scholarly journals SLAMP: A Rapid Fluorometric RT-LAMP Assay for Sensitive and Specific Detection of SARS-CoV-2 from Human Saliva

2021 ◽  
Author(s):  
Dimitri Athan Bikos ◽  
Chiachi Hwang ◽  
Kristen A Brileya ◽  
Albert Parker ◽  
Emma Kate Loveday ◽  
...  
Keyword(s):  
Reverse Transcription ◽  
Quantitative Polymerase Chain Reaction ◽  
Lamp Assay ◽  
High Sensitivity ◽  
Human Saliva ◽  
The Body ◽  
Proteinase K ◽  
Rapid Screening ◽  
Screening Methods ◽  
Asymptomatic Individuals

Rapid testing methods can identify outbreaks and trigger preventive strategies for slowing the spread of SARS-CoV-2, the virus that causes COVID-19. The gold-standard detection method for SARS-CoV-2 is reverse transcription quantitative polymerase chain reaction (RT-qPCR) performed on samples collected using a nasopharyngeal (NP) swab. While NP RT-qPCR provides high sensitivity, it requires trained personnel to administer and suffers from lengthy time-to-result. Recently, the testing community has turned to rapid saliva-based screening methods including saliva-to-RT-qPCR and/or saliva-to-RT-LAMP (reverse transcription loop-mediated isothermal amplification) to identify infected individuals regardless of symptomatic presentation. Here, we report a simple and rapid RT-LAMP fluorometric assay performed directly on heat-inactivated saliva, without the addition of buffers or proteinase K treatments we call saliva LAMP (SLAMP). Over the course of two days, a total of 243 individuals were tested using NP RT-qPCR, saliva-based qPCR, and saliva-based RT-LAMP. Of the 243 NP RT-qPCR tests, 65 were positive, 178 were negative, and SLAMP demonstrated a 91% sensitivity and 98% specificity. SLAMP sensitivity becomes 95% when samples negative in saliva tests while positive in NP RT-qPCR are excluded from evaluation, potentially indicating significant differences in viral titer between collection sites on the body. SLAMP is performed in triplicates and takes 45 min to run in the laboratory, requiring less technician time and instrument run time than NP RT-qPCR. These results demonstrate that saliva-based RT-LAMP can enable frequent and rapid screening of large numbers of people to identify pre-symptomatic and asymptomatic individuals thereby controlling outbreaks.

scholarly journals Development and validation of a one-step reverse transcription loop-mediated isothermal amplification (RT-LAMP) for rapid detection of ZIKV in patient samples from Brazil

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Severino Jefferson Ribeiro da Silva ◽  
Keith Pardee ◽  
Udeni B. R. Balasuriya ◽  
Lindomar Pena
Keyword(s):  
Reverse Transcription ◽  
Rapid Detection ◽  
Quantitative Polymerase Chain Reaction ◽  
Rna Extraction ◽  
Lamp Assay ◽  
Isothermal Amplification ◽  
Serum Samples ◽  
Low Resource ◽  
Loop Mediated Isothermal Amplification ◽  
One Step

AbstractWe have previously developed and validated a one-step assay based on reverse transcription loop-mediated isothermal amplification (RT-LAMP) for rapid detection of the Zika virus (ZIKV) from mosquito samples. Patient diagnosis of ZIKV is currently carried out in centralized laboratories using the reverse transcription-quantitative polymerase chain reaction (RT-qPCR), which, while the gold standard molecular method, has several drawbacks for use in remote and low-resource settings, such as high cost and the need of specialized equipment. Point-of-care (POC) diagnostic platforms have the potential to overcome these limitations, especially in low-resource countries where ZIKV is endemic. With this in mind, here we optimized and validated our RT-LAMP assay for rapid detection of ZIKV from patient samples. We found that the assay detected ZIKV from diverse sample types (serum, urine, saliva, and semen) in as little as 20 min, without RNA extraction. The RT-LAMP assay was highly specific and up to 100 times more sensitive than RT-qPCR. We then validated the assay using 100 patient serum samples collected from suspected cases of arbovirus infection in the state of Pernambuco, which was at the epicenter of the last Zika epidemic. Analysis of the results, in comparison to RT-qPCR, found that the ZIKV RT-LAMP assay provided sensitivity of 100%, specificity of 93.75%, and an overall accuracy of 95.00%. Taken together, the RT-LAMP assay provides a straightforward and inexpensive alternative for the diagnosis of ZIKV from patients and has the potential to increase diagnostic capacity in ZIKV-affected areas, particularly in low and middle-income countries.

