scholarly journals Rapid and extraction-free detection of SARS-CoV-2 from saliva with colorimetric LAMP

2020 ◽  
Author(s):  
Matthew A. Lalli ◽  
Xuhua Chen ◽  
S. Joshua Langmade ◽  
Catrina C. Fronick ◽  
Christopher S. Sawyer ◽  
...  
Keyword(s):  
High Performance ◽  
Colorimetric Assay ◽  
Rna Extraction ◽  
Human Saliva ◽  
Clinical Samples ◽  
Treatment Protocols ◽  
Diagnostic Assays ◽  
Viral Genomes ◽  
Nasal Swabs ◽  
Pre Treatment

AbstractRapid, reliable, and widespread testing is required to curtail the ongoing COVID-19 pandemic. Current gold standard diagnostic assays are hampered by supply shortages in critical reagents including nasal swabs, RNA extraction kits, personal protective equipment (PPE), instrumentation, and labor. Here we present an approach to overcome these challenges with the development of a rapid colorimetric assay using reverse-transcription loop-mediated isothermal amplification (RT-LAMP) optimized on human saliva samples without an RNA purification step. We describe our optimizations of the LAMP reaction and saliva pre-treatment protocols that enabled rapid and sensitive detection of < 102 viral genomes per reaction in contrived saliva controls. We also observed high performance of this assay on a limited number of clinical saliva samples. While thorough validation on additional clinical samples will be needed before such an assay can be widely used, these preliminary results demonstrate a promising approach to overcome the current bottlenecks limiting widespread testing.

scholarly journals Rapid and Extraction-Free Detection of SARS-CoV-2 from Saliva by Colorimetric Reverse-Transcription Loop-Mediated Isothermal Amplification

2020 ◽  
Author(s):  
Matthew A Lalli ◽  
Joshua S Langmade ◽  
Xuhua Chen ◽  
Catrina C Fronick ◽  
Christopher S Sawyer ◽  
...  
Keyword(s):  
Reverse Transcription ◽  
Limit Of Detection ◽  
Colorimetric Assay ◽  
Quantitative Polymerase Chain Reaction ◽  
Rna Extraction ◽  
Lamp Assay ◽  
Isothermal Amplification ◽  
Clinical Samples ◽  
Viral Detection ◽  
Loop Mediated Isothermal Amplification

Abstract Background Rapid, reliable, and widespread testing is required to curtail the ongoing COVID-19 pandemic. Current gold-standard nucleic acid tests are hampered by supply shortages in critical reagents including nasal swabs, RNA extraction kits, personal protective equipment, instrumentation, and labor. Methods To overcome these challenges, we developed a rapid colorimetric assay using reverse-transcription loop-mediated isothermal amplification (RT-LAMP) optimized on human saliva samples without an RNA purification step. We describe the optimization of saliva pretreatment protocols to enable analytically sensitive viral detection by RT-LAMP. We optimized the RT-LAMP reaction conditions and implemented high-throughput unbiased methods for assay interpretation. We tested whether saliva pretreatment could also enable viral detection by conventional reverse-transcription quantitative polymerase chain reaction (RT-qPCR). Finally, we validated these assays on clinical samples. Results The optimized saliva pretreatment protocol enabled analytically sensitive extraction-free detection of SARS-CoV-2 from saliva by colorimetric RT-LAMP or RT-qPCR. In simulated samples, the optimized RT-LAMP assay had a limit of detection of 59 (95% confidence interval: 44–104) particle copies per reaction. We highlighted the flexibility of LAMP assay implementation using 3 readouts: naked-eye colorimetry, spectrophotometry, and real-time fluorescence. In a set of 30 clinical saliva samples, colorimetric RT-LAMP and RT-qPCR assays performed directly on pretreated saliva samples without RNA extraction had accuracies greater than 90%. Conclusions Rapid and extraction-free detection of SARS-CoV-2 from saliva by colorimetric RT-LAMP is a simple, sensitive, and cost-effective approach with broad potential to expand diagnostic testing for the virus causing COVID-19.

scholarly journals MINERVA: A facile strategy for SARS-CoV-2 whole genome deep sequencing of clinical samples

2020 ◽  
Author(s):  
Chen Chen ◽  
Jizhou Li ◽  
Lin Di ◽  
Qiuyu Jing ◽  
Pengcheng Du ◽  
...  
Keyword(s):  
High Performance ◽  
Vaccine Development ◽  
Viral Evolution ◽  
High Sensitivity ◽  
High Yield ◽  
Clinical Samples ◽  
High Coverage ◽  
Preparation Methods ◽  
Viral Genomes ◽  
Viral Spread

