNA RT-LAMP assay is comparable to the real-time PCR assay which is routinely used for molecular detection of SARS-CoV-2. The results of the clinical evaluation are summarized in Table 3.
Comparative sensitivity of the PNA RT-LAMP assay and other molecular tests. The analytical sensitivity of the PNA RT-LAMP assay was evaluated using RNA extracts from heat-inactivated SARS-CoV-2 (USA-WA1/2020, ZeptoMetrix, USA) at 10-fold dilution series spiked in SARS-CoV-2 negative NP swabs comparing with commercially available FDA EUA authorized real-time RT-PCR test (SS-9930, Seasun Biomaterials) and Colorimetric LAMP SARS-CoV-2 assay (E2019S, NEB). All three methods showed identical analytical sensitivities which exhibited the lowest detection limit of approximately 1 genomic copy of SARS-CoV-2 per microliter of RNA extract, indicating that the analytical sensitivity of the PNA RT-LAMP assay was comparable to the real-time PCR and traditional colorimetric LAMP methods (Table 4, Supplementary Fig. 1).
For further evaluation, positive detection rates of PNA RT-LAMP and Colorimetric LAMP assays were evaluated using 15 clinical individual positive NP swabs including five (5) high positives which exhibited Ct values up to 30 cycles, five (5) moderate positives which exhibited Ct values between 31 to 34 cycles, and five (5) low positives which exhibited Ct values higher than 35 cycles for both SARS-CoV-2 ORF1ab and N genes that were previously identified using the Real-time RT PCR test (SS-9930, Seasun Biomaterials). The PNA RT-LAMP assay successfully detected all 15 samples from the three positive groups whereas the colorimetric LAMP test has missed 2 low positives which exhibited Ct values over 37 cycles for both ORF1ab and N genes on the real-time PCR assay (Table 5, Supplementary Fig. 2).
Those results show that the sensitivity of the PNA RT-LAMP assay is higher than the colorimetric LAMP assay and identical to the real-time PCR method even testing the low positive samples showed late amplification rates on the real-time PCR method.
PNA RT-LMAP testing on a portable isothermal amplifier. Finally, we tested the 15 clinical high, moderate, low positive NP swabs on the PNA RT-LAMP test using a portable isothermal amplifier SMARTAMP (SS-7010, Seasun Biomaterials) which could collect fluorescence signals of FAM and HEX (excitation at 490–540 and emission at 515–555) reporter dyes in real-time manner. The device is fully portable and compatible with tablet computers with an easy-to-use operating system that can be applicable at POC testing (Fig. 1). All 15 samples with various viral loads were detectable 100% within 15 minutes when testing with the same run condition as in real-time PCR instrument (Table 5). This data shows that the PNA RT-LAMP assay can be applicable at POC testing even further evaluations with increasing clinical sample numbers are required.