ABSTRACT The Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) pandemic demanded rapid diagnosis to isolate new COVID-19 cases and prevent disease transmission. Quantitative Real-Time Polymerase Chain Reaction (qRT-PCR) rapidly became the gold standard for diagnosis. However, due to the high cost and delay of the results, other types of diagnosis were implemented, such as COVID-19 Ag Rapid Tests and Reverse Transcription Technique followed by Loop-Mediated isothermal Amplification (RT-LAMP). In this work, we validated the use of RT-LAMP in saliva samples rather than nasopharyngeal swabs, as the collection is more comfortable. First, we selected 5 primer sets based on the limit of detection for SARS-CoV-2 RNA, then validated their sensitivity and specificity in patient samples. A total of 117 samples were analyzed by fluorometric RT-LAMP and compared with qRT-PCR results. Our results show that the use of a high-sensitive primer ORF1-a, together with a low-sensitive primer set Gene E (time to threshold of 22.9 and 36.4 minutes, respectively, using 200 copies of viral RNA), achieved sensitivity in purified RNA from saliva samples of 95.2% (95% CI 76.1-99.8) with 90.5% specificity (95% CI 69.6-98.8) (n = 42).As RNA purification increases the turnaround time, we tested the outcome of RT-LAMP utilizing raw saliva samples without purification. The test achieved a sensitivity of 81.8% (95% CI 59.7-94.8) and a specificity of 90.9% (95% CI 70.8-98.8). As a result, the accuracy of 92.9% (95% CI 80.5-98.5) in purified RNA-saliva samples was lowered to an acceptable level of 86.4% (95% CI 72.6-94.8) in raw saliva. Although mass vaccination has been implemented, new strains and low vaccination progress helped to spread COVID-19. This study shows that it is feasible to track new COVID-19 cases in a large population with the use of raw saliva as sample in RT-LAMP assay which yields accurate results and offers a less invasive test. Coronavirus2 Coronavirus 2 Coronavirus- SARSCoV2 SARSCoV SARS CoV (SARS-CoV-2 COVID19 COVID 19 COVID-1 transmission RealTime Real Time qRTPCR qRT PCR (qRT-PCR However implemented LoopMediated Loop Mediated RTLAMP. RTLAMP RT LAMP . (RT-LAMP) work swabs comfortable First SARS-CoV- 11 highsensitive sensitive ORF1a, ORF1a ORFa ORF1 a, ORF ORF1-a lowsensitive time 229 22 9 22. 364 36 4 36. minutes respectively 20 , RNA) 952 95 95.2 95% (95 76.199.8 761998 76.1 99.8 76 1 99 8 76.1-99.8 905 90 90.5 69.698.8 696988 69.6 98.8 69 6 98 69.6-98.8 n 42.As 42As 42 .As 818 81 81.8 59.794.8 597948 59.7 94.8 59 7 94 59.7-94.8 909 90.9 70.898.8. 708988 70.8 70 70.8-98.8) result 929 92 92.9 80.598.5 805985 80.5 98.5 80 80.5-98.5 RNAsaliva 864 86 86.4 72.694.8 726948 72.6 72 72.6-94.8 COVID19. 19. (SARS-CoV- COVID1 COVID- (RT-LAMP SARS-CoV 3 95. (9 199 76.199. 76199 761 76. 998 99. 76.1-99. 90. 698 69.698. 69698 696 69. 988 98. 69.6-98. 81. 794 59.794. 59794 597 59. 948 94. 59.7-94. 898 70.898.8 70898 708 70. 70.8-98.8 92. 598 80.598. 80598 805 80. 985 80.5-98. 86. 694 72.694. 72694 726 72. 72.6-94. (SARS-CoV ( 76.199 7619 76.1-99 69.698 6969 69.6-98 79 59.794 5979 59.7-94 89 70.898. 7089 70.8-98. 80.598 8059 80.5-98 72.694 7269 72.6-94 76.19 76.1-9 69.69 69.6-9 59.79 59.7-9 70.898 70.8-98 80.59 80.5-9 72.69 72.6-9 76.1- 69.6- 59.7- 70.89 70.8-9 80.5- 72.6- 70.8-

ABSTRACT Introduction: In the current standard of care (SoC) RT-PCR method for COVID-19, the patient’s swab was extracted in viral transport media (VTM). For the PanbioTM COVID-19 Ag Rapid Test, the patient swab is flushed out in extraction buffer, of which a small fraction is used for testing, leaving more than half the sample unused. This study was designed to show that RT-PCR results from the residual sample of the PanbioTM COVID-19 Ag Rapid Test (called Novel RT-PCR) are not worse than the SoC RT-PCR result. Methods: The study was performed using (1) dilution series of five patient samples, and (2) 413 patient samples comparing SOC versus Novel RT-PCR results. Results: For the dilution series samples, all tested positive by both methods. The bias between Ct values of Novel RT-PCR and SoC RT-PCR did not exceed 3.00 Ct using primers N1 and N2. A total of 413 COVID symptomatic patients seeking COVID testing were tested, of which 89 patients tested positive and 324 tested negative with SoC RT-PCR. In 324 patients who tested negative with SoC RT-PCR, 323 tested negative with Novel RT-PCR, and one (1) tested positive. Out of 89 who tested positive with SoC RT-PCR, 80 tested positive with the Novel RT-PCR, and nine patients showed a negative test result. The Overall Percent Agreement for the 413 valid patient sample pairs was 97.5 [95% CI 97 to 98]. Conclusion: The study demonstrated that the performance of the Novel RT-PCR method is acceptable compared to the SoC RT-PCR method and can be a useful tool to perform RTPCR without the need for new swab collections.