AbstractThe novel coronavirus disease 2019 (COVID-19) pandemic poses a serious public health risk. Analyzing the genome of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) from clinical samples is crucial for the understanding of viral spread and viral evolution, as well as for vaccine development. Existing sample preparation methods for viral genome sequencing are demanding on user technique and time, and thus not ideal for time-sensitive clinical samples; these methods are also not optimized for high performance on viral genomes. We have developed MetagenomIc RNA EnRichment VirAl sequencing (MINERVA), a facile, practical, and robust approach for metagenomic and deep viral sequencing from clinical samples. This approach uses direct tagmentation of RNA/DNA hybrids using Tn5 transposase to greatly simplify the sequencing library construction process, while subsequent targeted enrichment can generate viral genomes with high sensitivity, coverage, and depth. We demonstrate the utility of MINERVA on pharyngeal, sputum and stool samples collected from COVID-19 patients, successfully obtaining both whole metatranscriptomes and complete high-depth high-coverage SARS-CoV-2 genomes from these clinical samples, with high yield and robustness. MINERVA is compatible with clinical nucleic extracts containing carrier RNA. With a shortened hands-on time from sample to virus-enriched sequencing-ready library, this rapid, versatile, and clinic-friendly approach will facilitate monitoring of viral genetic variations during outbreaks, both current and future.

scholarly journals Validating Anti-Infective Activity of Pleurotus Opuntiae via Standardization of Its Bioactive Mycoconstituents through Multimodal Biochemical Approach

Coatings ◽  
2021 ◽  
Vol 11 (4) ◽  
pp. 484
Author(s):  
Aprajita Tiwari Pandey ◽  
Ishan Pandey ◽  
Anurag Kanase ◽  
Amita Verma ◽  
Beatriz Garcia-Canibano ◽  
...  
Keyword(s):  
Bioactive Compounds ◽  
High Performance ◽  
Colorimetric Assay ◽  
Diffusion Method ◽  
Therapeutic Effects ◽  
Medication Regimen ◽  
Methanol Extracts ◽  
Retention Factors ◽  
Before And After ◽  
Solvent Systems

Mushrooms produce a variety of bioactive compounds that are known to have anti-pathogenic properties with safer and effective therapeutic effects in human disease prognosis. The antibacterial activity of ethanol and methanol extracts of Pleurotus opuntiae were checked against pathogenic microorganisms viz. Pseudomonas aeruginosa ATCC 27853, Proteus mirabilis NCIM 2300, Proteus vulgaris NCIM 5266, Serratia marcescens NCIM 2078, Shigella flexeneri NCIM 5265, Moraxella sp. NCIM 2795, Staphylococcus aureus ATCC 25923 by agar well diffusion method at different concentrations of the extracts. Minimum Inhibitory Concentration (MIC) and Minimum Bactericidal Concentration (MBC) of the extracts was determined by INT (Iodonitrotetrazolium chloride) colorimetric assay. Extracts were standardized by thin layer chromatography (TLC) in different solvent systems. The Retention factors (Rf) of different compounds were calculated by high performance TLC (HPTLC) fingerprinting at UV 254, 366, and 540 nm before and after derivatization. The ethanol and methanol extracts of P. opuntiae showed bactericidal activity against all the test pathogens at MIC values of 15.6 to 52.08 mg/mL and 20.81 to 52.08 mg/mL respectively. Whereas the MBC values for ethanol and methanol extract of P. opuntiae against all pathogens were recorded as 26.03 to 62.5 mg/mL and 125 mg/mL respectively. Preliminary mycochemical screening of both the extracts revealed high contents of bioactive compounds. Amongst all the solvent systems used in TLC, the best result was given by chloroform + hexane (8:2) which eluted out 5 different compounds (spots). HPTLC results revealed spots with different Rf values for all the 24 compounds present. Thus, it can be inferred from the present investigation that the mycoconstituents could be an alternative medication regimen and could play a role in new drug discoveries against different infections. Further, the antimicrobial components of these mushrooms can be transformed to bioengineered antimicrobial coatings for surfaces, drug and other hybrid systems for public health implications in combating persistent infections.

scholarly journals Direct diagnostic testing of SARS-CoV-2 without the need for prior RNA extraction

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Shan Wei ◽  
Esther Kohl ◽  
Alexandre Djandji ◽  
Stephanie Morgan ◽  
Susan Whittier ◽  
...  
Keyword(s):  
Limit Of Detection ◽  
Point Of Care ◽  
Diagnostic Testing ◽  
Rna Extraction ◽  
Cost Effective ◽  
Clinical Samples ◽  
Nasopharyngeal Swab ◽  
Percentage Agreement ◽  
Testing Method ◽  
Viral Transport

AbstractThe COVID-19 pandemic has resulted in an urgent need for a rapid, point of care diagnostic testing that could be rapidly scaled on a worldwide level. We developed and tested a highly sensitive and robust assay based on reverse transcription loop mediated isothermal amplification (RT-LAMP) that uses readily available reagents and a simple heat block using contrived spike-in and actual clinical samples. RT-LAMP testing on RNA-spiked samples showed a limit of detection (LoD) of 2.5 copies/μl of viral transport media. RT-LAMP testing directly on clinical nasopharyngeal swab samples in viral transport media had an 85% positive percentage agreement (PPA) (17/20), and 100% negative percentage agreement (NPV) and delivered results in 30 min. Our optimized RT-LAMP based testing method is a scalable system that is sufficiently sensitive and robust to test for SARS-CoV-2 directly on clinical nasopharyngeal swab samples in viral transport media in 30 min at the point of care without the need for specialized or proprietary equipment or reagents. This cost-effective and efficient one-step testing method can be readily available for COVID-19 testing world-wide, especially in resource poor settings.

