High-Surety Isothermal Amplification and Detection of SARS-CoV-2.

Isothermal nucleic acid amplification tests (iNATs), such as loop-mediated isothermal amplification (LAMP), are good alternatives to PCR-based amplification assays, especially for point-of-care and low-resource use, in part because they can be carried out with relatively simple instrumentation. However, iNATs can often generate spurious amplicons, especially in the absence of target sequences, resulting in false-positive results. This is especially true if signals are based on non-sequence-specific probes, such as intercalating dyes or pH changes. In addition, pathogens often prove to be moving, evolving targets and can accumulate mutations that will lead to inefficient primer binding and thus false-negative results. Multiplex assays targeting different regions of the analyte and logical signal readout using sequence-specific probes can help to reduce both false negatives and false positives. Here, we describe rapid conversion of three previously described SARS-CoV-2 LAMP assays that relied on a non-sequence-specific readout into individual and multiplex one-pot assays that can be visually read using sequence-specific oligonucleotide strand exchange (OSD) probes. We describe both fluorescence-based and Boolean logic-gated colorimetric lateral flow readout methods and demonstrate detection of SARS-CoV-2 virions in crude human saliva.IMPORTANCE One of the key approaches to treatment and control of infectious diseases, such as COVID-19, is accurate and rapid diagnostics that is widely deployable in a timely and scalable manner. To achieve this, it is essential to go beyond the traditional gold standard of quantitative PCR (qPCR) that is often faced with difficulties in scaling due to the complexity of infrastructure and human resource requirements. Isothermal nucleic acid amplification methods, such as loop-mediated isothermal amplification (LAMP), have been long pursued as ideal, low-tech alternatives for rapid, portable testing. However, isothermal approaches often suffer from false signals due to employment of nonspecific readout methods. We describe general principles for rapidly converting nonspecifically read LAMP assays into assays that are read in a sequence-specific manner by using oligonucleotide strand displacement (OSD) probes. We also demonstrate that inclusion of OSD probes in LAMP assays maintains the simplicity of one-pot assays and a visual yes/no readout by using fluorescence or colorimetric lateral-flow dipsticks while providing accurate sequence-specific readout and the ability to logically query multiplex amplicons for redundancy or copresence. These principles not only yielded high-surety isothermal assays for SARS-CoV-2 but might also aid in the design of more sophisticated molecular assays for other analytes.

Bhadra S ，Riedel TE ，Lakhotia S ，Tran ND ，Ellington AD ... -  《mSphere》

Comparative analysis of loop-mediated isothermal amplification (LAMP)-based assays for rapid detection of SARS-CoV-2 genes.

The COVID-19 pandemic caused by SARS-CoV-2 has infected millions worldwide, therefore there is an urgent need to increase our diagnostic capacity to identify infected cases. Although RT-qPCR remains the gold standard for SARS-CoV-2 detection, this method requires specialised equipment in a diagnostic laboratory and has a long turn-around time to process the samples. To address this, several groups have recently reported the development of loop-mediated isothermal amplification (LAMP) as a simple, low cost and rapid method for SARS-CoV-2 detection. Herein we present a comparative analysis of three LAMP-based assays that target different regions of the SARS-CoV-2: ORF1ab RdRP, ORF1ab nsp3 and Gene N. We perform a detailed assessment of their sensitivity, kinetics and false positive rates for SARS-CoV-2 diagnostics in LAMP or RT-LAMP reactions, using colorimetric or fluorescent detection. Our results independently validate that all three assays can detect SARS-CoV-2 in 30 min, with robust accuracy at detecting as little as 1000 RNA copies and the results can be visualised simply by color changes. Incorporation of RT-LAMP with fluorescent detection further increases the detection sensitivity to as little as 100 RNA copies. We also note the shortcomings of some LAMP-based assays, including variable results with shorter reaction time or lower load of SARS-CoV-2, and false positive results in some experimental conditions and clinical saliva samples. Overall for RT-LAMP detection, the ORF1ab RdRP and ORF1ab nsp3 assays have faster kinetics for detection but varying degrees of false positives detection, whereas the Gene N assay exhibits no false positives in 30 min reaction time, which highlights the importance of optimal primer design to minimise false-positives in RT-LAMP. This study provides validation of the performance of LAMP-based assays as a rapid, highly sensitive detection method for SARS-CoV-2, which have important implications in development of point-of-care diagnostics for SARS-CoV-2.

Urrutia-Cabrera D ，Liou RH ，Wang JH ，Chan J ，Hung SS ，Hewitt AW ，Martin KR ，Edwards TL ，Kwan P ，Wong RC ... -  《Scientific Reports》

Detection of SARS-CoV-2 RNA using RT-LAMP and molecular beacons.

Sherrill-Mix S ，Hwang Y ，Roche AM ，Glascock A ，Weiss SR ，Li Y ，Haddad L ，Deraska P ，Monahan C ，Kromer A ，Graham-Wooten J ，Taylor LJ ，Abella BS ，Ganguly A ，Collman RG ，Van Duyne GD ，Bushman FD ... -  《GENOME BIOLOGY》

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 ... -  《-》

Multisite Clinical Validation of Isothermal Amplification-Based SARS-CoV-2 Detection Assays Using Different Sampling Strategies.

Desai KT ，Alfaro K ，Mendoza L ，Faron M ，Mesich B ，Maza M ，Dominguez R ，Valenzuela A ，Acosta CD ，Martínez M ，Felix JC ，Masch R ，Smith JS ，Gabrilovich S ，Wu T ，Plump M ，Novetsky AP ，Einstein MH ，Douglas NC ，Cremer M ，Wentzensen N ... -  《Microbiology Spectrum》