A highly effective reverse-transcription loop-mediated isothermal amplification (RT-LAMP) assay for the rapid detection of SARS-CoV-2 infection.

The COVID-19 pandemic has illustrated the importance of simple, rapid and accurate diagnostic testing. This study describes the validation of a new rapid SARS-CoV-2 RT-LAMP assay for use on extracted RNA or directly from swab offering an alternative diagnostic pathway that does not rely on traditional reagents that are often in short supply during a pandemic. Analytical specificity (ASp) of this new RT-LAMP assay was 100% and analytical sensitivity (ASe) was between 1 × 101 and 1 × 102 copies per reaction when using a synthetic DNA target. The overall diagnostic sensitivity (DSe) and specificity (DSp) of RNA RT-LAMP was 97% and 99% respectively, relative to the standard of care rRT-PCR. When a CT cut-off of 33 was employed, above which increasingly evidence suggests there is a low risk of patients shedding infectious virus, the diagnostic sensitivity was 100%. The DSe and DSp of Direct RT-LAMP (that does not require RNA extraction) was 67% and 97%, respectively. When setting CT cut-offs of ≤33 and ≤25, the DSe increased to 75% and 100%, respectively, time from swab-to-result, CT < 25, was < 15 min. We propose that RNA RT-LAMP could replace rRT-PCR where there is a need for increased sample throughput and Direct RT-LAMP as a near-patient screening tool to rapidly identify highly contagious individuals within emergency departments and care homes during times of increased disease prevalence ensuring negative results still get laboratory confirmation.

Fowler VL ，Armson B ，Gonzales JL ，Wise EL ，Howson ELA ，Vincent-Mistiaen Z ，Fouch S ，Maltby CJ ，Grippon S ，Munro S ，Jones L ，Holmes T ，Tillyer C ，Elwell J ，Sowood A ，de Peyer O ，Dixon S ，Hatcher T ，Patrick H ，Laxman S ，Walsh C ，Andreou M ，Morant N ，Clark D ，Moore N ，Houghton R ，Cortes NJ ，Kidd SP ... -  《-》

SARS-CoV-2 detection by fluorescence loop-mediated isothermal amplification with and without RNA extraction.

Rapid and simple point-of-care detection of SARS-CoV-2 is an urgent need to prevent pandemic. Reverse transcription loop-mediated isothermal amplification (RT-LAMP) can detect SARS-CoV-2 more rapidly than RT-PCR. Saliva is non-invasive specimen suitable for mass-screening, but data comparing utility of nasopharyngeal swab (NPS) and saliva in RT-LAMP test are lacking and it remains unclear whether SARS-CoV-2 could be detected by direct processing of samples without the need for prior RNA extraction saliva. In this study, we compared utility of saliva and NPS samples for the detection of SARS-CoV-2 by a novel RT-fluorescence LAMP (RT-fLAMP). The sensitivity and specificity of the RT-fLAMP with RNA extraction were 97% and 100%, respectively, with equivalent utility of NPS and saliva. However, sensitivity was decreased to 71% and 47% in NPS and saliva samples without RNA extraction, respectively, suggesting that RNA extraction process may be critical for the virus detection by RT-fLAMP.

Taki K ，Yokota I ，Fukumoto T ，Iwasaki S ，Fujisawa S ，Takahashi M ，Negishi S ，Hayasaka K ，Sato K ，Oguri S ，Nishida M ，Sugita J ，Konno S ，Saito T ，Teshima T ... -  《-》

Preliminary optimisation of a simplified sample preparation method to permit direct detection of SARS-CoV-2 within saliva samples using reverse-transcription loop-mediated isothermal amplification (RT-LAMP).

We describe the optimisation of a simplified sample preparation method which permits rapid and direct detection of SARS-CoV-2 RNA within saliva, using reverse-transcription loop-mediated isothermal amplification (RT-LAMP). Treatment of saliva samples prior to RT-LAMP by dilution 1:1 in Mucolyse™, followed by dilution in 10 % (w/v) Chelex© 100 Resin and a 98 °C heat step for 2 min enabled detection of SARS-CoV-2 RNA in positive saliva samples. Using RT-LAMP, SARS-CoV-2 RNA was detected in as little as 05:43 min, with no amplification detected in 3097 real-time reverse transcription PCR (rRT-PCR) negative saliva samples from staff tested within a service evaluation study, or for other respiratory pathogens tested (n = 22). Saliva samples can be collected non-invasively, without the need for skilled staff and can be obtained from both healthcare and home settings. Critically, this approach overcomes the requirement for, and validation of, different swabs and the global bottleneck in obtaining access to extraction robots and reagents to enable molecular testing by rRT-PCR. Such testing opens the possibility of public health approaches for effective intervention during the COVID-19 pandemic through regular SARS-CoV-2 testing at a population scale, combined with isolation and contact tracing.

Howson ELA ，Kidd SP ，Armson B ，Goring A ，Sawyer J ，Cassar C ，Cross D ，Lewis T ，Hockey J ，Rivers S ，Cawthraw S ，Banyard A ，Anderson P ，Rahou S ，Andreou M ，Morant N ，Clark D ，Walsh C ，Laxman S ，Houghton R ，Slater-Jefferies J ，Costello P ，Brown I ，Cortes N ，Godfrey KM ，Fowler VL ... -  《-》

Colorimetric reverse transcription loop-mediated isothermal amplification (RT-LAMP) as a visual diagnostic platform for the detection of the emerging coronavirus SARS-CoV-2.

Nawattanapaiboon K ，Pasomsub E ，Prombun P ，Wongbunmak A ，Jenjitwanich A ，Mahasupachai P ，Vetcho P ，Chayrach C ，Manatjaroenlap N ，Samphaongern C ，Watthanachockchai T ，Leedorkmai P ，Manopwisedjaroen S ，Akkarawongsapat R ，Thitithanyanont A ，Phanchana M ，Panbangred W ，Chauvatcharin S ，Srikhirin T ... -  《-》

Extraction-free RT-LAMP to detect SARS-CoV-2 is less sensitive but highly specific compared to standard RT-PCR in 101 samples.

The current scale of public and private testing cannot be expected to meet the emerging need for higher levels of community-level and repeated screening of asymptomatic Canadians for SARS-CoV-2. Rapid point-of-care techniques are increasingly being offered to fill the gap in screening levels required to identify undiagnosed individuals with high viral loads. However, rapid, point-of-care tests often have lower sensitivity in practice. Reverse transcription loop-mediated isothermal amplification (RT-LAMP) for SARS-CoV-2 has proven sensitive and specific and provides visual results in minutes. Using a commercially available kit for RT-LAMP and primer set targetting nucleocapsid (N), we tested a blinded set of 101 archived nasopharyngeal (NP) swab samples with known RT-PCR results. RT-LAMP reactions were incubated at 65 °C for 30 min, using heat-inactivated nasopharyngeal swab sample in viral transport medium, diluted tenfold in water, as input. RT-LAMP agreed with all RT-PCR defined negatives (N = 51), and all positives with cycle threshold (Ct) less than 20 (N = 24), 65% of positives with Ct between 20-30 (N = 17), and no positives with Ct greater than 30 (N = 9). RT-LAMP requires fewer and different core components, so may not compete directly with the mainline testing workflow, preserving precious central laboratory resources for those with the greatest need. Careful messaging must be provided when using less-sensitive tests, so that people are not falsely reassured by negative results, but this caveat must be weighed against the clear benefits of reliably identifying those with high levels of virus in prioritized samples at the point of care.

Schellenberg JJ ，Ormond M ，Keynan Y 《-》