scholarly journals Toxoplasma gondii and pre-treatment protocols for polymerase chain reaction analysis of milk samples: a field trial in sheep from Southern Italy

2017 ◽  
Vol 6 (1) ◽  
Author(s):  
Alice Vismarra ◽  
Elena Barilli ◽  
Maura Miceli ◽  
Carlo Mangia ◽  
Cristina Bacci ◽  
...  
Keyword(s):  
Toxoplasma Gondii ◽  
Real Time ◽  
Dna Extraction ◽  
Real Time Pcr ◽  
Southern Italy ◽  
Raw Milk ◽  
Type I ◽  
Treatment Protocols ◽  
Milk Samples ◽  
Pre Treatment

Toxoplasmosis is a zoonotic disease caused by the protozoan <em>Toxoplasma gondii</em>. Ingestion of raw milk has been suggested as a risk for transmission to humans. Here the authors evaluated pre-treatment protocols for DNA extraction on <em>T. gondii</em> tachyzoite-spiked sheep milk with the aim of identifying the method that resulted in the most rapid and reliable PCR positivity. This protocol was then used to analyze milk samples form sheep from three different farms in southern Italy, including Real Time PCR for DNA quantification and PCR-RFLP for genotyping. The pre-treatment protocol using EDTA and Tris-HCl to remove casein gave the best results in the least amount of time compared to the others on spiked milk samples. One sample of 21 collected from sheep farms was positive on one-step PCR, Real Time PCR and resulted in a Type I genotype at one locus (SAG3). Milk usually contains a low number of tachyzoites and this could be a limiting factor for molecular identification. Our preliminary data has evaluated a rapid, cost-effective and sensitive protocol to treat milk before DNA extraction. The results of the present study also confirm the possibility of <em>T. gondii</em> transmission through consumption of raw milk and its unpasteurized derivatives.

Use of a flow-injection sample manipulator as an interface between a "high-performance" liquid chromatograph and an atomic absorption spectrophotometer.

1981 ◽  
Vol 27 (9) ◽  
pp. 1546-1550 ◽  
Author(s):  
B W Renoe ◽  
C E Shideler ◽  
J Savory
Keyword(s):  
Atomic Absorption ◽  
Flow Injection ◽  
Metal Binding ◽  
High Performance ◽  
Atomic Absorption Spectrophotometry ◽  
Clinical Samples ◽  
High Performance Liquid Chromatograph ◽  
Liquid Chromatograph ◽  
Flame Atomic Absorption ◽  
Column Eluate

Abstract We describe an integrated, molecular-absorbance, atomic absorption instrument for studying metal/ligand binding in clinical samples. For an interface between the "high-performance" liquid chromatograph and the atomic absorption instrument we used a flow-injection sample manipulator, thus allowing both the chromatograph and the atomic absorption detector to operate at their separate optimum conditions. After specimen separation with a gel permeation column, we measured the molecular components of the column eluate by molecular absorbance spectrometry and the atomic components (calcium and magnesium) by flame atomic absorption spectrophotometry. This instrument system is capable of separating and analyzing multiple components within 20 min of injection of the sample on the column. The chromatograms presented demonstrate the utility of the system for investigating metal binding to a variety of ligands in clinical samples.

Analysis of caffeine and paraxanthine in human saliva with ultra-high-performance liquid chromatography for CYP1A2 phenotyping

2015 ◽  
Vol 995-996 ◽  
pp. 70-73 ◽  
Author(s):  
Nan Yeun Jordan ◽  
Jolet Y. Mimpen ◽  
Willie J.M. van den Bogaard ◽  
Frits M. Flesch ◽  
Michiel H.M. van de Meent ◽  
...  
Keyword(s):  
High Performance Liquid Chromatography ◽  
Liquid Chromatography ◽  
High Performance ◽  
Human Saliva

scholarly journals SARS-CoV-2 Direct Detection Without RNA Isolation With Loop-Mediated Isothermal Amplification (LAMP) and CRISPR-Cas12

2021 ◽  
Vol 8 ◽  
Author(s):  
Alfredo Garcia-Venzor ◽  
Bertha Rueda-Zarazua ◽  
Eduardo Marquez-Garcia ◽  
Vilma Maldonado ◽  
Angelica Moncada-Morales ◽  
...  
Keyword(s):  
Limit Of Detection ◽  
Direct Detection ◽  
Direct Method ◽  
Rna Extraction ◽  
Rna Isolation ◽  
High Sensitivity ◽  
High Specificity ◽  
Isothermal Amplification ◽  
Clinical Samples ◽  
Loop Mediated Isothermal Amplification

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/μL 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.

Sign in / Sign up

Export Citation Format

Share Document