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 A Rapid Screening Test for the Diagnosis of Influenza Infection Incubation Period Using Coincidence Analysis of Pulse Waves

2017 ◽  
Author(s):  
Po-Ying Chen ◽  
Keng-Chang Tu ◽  
Shyr-Shen Yu ◽  
Wen-Kuan Yeh
Keyword(s):  
Incubation Period ◽  
Common Cold ◽  
Pulse Wave ◽  
Viral Infections ◽  
Critical Role ◽  
The Body ◽  
Rapid Screening ◽  
Screening Methods ◽  
Viral Spread ◽  
Pulse Waves

Viral infections have long been the biggest threat to human survival, and from a medical perspective, the development of noninvasive high-throughput screening methods that target the incubation period to either treat diseases or limit viral spread would be strikingly effective. Using this technology to target viral incubation periods would also be inexpensive to perform. The current study proposes to transform pulse signals into a rapid diagnostic test using “coincidence analysis” in the hope of preventing or reducing the symptoms of viral infections. The heart plays a critical role in calculating and supplying the needs of all tissues of the body. Pulse waves are pressurized signals in response to heart’s calculations and include all phases of the cardiac cycle, which maintains life and provides energy needed to perform tasks. Any small movement gives a corresponding signal to pulse waves. The current study investigated conclusive data on self-limiting infections, such as common cold. We used pulse wave, coincidence analysis technology to capture signals from individuals with common cold during the incubation period and investigated if particular characteristic signals could be applied to influenza during the incubation period. Preliminary work demonstrated that pulse waves could generate signals using this technology that would be worthwhile for future research. A small amount of analytical data from common cold existed previously. The data structure is based on the idea that a single pulse wave at differing physiological conditions would have slight modifications that would be amplified and presented by various geometrical shapes after extensive data are accumulated. These geometric shapes can then be sliced vertically or horizontally to extract data during different illness stages. The significance of these findings are impressive; from a personal or a public hygiene perspective, this analytical technology provides many benefits, such as rapid and precise decision-making that can be directly visualized or can be analyzed using software programs. Also, this technology also uses futuristic wearable technology that brings practical problem solving to physiology.

scholarly journals Comparative Evaluation of LAMP, qPCR, Conventional PCR, and ELISA to Detect Ralstonia solanacearum in Kenyan Potato Fields

Plant Disease ◽  
2019 ◽  
Vol 103 (5) ◽  
pp. 959-965 ◽  
Author(s):  
Lilian A. Okiro ◽  
Matthew A. Tancos ◽  
Steven G. Nyanjom ◽  
Christine D. Smart ◽  
Monica L. Parker
Keyword(s):  
Late Blight ◽  
Seed Potato ◽  
Ralstonia Solanacearum ◽  
Quantitative Polymerase Chain Reaction ◽  
Lamp Assay ◽  
High Sensitivity ◽  
Sub Saharan Africa ◽  
Conventional Pcr ◽  
Specificity And Sensitivity ◽  
Field Samples

Bacterial wilt caused by Ralstonia solanacearum is considered among the most damaging diseases of potato in Sub-Saharan Africa and the most significant biotic constraint of potato production alongside late blight. Unlike late blight, which can be managed by chemical means, R. solanacearum can only be managed through cultural methods and clean seed. Laboratory testing to certify seed before planting is required to confirm the absence of the pathogen in Kenya. A loop-mediated isothermal amplification (LAMP) assay was developed using the UDP-(3-O-acyl)-N-acetylglucosamine deacetylase gene (IpxC) to screen seed potato for R. solanacearum strains. The assay was assessed using DNA extracted from R. solanacearum and other soil and potato pathogens to demonstrate specificity and sensitivity. The LAMP assay was validated using field samples from different potato growing regions of Kenya collected over two growing seasons and compared with established nucleic acid and protein-based assays. The IpxC LAMP assay was found to be specific and sensitive to R. solanacearum, detecting as low as 2.5 pg/µl of R. solanacearum DNA. Of the 47 potentially infected field samples collected, both IpxC LAMP and quantitative polymerase chain reaction (PCR) detected R. solanacearum DNA in 90% of the samples, followed by conventional PCR (86%) and ELISA (75%). This IpxC LAMP assay is a promising diagnostic tool to rapidly screen for R. solanacearum in seed potato with high sensitivity in Kenya. [Formula: see text] Copyright © 2019 The Author(s). This is an open access article distributed under the CC BY 4.0 International license .

scholarly journals CovidArray: A Microarray-Based Assay with High Sensitivity for the Detection of Sars-Cov-2 in Nasopharyngeal Swabs

Sensors ◽  
2021 ◽  
Vol 21 (7) ◽  
pp. 2490
Author(s):  
Francesco Damin ◽  
Silvia Galbiati ◽  
Stella Gagliardi ◽  
Cristina Cereda ◽  
Francesca Dragoni ◽  
...  
Keyword(s):  
Reverse Transcription ◽  
Solid Phase ◽  
Quantitative Polymerase Chain Reaction ◽  
False Negative ◽  
Rna Extraction ◽  
High Sensitivity ◽  
Great Sensitivity ◽  
Viral Rna ◽  
Analytical Sensitivity ◽  
Negative Results

