scholarly journals A Simple, Affordable, Rapid, Stabilized, Colorimetric, Versatile RT-LAMP Assay to Detect SARS-CoV-2

Diagnostics ◽  
2021 ◽  
Vol 11 (3) ◽  
pp. 438
Author(s):  
Juan García-Bernalt Diego ◽  
Pedro Fernández-Soto ◽  
Marta Domínguez-Gil ◽  
Moncef Belhassen-García ◽  
Juan Luis Muñoz Bellido ◽  
...  
Keyword(s):  
Low Income ◽  
Reverse Transcription ◽  
Lamp Assay ◽  
Virus Genome ◽  
Low Income Countries ◽  
Molecular Diagnostic ◽  
Clinical Samples ◽  
The Novel ◽  
Technological Infrastructure ◽  
One Step

The SARS-CoV-2 pandemic has forced all countries worldwide to rapidly develop and implement widespread testing to control and manage the Coronavirus Disease 2019 (COVID-19). reverse-transcription (RT)-qPCR is the gold standard molecular diagnostic method for COVID-19, mostly in automated testing platforms. These systems are accurate and effective, but also costly, time-consuming, high-technological, infrastructure-dependent, and currently suffer from commercial reagent supply shortages. The reverse-transcription loop-mediated isothermal amplification (RT-LAMP) can be used as an alternative testing method. Here, we present a novel versatile (real-time and colorimetric) RT-LAMP for the simple (one-step), affordable (~1.7 €/sample), and rapid detection of SARS-CoV-2 targeting both ORF1ab and N genes of the novel virus genome. We demonstrate the assay on RT-qPCR-positive clinical samples, obtaining most positive results under 25 min. In addition, a novel 30-min one-step drying protocol has been developed to stabilize the RT-LAMP reaction mixtures, allowing them to be stored at room temperature functionally for up to two months, as predicted by the Q10. This Dry-RT-LAMP methodology is suitable for potentially ready-to-use COVID-19 diagnosis. After further testing and validation, it could be easily applied both in developed and in low-income countries yielding rapid and reliable results.

scholarly journals A Simple, Affordable, Rapid, Stabilized, Colorimetric, Versatile RT-LAMP assay to detect SARS-CoV-2

2020 ◽  
Author(s):  
Juan García-Bernalt Diego ◽  
Pedro Fernández-Soto ◽  
Marta Domínghez-Gil ◽  
Moncef Belhassen-García ◽  
Juan Luis Muñoz Bellido ◽  
...  
Keyword(s):  
Low Income ◽  
Lamp Assay ◽  
Low Income Countries ◽  
Molecular Diagnostic ◽  
Clinical Samples ◽  
Technological Infrastructure ◽  
Testing Method ◽  
One Step ◽  
Alternative Testing ◽  
Temperature Storage

Abstract The SARS-Cov-2 pandemic has forced all countries worldwide to rapidly develop and implement widespread testing to control and manage the Coronavirus Disease 2019 (COVID-19). RT-qPCR is the gold standard molecular diagnostic method for COVID-19, mostly in automated testing platforms. These systems are accurate and effective, but also costly, time-consuming, high technological, infrastructure dependent and currently suffer from commercial reagent supply shortages. The reverse-transcription loop-mediated isothermal amplification (RT-LAMP) can be used as alternative testing method. Here, we present a novel versatile (real-time and colorimetric) RT-LAMP for the simple (one-step) and rapid (as soon as 9 min) detection of SARS-CoV-2 and demonstrate the assay on RT-qPCR-positive clinical samples. We further transformed the RT-LAMP into a dry format for room-temperature storage suitable for potentially ready-to-use COVID-19 diagnosis. After further testing and validation, the Dry-RT-LAMP could be easily applied both in developed and in low-income countries yielding rapid and reliable results.

scholarly journals Molecular and Immunological Diagnostic Techniques of Medical Viruses

2020 ◽  
Vol 2020 ◽  
pp. 1-19
Author(s):  
Daniel Hussien Reta ◽  
Tesfaye Sisay Tessema ◽  
Addis Simachew Ashenef ◽  
Adey Feleke Desta ◽  
Wajana Lako Labisso ◽  
...  
Keyword(s):  
Low Income ◽  
Viral Infections ◽  
Diagnostic Techniques ◽  
Diagnostic Methods ◽  
Low Income Countries ◽  
Molecular Diagnostic ◽  
Clinical Samples ◽  
Molecular Diagnostic Techniques ◽  
Global Public Health ◽  
Patient Sample

