scholarly journals 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)

2020 ◽  
pp. 114048
Author(s):  
Emma L.A. Howson ◽  
Stephen P. Kidd ◽  
Bryony Armson ◽  
Alice Goring ◽  
Jason Sawyer ◽  
...  
Keyword(s):  
Sample Preparation ◽  
Reverse Transcription ◽  
Preparation Method ◽  
Direct Detection ◽  
Isothermal Amplification ◽  
Sample Preparation Method ◽  
Loop Mediated Isothermal Amplification

scholarly journals 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)

2020 ◽  
Author(s):  
Emma Howson ◽  
Stephen Kidd ◽  
Jason Sawyer ◽  
Claire Cassar ◽  
David Cross ◽  
...  
Keyword(s):  
Sample Preparation ◽  
Reverse Transcription ◽  
Preparation Method ◽  
Direct Detection ◽  
Saliva Sample ◽  
Rna Extraction ◽  
Isothermal Amplification ◽  
Molecular Testing ◽  
Sample Preparation Method ◽  
Loop Mediated Isothermal Amplification

We describe the optimization 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 MucolyseTM, followed by dilution (within the range of 1 in 5 to 1 in 40) in 10% (w/v) Chelex 100 Resin and a 98oC heat step for 2 minutes enabled detection of SARS-CoV-2 RNA in all positive saliva samples tested, with no amplification detected in pooled negative saliva. The time to positivity for which SARS-CoV-2 RNA was detected in these positive saliva samples was proportional to the real-time reverse-transcriptase PCR cycle threshold (CT), with SARS-CoV-2 RNA detected in as little as 05:43 (CT 21.08), 07:59 (CT 24.47) and 08:35 (CT 25.27) minutes, respectively. The highest CT where direct RT-LAMP detected SARS-CoV-2 RNA was 31.39 corresponding to a 1 in 40 dilution of a positive saliva sample (1:1 in MucolyseTM) with a starting CT of 25.27. When RT-LAMP was performed on pools of SARS-CoV-2 negative saliva samples spiked with whole inactivated SARS-CoV-2 virus, RNA was detected at dilutions spanning 1 in 5 to 1 in 160 representing CTs spanning 22.49-26.43. Here we describe a simple but critical rapid sample preparation method which can be used up front of RT-LAMP to permit direct detection of SARS-CoV-2 within saliva samples. Saliva is a sample which can be collected non-invasively without the use of highly skilled staff and critically can be obtained from both healthcare and home settings. Critically, this approach overcomes both the requirement and validation of different swabs and the global bottleneck in obtaining RNA extraction robots and reagents to enable molecular testing by PCR. Such testing opens the possibility of public health approaches for effective intervention to control the COVID-19 pandemic through regular SARS-CoV-2 testing at a population scale, combined with isolation and contact tracing for positive cases.

scholarly journals Two extraction-free reverse transcription loop-mediated isothermal amplification assays for detection of SARS-CoV-2

2021 ◽  
Vol 21 (1) ◽  
Author(s):  
Meng Yee Lai ◽  
Fatma Diyana Mohd Bukhari ◽  
Nur Zulaikha Zulkefli ◽  
Ilyiana Ismail ◽  
Nur Izati Mustapa ◽  
...  
Keyword(s):  
Reverse Transcription ◽  
Virus Isolation ◽  
Direct Detection ◽  
Isothermal Amplification ◽  
Rapid Screening ◽  
Nasopharyngeal Swab ◽  
Purification Step ◽  
Simple Method ◽  
Loop Mediated Isothermal Amplification ◽  
Rna Purification

Abstract Background Current assays for detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) rely on time consuming, costly and laboratory based methods for virus isolation, purification and removing inhibitors. To address this limitation, we propose a simple method for testing RNA from nasopharyngeal swab samples that bypasses the RNA purification step. Methods In the current project, we have described two extraction-free reverse transcription loop-mediated isothermal amplification (RT-LAMP) assays for the detection of SARS-CoV-2 by using E gene and RdRp gene as the targets. Results Here, results showed that reverse transcription loop-mediated isothermal amplification assays with 88.4% sensitive (95% CI: 74.9–96.1%) and 67.4% sensitive (95% CI: 51.5–80.9%) for E gene and RdRp gene, respectively. Conclusion Without the need of RNA purification, our developed RT-LAMP assays for direct detection of SARS-CoV-2 from nasopharyngeal swab samples could be turned into alternatives to qRT-PCR for rapid screening.