A new coronavirus (SARS-CoV-2) caused the current coronavirus disease (Covid-19) epidemic. Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) is used as the gold standard for clinical detection of SARS-CoV-2. Under ideal conditions, RT-qPCR Covid-19 assays have analytical sensitivity and specificity greater than 95%. However, when the sample panel is enlarged including asymptomatic individuals, the sensitivity decreases and false negatives are reported. Moreover, RT-qPCR requires up to 3–6 h with most of the time involved in RNA extraction from swab samples. We introduce CovidArray, a microarray-based assay, to detect SARS-CoV-2 markers N1 and N2 in the nasopharyngeal swabs. The method is based on solid-phase hybridization of fluorescently-labeled amplicons upon RNA extraction and reverse transcription. This approach combines the physical-optical properties of the silicon substrate with the surface chemistry used to coat the substrate to obtain a diagnostic tool of great sensitivity. Furthermore, we used an innovative approach, RNAGEM, to extract and purify viral RNA in less than 15 min. We correctly assigned 12 nasopharyngeal swabs, previously analyzed by RT-qPCR. Thanks to the CovidArray sensitivity we were able to identify a false-negative sample. CovidArray is the first DNA microarray-based assay to detect viral genes in the swabs. Its high sensitivity and the innovative viral RNA extraction by RNAGEM allows the reduction of both the amount of false-negative results and the total analysis time to about 2 h.

scholarly journals Detection of Carbapenemase Production in a Collection of Enterobacteriaceae with Characterized Resistance Mechanisms from Clinical and Environmental Origins by Use of Both Carba NP and Blue-Carba Tests: TABLE 1

2015 ◽  
Vol 54 (2) ◽  
pp. 464-466 ◽  
Author(s):  
Sergio García-Fernández ◽  
María-Isabel Morosini ◽  
Desirèe Gijón ◽  
Lorena Beatobe ◽  
Patricia Ruiz-Garbajosa ◽  
...  
Keyword(s):  
High Sensitivity ◽  
Cost Effective ◽  
Multidrug Resistant ◽  
Resistance Mechanisms ◽  
Rapid Screening ◽  
Resistant Bacteria ◽  
Screening Methods ◽  
Routine Laboratory ◽  
Multidrug Resistant Bacteria ◽  
Content Type

Rapid-screening methods to confirm the presence of resistance mechanisms in multidrug-resistant bacteria are currently recommended. Carba NP and Blue-Carba tests were evaluated in carbapenemase-producingEnterobacteriaceaefrom hospital (n= 102) and environmental (n= 57) origins for detecting the different molecular classes among them. Both methods showed to be fast and cost-effective, with high sensitivity (98% to 100%) and specificity (100%), and may be easily introduced in the routine laboratory.

scholarly journals Detection of SARS-CoV-2 in Saliva by RT-LAMP During a Screening of Workers in Brazil, Including Pre-Symptomatic Carriers

2021 ◽  
Author(s):  
Carlos dos Santos ◽  
Kézia de Oliveira ◽  
Geovana Mendes ◽  
Lívia Silva ◽  
Marcio de Souza Jr. ◽  
...  
Keyword(s):  
Reverse Transcription ◽  
Limit Of Detection ◽  
Asymptomatic Carrier ◽  
Point Of Care ◽  
Quantitative Polymerase Chain Reaction ◽  
Lamp Assay ◽  
Diagnostic Methods ◽  
Public And Private ◽  
Polymerase Chain ◽  
Increasing Demand

The coronavirus pandemic has been causing damage to many nations, as public and private health systems deteriorate by the increasing demand. Some infected patients have culturable severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) even though not presenting any symptoms, and therefore, are probably able to transmit it. Correctly diagnosing and isolating infected patients is an important step towards preventing new infections. Current diagnostic methods rely mainly on reverse transcription quantitative polymerase chain reaction (RT-qPCR). Methods such as reverse transcription loop-mediated isothermal amplification (RT-LAMP) have risen as viable alternatives, as they are cheaper and require less infrastructure, they have the potential to be applied in low-resource scenarios and even at point-of-care. Here we report a colorimetric RT‑LAMP assay capable of detecting SARS-CoV-2 in ribonucleic acid (RNA) from saliva. In some cases, the test was able to detect viral RNA before symptom onset and even in a self-reported asymptomatic carrier. It had a limit of detection of 300 copies per reaction and showed a sensitivity of 80%, a specificity of 100%, a general accuracy of 99.59%, and a Cohen’s kappa of 0.887. The possibility of detecting positive cases even before the clinical manifestation shows great potential and can contribute to controlling the pandemic.