Viral infections are causing serious problems in human population worldwide. The recent outbreak of coronavirus disease 2019 caused by SARS-CoV-2 is a perfect example how viral infection could pose a great threat to global public health and economic sectors. Therefore, the first step in combating viral pathogens is to get a timely and accurate diagnosis. Early and accurate detection of the viral presence in patient sample is crucial for appropriate treatment, control, and prevention of epidemics. Here, we summarize some of the molecular and immunological diagnostic approaches available for the detection of viral infections of humans. Molecular diagnostic techniques provide rapid viral detection in patient sample. They are also relatively inexpensive and highly sensitive and specific diagnostic methods. Immunological-based techniques have been extensively utilized for the detection and epidemiological studies of human viral infections. They can detect antiviral antibodies or viral antigens in clinical samples. There are several commercially available molecular and immunological diagnostic kits that facilitate the use of these methods in the majority of clinical laboratories worldwide. In developing countries including Ethiopia where most of viral infections are endemic, exposure to improved or new methods is highly limited as these methods are very costly to use and also require technical skills. Since researchers and clinicians in all corners of the globe are working hard, it is hoped that in the near future, they will develop good quality tests that can be accessible in low-income countries.

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 One-Step Reverse-Transcription Recombinase Polymerase Amplification Using Lateral Flow Strips for the Detection of Coxsackievirus A6

2021 ◽  
Vol 12 ◽  
Author(s):  
Jia Xie ◽  
Xiaohan Yang ◽  
Lei Duan ◽  
Keyi Chen ◽  
Pan Liu ◽  
...  
Keyword(s):  
Reverse Transcription ◽  
Clinical Performance ◽  
Quantitative Polymerase Chain Reaction ◽  
Lateral Flow ◽  
Recombinase Polymerase Amplification ◽  
Clinical Samples ◽  
Effective Vaccine ◽  
Lateral Flow Strip ◽  
Coxsackievirus A6 ◽  
One Step

Hand, foot, and mouth disease (HFMD) is a common infectious disease affecting mainly children under 5 years of age. Coxsackievirus A6 (CVA-6), a major causative pathogen of HFMD, has caused outbreaks in recent years. Currently, no effective vaccine or antiviral treatments are available. In this study, one-step reverse-transcription recombinase polymerase amplification (RT-RPA), combined with a disposable lateral flow strip (LFS) assay, was developed to detect CVA-6. This assay can be performed in less than 35 min at 37°C without expensive instruments, and the result can be observed directly with the naked eye. The sensitivity of the RT-RPA-LFS was 10 copies per reaction, which was comparable to that of the conventional real-time quantitative polymerase chain reaction (qPCR) assays. Moreover, the assay specificity was 100%. The clinical performance of the RT-RPA-LFS assay was evaluated using 142 clinical samples, and the coincidence rate between RT-RPA-LFS and qPCR was 100%. Therefore, our RT-RPA-LFS assay provides a simple and rapid approach for point-of-care CVA-6 diagnosis.

scholarly journals A reverse transcription loop-mediated isothermal amplification for broad coverage detection of Asian and African Zika virus lineages

2020 ◽  
Author(s):  
Boon-Teong Teoh ◽  
Kim-Ling Chin ◽  
Nur-Izyan Samsudin ◽  
Shih-Keng Loong ◽  
Sing-Sin Sam ◽  
...  
Keyword(s):  
Detection Limit ◽  
Sensitivity And Specificity ◽  
Reverse Transcription ◽  
Zika Virus ◽  
Lamp Assay ◽  
Isothermal Amplification ◽  
Clinical Samples ◽  
Pcr Assay ◽  
Zikv Infection ◽  
Qrt Pcr

Abstract Background: Early detection of Zika virus (ZIKV) infection during the viremia and viruria facilitates proper patient management and mosquito control measurement to prevent disease spread. Therefore, a cost-effective nucleic acid detection method for the diagnosis of ZIKV infection, especially in resource-deficient settings, is highly required.Methods: In the present study, a single-tube reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay was developed for the detection of both the Asian and African-lineage ZIKV. The detection limit, strain coverage and cross-reactivity of the ZIKV RT-LAMP assay was evaluated. The sensitivity and specificity of the RT-LAMP were also evaluated using a total of 24 simulated clinical samples. The ZIKV quantitative reverse transcription-polymerase chain reaction (qRT-PCR) assay was used as the reference assay.Results: The detection limit of the RT-LAMP assay was 3.73 ZIKV RNA copies (probit analysis, P ≤ 0.05). The RT-LAMP assay detected the ZIKV genomes of both the Asian and African lineages without cross-reacting with other arthropod-borne viruses. The sensitivity and specificity of the RT-LAMP assay were 90% (95% CI = 59.6 – 98.2) and 100% (95% CI = 78.5 – 100.0), respectively. The RT-LAMP assay detected ZIKV genome in 9 of 24 (37.5%) of the simulated clinical samples compared to 10 of 24 (41.7%) by qRT-PCR assay with a high level of concordance (k = 0.913, P < 0.001).Conclusion: The RT-LAMP assay is applicable for the broad coverage detection of both the Asian and African ZIKV strains in resource-deficient settings.

