Aligner-Mediated Cleavage-Based Isothermal Amplification for SARS-CoV-2 RNA Detection.
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ABSTRACT:
SUBMITTER: Zhang C
PROVIDER: S-EPMC7931623 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:We herein describe a simple and versatile approach to use conventional nicking endonuclease (NEase) for programmable sequence-specific cleavage of DNA, termed aligner-mediated cleavage (AMC), and its application to DNA isothermal exponential amplification (AMC-based strand displacement amplification, AMC-SDA). AMC uses a hairpin-shaped DNA aligner (DA) that contains a recognition site in its stem and two side arms complementary to target DNA. Thus, it enables the loading of an NEase on DA's stem, localization to a specific locus through hybridization of the side arms with target DNA, and cleavage thereof. By using just one NEase, it is easy to make a break at any specific locus and tune the cleavage site to the single-nucleotide scale. This capability also endows the proposed AMC-SDA with excellent universality, since the cleavage of target DNA, followed by a polymerase-catalyzed extension along a particular primer as a key step for initiating SDA, no longer relies on any special sequence. Moreover, this manner of initiation facilitates the adoption of 3'-terminated primers, thus making AMC-SDA highly sensitive and highly specific, as well as simple primer design.
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Project description: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.
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Project description:As to date, more than 49 million confirmed cases of Coronavirus Disease 19 (COVID-19) have been reported worldwide. Current diagnostic protocols use qRT-PCR for viral RNA detection, which is expensive and requires sophisticated equipment, trained personnel and previous RNA extraction. For this reason, we need a faster, direct and more versatile detection method for better epidemiological management of the COVID-19 outbreak. In this work, we propose a direct method without RNA extraction, based on the Loop-mediated isothermal amplification (LAMP) and Clustered Regularly Interspaced Short Palindromic Repeats-CRISPR associated protein (CRISPR-Cas12) technique that allows the fast detection of SARS-CoV-2 from patient samples with high sensitivity and specificity. We obtained a limit of detection of 16 copies/?L with high specificity and at an affordable cost. The diagnostic test readout can be done with a real-time PCR thermocycler or with the naked eye in a blue-light transilluminator. Our method has been evaluated on a small set of clinical samples with promising results.
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Project description:BackgroundCurrent diagnosis of SARS-CoV-2 infection relies on RNA purification prior to amplification. Typical extraction methods limit the processing speed and turnaround time for SARS-CoV-2 diagnostic testing.MethodsHere, we applied reverse transcription loop-mediated isothermal amplification directly onto human clinical swabs samples to amplify the RNA from SARS-CoV-2 swab samples after processing with chelating resin.ResultsBy testing our method on 64 samples, we managed to develop an RT-LAMP assay with 95.9% sensitivity (95% CI 86 to 99.5%) and 100% specificity (95% CI 78.2-100%).ConclusionThe entire process including sample processing can be completed in approximately 50 min. This method has promising potential to be applied as a fast, simple and inexpensive diagnostic tool for the detection of SARS-CoV-2.
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Project description:In the fight against the recent COVID-19 pandemics, testing is crucial. Nasopharyngeal swabs and real-time RT-PCR are used for the detection of the viral RNA. The collection of saliva is non-invasive, pain-free and does not require trained personnel. An alternative to RT-PCR is loop-mediated isothermal amplification coupled with reverse transcription (RT-LAMP) that is easy to perform, quick and does not require a thermal cycler. The aim of this study was to test whether SARS-CoV-2 RNA can be detected directly in saliva using RT-LAMP. We have tested 16 primer mixes from the available literature in three rounds of sensitivity assays. The selected RT-LAMP primer mix has a limit of detection of 6 copies of viral RNA per reaction in comparison with RT-PCR with 1 copy per reaction. Whole saliva, as well as saliva collected using Salivette collection tubes, interfered with the RT-LAMP analysis. Neither Chelex-100 nor protease treatment of saliva prevented the inhibitory effect of saliva. With the addition of the ribonuclease inhibitor, the sensitivity of the RT-LAMP assay was 12 copies per reaction of RNA in Salivette® saliva samples and 6 copies per reaction of RNA in whole saliva samples. This study shows that it is possible to combine the use of saliva and RT-LAMP for SARS-CoV-2 RNA detection without RNA extraction which was confirmed on a small set of correctly diagnosed clinical samples. Further studies should prove whether this protocol is suitable for point of care testing in the clinical setting.
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Project description:The availability of simple SARS-CoV-2 detection methods is crucial to contain the COVID-19 pandemic. This study examined whether a commercial LAMP assay can reliably detect SARS-CoV-2 genomes directly in respiratory samples without having to extract nucleic acids (NA) beforehand. Nasopharyngeal swabs (NPS, n = 220) were tested by real-time reverse transcription (RT)-PCR and with the LAMP assay. For RT-PCR, NA were investigated. For LAMP, NA from 26 NPS in viral transport medium (VTM) were tested. The other 194 NPS were analyzed directly without prior NA extraction (140 samples in VTM; 54 dry swab samples stirred in phosphate buffered saline). Ten NPS were tested directly by LAMP using a sous-vide cooking unit. The isothermal assay demonstrated excellent specificity (100%) but moderate sensitivity (68.8%), with a positive predictive value of 1 and a negative predictive value of 0.65 for direct testing of NPS in VTM. The use of dry swabs, even without NA extraction, improved the analytical sensitivity; up to 6% of samples showed signs of inhibition. LAMP could be performed successfully with a sous-vide cooking unit. This technique is very fast, requires little laboratory resources, and can replace rapid antigen tests or verify reactive rapid tests on-site.
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