scholarly journals Development of a saliva-optimized RT-LAMP assay for SARS-CoV-2

2021 ◽  
Author(s):  
Albert Dayuan Yu ◽  
Kristina Galatsis ◽  
Jian Zheng ◽  
Jasmine Quynh Le ◽  
Dingbang Ma ◽  
...  
Keyword(s):  
Reverse Transcription ◽  
Low Cost ◽  
Quantitative Polymerase Chain Reaction ◽  
Diagnostic Testing ◽  
Lamp Assay ◽  
Magnetic Bead ◽  
Turnaround Time ◽  
The Novel ◽  
Rna Purification ◽  
Polymerase Chain

AbstractConventional reverse transcription quantitative polymerase chain reaction (RT-qPCR) technology has struggled to fulfill the unprecedented need for diagnostic testing created by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic. Complexity and cost hinder access to testing, and long turnaround-time decreases its utility. To ameliorate these issues, we focus on saliva and introduce several advances to colorimetric reverse-transcription loop-mediated isothermal amplification (RT-LAMP) technology; RT-LAMP offers a minimal equipment alternative to RT-qPCR. First, we validated the use of the novel dye LAMPShade Violet (LSV), which improves the visual clarity and contrast of the colorimetric readout. Second, we compared different inactivation conditions on infectivity and RNA yield from saliva. Third, we developed a ten-minute RNA purification protocol from saliva. We call this magnetic bead protocol SalivaBeads. Finally, we developed a magnetic stick, StickLAMP, which provides reliable bead-based RNA purification as well as simple and low-cost access to scalable testing from saliva.

scholarly journals CovidArray: a microarray-based assay with high sensitivity for the detection of SARS-CoV-2 in nasopharyngeal swabs

2021 ◽  
Author(s):  
Francesco Damin ◽  
Silvia Galbiati ◽  
Stella Gagliardi ◽  
Cristina Cereda ◽  
Francesca Dragoni ◽  
...  
Keyword(s):  
Reverse Transcription ◽  
Solid Phase ◽  
Quantitative Polymerase Chain Reaction ◽  
False Negative ◽  
Rna Extraction ◽  
High Sensitivity ◽  
Digital Pcr ◽  
Great Sensitivity ◽  
Viral Rna ◽  
Analytical Sensitivity

AbstractBackgroundA new coronavirus (SARS-CoV-2) caused the current Covid-19 epidemic. Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) is used as the gold standard for clinical detection of SARS-CoV-2. Under ideal conditions RT-qPCR Covid-19 assays have analytical sensitivity and specificity greater than 95%. However, when the sample panel is enlarged including asymptomatic individuals, the sensitivity decreases and false-negative are reported. Moreover, RT-qPCR requires up to 3-6 hours with most of the time involved in RNA extraction from swab samples.MethodsWe introduce CovidArray, a microarray-based assay, to detect SARS-CoV-2 markers N1 and N2 in the nasopharyngeal swabs. The method is based on solid phase hybridization of fluorescently labelled amplicons upon RNA extraction and reverse transcription. This approach combines the physical-optical properties of the silicon substrate with the surface chemistry used to coat the substrate to obtain a diagnostic tool of great sensitivity. Furthermore, we used an innovative approach, RNAGEM, to extract and purify viral RNA in less than 15 minutes. To validate the CovidArray results, we exploited the high sensitivity of the droplet digital PCR (ddPCR) technique.ResultWe correctly assigned 12 nasopharyngeal swabs, previously analyzed by RT-qPCR. Thanks to the CovidArray sensitivity that matches that of the ddPCR, we were able to identify a false-negative sample.ConclusionsCovidArray is the first DNA microarray-based assay to detect viral genes in the swabs. Its high sensitivity and the innovative viral RNA extraction by RNAGEM allows to reduce both the amount of false negative results and the total analysis time to about 2 hours.

Rapid Screening Methods for Bleeding Disorders. A Three Year Survey

1964 ◽  
Vol 11 (02) ◽  
pp. 506-512 ◽  
Author(s):  
V. A Lovric ◽  
J Margolis
Keyword(s):  
Laboratory Tests ◽  
Capillary Blood ◽  
Screening Tests ◽  
Rapid Screening ◽  
Screening Methods ◽  
Bleeding Disorders ◽  
Routine Laboratory ◽  
Clotting Time ◽  
Kaolin Clotting Time ◽  
In Infants And Children

SummaryAn adaptation of “kaolin clotting time” and prothrombin time for use on haemolysed capillary blood provided simple and sensitive screening tests suitable for use in infants and children. A survey of three year’s experience shows that these are reliable routine laboratory tests for detection of latent coagulation disorders.

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