scholarly journals Allogenous Selection of Mutational Collateral Resistance: Old Drugs Select for New Resistances Within Antibiotic Families

2021 ◽  
Author(s):  
Fernando Baquero ◽  
Jose-Luis martínez ◽  
Angela Silva-Novais ◽  
Jerónimo Rodríguez-Beltrán ◽  
Laura Martínez-García ◽  
...  
Keyword(s):  
Low Income ◽  
Antimicrobial Agents ◽  
Low Income Countries ◽  
Future Research ◽  
The Novel ◽  
Bacterial Populations ◽  
Minimal Inhibitory Concentrations ◽  
The Family ◽  
Old Drugs ◽  
Selection Of

Allogeneous selection occurs when an antibiotic selects for resistance to more advanced members of the same family. The mechanisms of allogenous selection are (a) collateral expansion, when the antibiotic expands the gene and gene-containing bacterial populations favoring the emergence of other mutations, inactivating the more advanced antibiotics; (b) collateral selection, when the old antibiotic selects its own resistance but also resistances to more modern drugs; (c) collateral hyper-resistance, when resistance to the old antibiotic selects in higher degree for populations resistant to other antibiotics of the family than to itself; and (d) collateral evolution, when the simultaneous or sequential use of antibiotics of the same family selects for new mutational combinations with novel phenotypes, generally with higher activity or broader spectrum. Note that in some cases, collateral selection derives from collateral evolution. In this study, examples of allogenous selection are provided for the major families of antibiotics. Improvements in minimal inhibitory concentrations with the newest drugs do not necessarily exclude “old” antibiotics of the same family of retaining some selective power for resistance to the newest agents. If this were true, the use of older members of the same drug family would facilitate the emergence of mutational resistance to the younger drugs of the family, which is frequently based on previously established resistance traits. The extensive use of old drugs (particularly in low-income countries and in farming) might be significant for the emergence and selection of resistances to the novel members of the family, becoming a growing source of variation and selection of resistance to the whole family. In terms of future research, it could be advisable to focus antimicrobial drug discovery more on the identification of new targets and new (unique) classes of antimicrobial agents, than on the perpetual chemical exploitation of classic existing ones.

scholarly journals A reverse transcription loop-mediated isothermal amplification for broad coverage detection of Asian and African Zika virus lineages

2020 ◽  
Author(s):  
Boon-Teong Teoh ◽  
Kim-Ling Chin ◽  
Nur-Izyan Samsudin ◽  
Shih-Keng Loong ◽  
Sing-Sin Sam ◽  
...  
Keyword(s):  
Detection Limit ◽  
Sensitivity And Specificity ◽  
Reverse Transcription ◽  
Zika Virus ◽  
Lamp Assay ◽  
Isothermal Amplification ◽  
Clinical Samples ◽  
Pcr Assay ◽  
Zikv Infection ◽  
Qrt Pcr

Abstract Background: Early detection of Zika virus (ZIKV) infection during the viremia and viruria facilitates proper patient management and mosquito control measurement to prevent disease spread. Therefore, a cost-effective nucleic acid detection method for the diagnosis of ZIKV infection, especially in resource-deficient settings, is highly required.Methods: In the present study, a single-tube reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay was developed for the detection of both the Asian and African-lineage ZIKV. The detection limit, strain coverage and cross-reactivity of the ZIKV RT-LAMP assay was evaluated. The sensitivity and specificity of the RT-LAMP were also evaluated using a total of 24 simulated clinical samples. The ZIKV quantitative reverse transcription-polymerase chain reaction (qRT-PCR) assay was used as the reference assay.Results: The detection limit of the RT-LAMP assay was 3.73 ZIKV RNA copies (probit analysis, P ≤ 0.05). The RT-LAMP assay detected the ZIKV genomes of both the Asian and African lineages without cross-reacting with other arthropod-borne viruses. The sensitivity and specificity of the RT-LAMP assay were 90% (95% CI = 59.6 – 98.2) and 100% (95% CI = 78.5 – 100.0), respectively. The RT-LAMP assay detected ZIKV genome in 9 of 24 (37.5%) of the simulated clinical samples compared to 10 of 24 (41.7%) by qRT-PCR assay with a high level of concordance (k = 0.913, P < 0.001).Conclusion: The RT-LAMP assay is applicable for the broad coverage detection of both the Asian and African ZIKV strains in resource-deficient settings.

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