Protocols in Current Issue
Protocols in Past Issues
0 Q&A 4902 Views Mar 20, 2021

During pandemics, such as the one caused by SARS-CoV-2 coronavirus, simple methods to rapidly test large numbers of people are needed. As a faster and less resource-demanding alternative to detect viral RNA by conventional qPCR, we used reverse transcription loop-mediated isothermal amplification (RT-LAMP). We previously established colorimetric RT-LAMP assays on both purified and unpurified SARS-CoV-2 clinical specimens and further developed a multiplexed sequencing protocol (LAMP-sequencing) to analyze the outcome of many RT-LAMP reactions at the same time (Dao Thi et al., 2020). Extending on this work, we hereby provide step-by-step protocols for both RT-LAMP assays and read-outs.

0 Q&A 4505 Views Nov 5, 2020
Standard diagnostic methods of Coronavirus Disease 2019 (COVID-19) rely on RT-qPCR technique which have limited point-of-care test (POCT) potential due to necessity of dedicated equipment and specialized personnel. LAMP, an isothermal nucleic acid amplification test (NAAT), is a promising technique that may substitute RT-qPCR for POCT of genomic materials. Here, we provide a protocol to perform reverse transcription LAMP targeting SARS-CoV-2. We adopted both real-time fluorescence detection and end-point colorimetric detection approaches. Our protocol would be useful for screening diagnosis of COVID-19 and be a baseline for development of improved POCT NAAT.
0 Q&A 3763 Views Oct 20, 2020
SARS-CoV-2 has quickly spread all around the globe causing illness and wide damages. Most countries were unprepared for such a rapid spread and crisis. This led to various strategies for effective control of the new pandemic. A key aspect in all countries was to effectively test the population for the virus. Most countries chose a lockdown strategy in which many workplaces and activities are completely closed, leading to substantial economy costs. Here, we present a protocol we recently developed that allows rapid and simple detection of SARS-CoV-2 for the large population, eliminating costs and involvement of professional teams and laboratories. This protocol is based on Reverse Transcribed Loop-Mediated Isothermal Amplification (RT-LAMP). We tested this protocol directly on patient samples, both nasal and throat clinical swabs as well as saliva. Notably, this protocol is simple, cheap and can be easily applied to other pathogens as well.
0 Q&A 5119 Views Nov 5, 2019
Loop-mediated isothermal amplification (LAMP) has been widely used in the detection of pathogens. However, there are usually numerous variants in one viral pathogen and primers employed in LAMP can hardly match all these variants. The mismatches between the primers and the viral genomes, especially those at the 3′-end of the primers, hinder LAMP reactions, leading to failure of the detection. Here, we present a mismatch-tolerant RT-LAMP protocol, which utilizes the 3′-5′ exonuclease activity of the Q5 high-fidelity DNA polymerase to remove potential mismatched bases at the 3′-end of the primers during LAMP amplification. Using HIV-1 as a proof-of-principle, we showed that this protocol could represent a promising tool for accurate detection of genetically unstable viruses in laboratory, hospital and field.

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