Sequence-specific and multiplex detection of COVID-19 virus (SARS-CoV-2) using proofreading enzyme-mediated probe cleavage coupled with isothermal amplification.

The outbreak of COVID-19 caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has been challenging human health worldwide. Loop-mediated isothermal amplification (LAMP) has been promptly applied to the detection of SARS-CoV-2 owing to its high amplification efficacy and less requirement of the thermal cycler. However, the vast majority of these LAMP-based assays depend on the non-specific detection of LAMP products, which can not discern the undesirable amplificons, likely to yield unreliable results. Herein, a sequence-specific LAMP assay was reported to detect SARS-CoV-2 using proofreading enzyme-mediated probe cleavage (named Proofman), which could realize real-time and visual detection without uncapping. This assay, introducing a proofreading enzyme and the fluorogenic probe to reverse-transcription LAMP (RT-Proofman-LAMP), can specifically detect the SARS-CoV-2 RNA with a detection limit of 100 copies. In addition to the real-time analysis, the assay is capable of endpoint visualization under a transilluminator within 50 min, providing a convenient reporting manner under the setting of point-of-care testing (POCT). In combination with different fluorophores, the one-pot multiplex assay was successfully achieved to detect multiple targets of SARS-CoV-2 and inner control simultaneously. In summary, the development of RT-Proofman-LAMP offers a versatile and highly-specific method for fast field screening and laboratory testing of SARS-CoV-2, making it a promising platform in COVID-19 diagnosis.

Ding S ，Chen G ，Wei Y ，Dong J ，Du F ，Cui X ，Huang X ，Tang Z ... -  《-》

opvCRISPR: One-pot visual RT-LAMP-CRISPR platform for SARS-cov-2 detection.

The 2019 novel coronavirus disease (COVID-19) pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has affected all aspects of human life. Rapid, accurate, sensitive and user friendly detection method is urgently needed to facilitate early intervention and control the spread of SARS-CoV-2. Here, we propose a one-pot visual SARS-CoV-2 detection system named "opvCRISPR" by integrating reverse transcription loop-mediated isothermal amplification (RT-LAMP) and Cas12a cleavage in a single reaction system. We demonstrate that the collateral activity against single-stranded DNA (ssDNA) reporters of activated Cas12a triggered by RT-LAMP amplicon increases detection sensitivity and makes detection results observable with naked eye. The opvCRISPR enables detection at nearly single molecule level in 45 min. We validate this method with 50 SARS-CoV-2 potentially infected clinical samples. The opvCRISPR diagnostic results provide 100% agreement with the Centers for Disease Control and Prevention (CDC)-approved quantitative RT-PCR assay. The opvCRISPR holds great potential for SARS-CoV-2 detection in next-generation point-of-care molecular diagnostics.

Wang R ，Qian C ，Pang Y ，Li M ，Yang Y ，Ma H ，Zhao M ，Qian F ，Yu H ，Liu Z ，Ni T ，Zheng Y ，Wang Y ... -  《-》

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》

Multiplex reverse transcription loop-mediated isothermal amplification combined with nanoparticle-based lateral flow biosensor for the diagnosis of COVID-19.

The ongoing global pandemic (COVID-19), caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has become a huge public health issue. Hence, we devised a multiplex reverse transcription loop-mediated isothermal amplification (mRT-LAMP) coupled with a nanoparticle-based lateral flow biosensor (LFB) assay (mRT-LAMP-LFB) for diagnosing COVID-19. Using two LAMP primer sets, the ORF1ab (opening reading frame 1a/b) and N (nucleoprotein) genes of SARS-CoV-2 were simultaneously amplified in a single-tube reaction, and detected with the diagnosis results easily interpreted by LFB. In presence of FITC (fluorescein)-/digoxin- and biotin-labeled primers, mRT-LAMP produced numerous FITC-/digoxin- and biotin-attached duplex amplicons, which were determined by LFB through immunoreactions (FITC/digoxin on the duplex and anti-FITC/digoxin on the test line of LFB) and biotin/treptavidin interaction (biotin on the duplex and strptavidin on the polymerase nanoparticle). The accumulation of nanoparticles leaded a characteristic crimson band, enabling multiplex analysis of ORF1ab and N gene without instrumentation. The limit of detection (LoD) of COVID-19 mRT-LAMP-LFB was 12 copies (for each detection target) per reaction, and no cross-reactivity was generated from non-SARS-CoV-2 templates. The analytical sensitivity of SARS-CoV-2 was 100% (33/33 oropharynx swab samples collected from COVID-19 patients), and the assay's specificity was also 100% (96/96 oropharynx swab samples collected from non-COVID-19 patients). The total diagnostic test can be completed within 1 h from sample collection to result interpretation. In sum, the COVID-19 mRT-LAMP-LFB assay is a promising tool for diagnosing SARS-CoV-2 infections in frontline public health field and clinical laboratories, especially from resource-poor regions.

Zhu X ，Wang X ，Han L ，Chen T ，Wang L ，Li H ，Li S ，He L ，Fu X ，Chen S ，Xing M ，Chen H ，Wang Y ... -  《-》

A semi-automated, isolation-free, high-throughput SARS-CoV-2 reverse transcriptase (RT) loop-mediated isothermal amplification (LAMP) test.

Shortages of reverse transcriptase (RT)-polymerase chain reaction (PCR) reagents and related equipment during the COVID-19 pandemic have demonstrated the need for alternative, high-throughput methods for severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2)-mass screening in clinical diagnostic laboratories. A robust, SARS-CoV-2 RT-loop-mediated isothermal amplification (RT-LAMP) assay with high-throughput and short turnaround times in a clinical laboratory setting was established and compared to two conventional RT-PCR protocols using 323 samples of individuals with suspected SARS-CoV-2 infection. Limit of detection (LoD) and reproducibility of the isolation-free SARS-CoV-2 RT-LAMP test were determined. An almost perfect agreement (Cohen's kappa > 0.8) between the novel test and two classical RT-PCR protocols with no systematic difference (McNemar's test, P > 0.05) was observed. Sensitivity and specificity were in the range of 89.5 to 100% and 96.2 to 100% dependent on the reaction condition and the RT-PCR method used as reference. The isolation-free RT-LAMP assay showed high reproducibility (Tt intra-run coefficient of variation [CV] = 0.4%, Tt inter-run CV = 2.1%) with a LoD of 95 SARS-CoV-2 genome copies per reaction. The established SARS-CoV-2 RT-LAMP assay is a flexible and efficient alternative to conventional RT-PCR protocols, suitable for SARS-CoV-2 mass screening using existing laboratory infrastructure in clinical diagnostic laboratories.

Schmidt J ，Berghaus S ，Blessing F ，Wenzel F ，Herbeck H ，Blessing J ，Schierack P ，Rödiger S ，Roggenbuck D ... -  《Scientific Reports》