Journal: Scientific Reports

Article Title: Validation of a rapid, saliva-based, and ultra-sensitive SARS-CoV-2 screening system for pandemic-scale infection surveillance

doi: 10.1038/s41598-022-08263-4

Figure Lengend Snippet: The presence of mineral oil markedly reduces the rate of production of false positive RT-LAMP reactions. Two assay chemistries were compared: NEB WarmStart Colorimetric LAMP with UDG (M1804) and Hayat Rapid Colorimetric & Fluorometric One Step LAMP SARS-CoV-2 Test Kit, each set up with and without 15 μl mineral oil overlay. Twenty-one identical replicate negative control reactions were set up per condition with a single saliva sample negative for SARS-CoV-2 diluted in VTM and AviSal at a ratio of 1:1:2 and heat inactivated for 10 min at 95 °C. The sample was added to give 5% (NEB) and 3.75% (Hayat) final concentrations of crude saliva in a 25 μl reaction volume. + M.O. with mineral oil overlay; − M.O. without mineral oil. In a typical 30-min reaction runtime, only Hayat chemistry resulted in no false positives and 100% specificity. For the NEB chemistry, false positives were observed after 20 min, even with a mineral oil overlay. Mineral oil overlay markedly reduces the false positive rate.

Article Snippet: Two assay chemistries were compared: NEB WarmStart Colorimetric LAMP with UDG (M1804) and Hayat Rapid Colorimetric & Fluorometric One Step LAMP SARS-CoV-2 Test Kit, each set up with and without 15 μl mineral oil overlay.

Techniques: Negative Control

Journal: Scientific Reports

Article Title: Validation of a rapid, saliva-based, and ultra-sensitive SARS-CoV-2 screening system for pandemic-scale infection surveillance

doi: 10.1038/s41598-022-08263-4

Figure Lengend Snippet: Stability of saliva samples during short-term storage determined by RT-PCR and RT-LAMP. Five sample pools were prepared from ten saliva samples to give a range of viral loads corresponding to predicted Ct values (from extracted RNA) from 24.6 to 35.0 for the N-gene and 25.8 to 39.1 for ORF1ab. Sample pools were diluted with an equal volume of AviSal buffer and stored short-term under the following test conditions prior to heat inactivation: room temperature (RT) for 0, 6, 24 and 48 h (t = 0, 6 h, 24 h and 48 h) or 4 °C for 48 h. Samples were then heat inactivated at 95 °C for 10 min and frozen. Upon thawing, samples were stored post inactivation at either 4 °C or RT for 2 h or 4 °C for 48 h (t = 0 only). The 4 °C incubation was taken as baseline as a proxy for no post-inactivation storage. RT-PCR (BGI Real-Time Fluorescent RT-PCR Kit for Detecting SARS-CoV-2), targeting ORF1ab ( A ) and the N gene ( B ), and RT-LAMP (Hayat Genetics) ( C , D ) assays were performed on samples subjected to each of these conditions. To understand the effect of freezing samples prior to heat inactivation, for the t = 0 timepoint, a fresh versus frozen comparison was performed. Viral loads (as measured by RT-PCR on extracted RNA) of sample pools #3, 4 and 5 are too low for 100% sensitivity of detection by RT-LAMP—all have Ct ≥ 33 for N gene. Hence, not all reactions were positive within the 30 min runtime ( D ). All Ct (Cycle Threshold) and TTT (Time to Threshold) values were calculated as the mean of technical duplicates.

Article Snippet: Two assay chemistries were compared: NEB WarmStart Colorimetric LAMP with UDG (M1804) and Hayat Rapid Colorimetric & Fluorometric One Step LAMP SARS-CoV-2 Test Kit, each set up with and without 15 μl mineral oil overlay.

Techniques: Reverse Transcription Polymerase Chain Reaction, Incubation