To Eliminate Curtain Effect of FIB TEM Samples by a Combination of Sample Dicing and Backside Milling

2014 ◽  
Author(s):  
Jian-Shing Luo ◽  
Hsiu Ting Lee
Keyword(s):  
Sample Preparation ◽  
Focused Ion Beam ◽  
Preparation Method ◽  
Low Cost ◽  
Ion Beam ◽  
Sample Preparation Method ◽  
Site Specific ◽  
Dual Beam ◽  
Transmission Electron

Abstract Several methods are used to invert samples 180 deg in a dual beam focused ion beam (FIB) system for backside milling by a specific in-situ lift out system or stages. However, most of those methods occupied too much time on FIB systems or requires a specific in-situ lift out system. This paper provides a novel transmission electron microscopy (TEM) sample preparation method to eliminate the curtain effect completely by a combination of backside milling and sample dicing with low cost and less FIB time. The procedures of the TEM pre-thinned sample preparation method using a combination of sample dicing and backside milling are described step by step. From the analysis results, the method has applied successfully to eliminate the curtain effect of dual beam FIB TEM samples for both random and site specific addresses.

Cross-Section Sample Preparation Method for Imaging Dopant Related Anomalies Using Scanning Probe Microscopy Techniques

2014 ◽  
Author(s):  
Swaminathan Subramanian ◽  
Khiem Ly ◽  
Tony Chrastecky
Keyword(s):  
Sample Preparation ◽  
Failure Analysis ◽  
Cross Section ◽  
Scanning Probe Microscopy ◽  
Preparation Method ◽  
Fault Isolation ◽  
Scanning Probe ◽  
Sample Preparation Method ◽  
Probe Microscopy ◽  
Section Sample

Abstract Visualization of dopant related anomalies in integrated circuits is extremely challenging. Cleaving of the die may not be possible in practical failure analysis situations that require extensive electrical fault isolation, where the failing die can be submitted of scanning probe microscopy analysis in various states such as partially depackaged die, backside thinned die, and so on. In advanced technologies, the circuit orientation in the wafer may not align with preferred crystallographic direction for cleaving the silicon or other substrates. In order to overcome these issues, a focused ion beam lift-out based approach for site-specific cross-section sample preparation is developed in this work. A directional mechanical polishing procedure to produce smooth damage-free surface for junction profiling is also implemented. Two failure analysis applications of the sample preparation method to visualize junction anomalies using scanning microwave microscopy are also discussed.

scholarly journals Application of the Dehydration Homogeneous Liquid–Liquid Extraction (DHLLE) Sample Preparation Method for Fingerprinting of Honey Volatiles

Molecules ◽  
2021 ◽  
Vol 26 (8) ◽  
pp. 2277
Author(s):  
Piotr M. Kuś ◽  
Igor Jerković
Keyword(s):  
Carboxylic Acid ◽  
Sample Preparation ◽  
Preparation Method ◽  
Liquid Extraction ◽  
Sample Preparation Method ◽  
Phenyllactic Acid ◽  
Homogeneous Liquid ◽  
Liquid Liquid Extraction ◽  
Wide Range ◽  
Chemical Groups

Recently, we proposed a new sample preparation method involving reduced solvent and sample usage, based on dehydration homogeneous liquid–liquid extraction (DHLLE) for the screening of volatiles and semi-volatiles from honey. In the present research, the method was applied to a wide range of honeys (21 different representative unifloral samples) to determine its suitability for detecting characteristic honey compounds from different chemical classes. GC-FID/MS disclosed 130 compounds from different structural and chemical groups. The DHLLE method allowed the extraction and identification of a wide range of previously reported specific and nonspecific marker compounds belonging to different chemical groups (including monoterpenes, norisoprenoids, benzene derivatives, or nitrogen compounds). For example, DHLLE allowed the detection of cornflower honey chemical markers: 3-oxo-retro-α-ionols, 3,4-dihydro-3-oxoedulan, phenyllactic acid; coffee honey markers: theobromine and caffeine; linden honey markers: 4-isopropenylcyclohexa-1,3-diene-1-carboxylic acid and 4-(2-hydroxy-2-propanyl)cyclohexa-1,3-diene-1-carboxylic acid, as well as furan derivatives from buckwheat honey. The obtained results were comparable with the previously reported data on markers of various honey varieties. Considering the application of much lower volumes of very common reagents, DHLLE may provide economical and ecological advantages as an alternative sample preparation method for routine purposes.

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