scholarly journals Validation of a single-step, single-tube reverse transcription loop-mediated isothermal amplification assay for rapid detection of SARS-CoV-2 RNA

2020 ◽  
Vol 69 (9) ◽  
pp. 1169-1178 ◽  
Author(s):  
Jean Y. H. Lee ◽  
Nickala Best ◽  
Julie McAuley ◽  
Jessica L. Porter ◽  
Torsten Seemann ◽  
...  
Keyword(s):  
Sensitivity And Specificity ◽  
Reverse Transcription ◽  
Rapid Detection ◽  
Lamp Assay ◽  
Single Step ◽  
Isothermal Amplification ◽  
Assay Sensitivity ◽  
Single Tube ◽  
Clinical Specimens ◽  
Loop Mediated Isothermal Amplification

Introduction. The SARS-CoV-2 pandemic of 2020 has resulted in unparalleled requirements for RNA extraction kits and enzymes required for virus detection, leading to global shortages. This has necessitated the exploration of alternative diagnostic options to alleviate supply chain issues. Aim. To establish and validate a reverse transcription loop-mediated isothermal amplification (RT- LAMP) assay for the detection of SARS-CoV-2 from nasopharyngeal swabs. Methodology. We used a commercial RT-LAMP mastermix from OptiGene in combination with a primer set designed to detect the CDC N1 region of the SARS-CoV-2 nucleocapsid (N) gene. A single-tube, single-step fluorescence assay was implemented whereby 1 µl of universal transport medium (UTM) directly from a nasopharyngeal swab could be used as template, bypassing the requirement for RNA purification. Amplification and detection could be conducted in any thermocycler capable of holding 65 °C for 30 min and measure fluorescence in the FAM channel at 1 min intervals. Results. Assay evaluation by assessment of 157 clinical specimens previously screened by E-gene RT-qPCR revealed assay sensitivity and specificity of 87 and 100%, respectively. Results were fast, with an average time-to-positive (Tp) for 93 clinical samples of 14 min (sd±7 min). Using dilutions of SARS-CoV-2 virus spiked into UTM, we also evaluated assay performance against FDA guidelines for implementation of emergency-use diagnostics and established a limit-of-detection of 54 Tissue Culture Infectious Dose 50 per ml (TCID50 ml−1), with satisfactory assay sensitivity and specificity. A comparison of 20 clinical specimens between four laboratories showed excellent interlaboratory concordance; performing equally well on three different, commonly used thermocyclers, pointing to the robustness of the assay. Conclusion. With a simplified workflow, The N1 gene Single Tube Optigene LAMP assay (N1-STOP-LAMP) is a powerful, scalable option for specific and rapid detection of SARS-CoV-2 and an additional resource in the diagnostic armamentarium against COVID-19.

scholarly journals Validation of a single-step, single-tube reverse transcription-loop-mediated isothermal amplification assay for rapid detection of SARS-CoV-2 RNA

2020 ◽  
Author(s):  
Jean Y. H. Lee ◽  
Nickala Best ◽  
Julie McAuley ◽  
Jessica L. Porter ◽  
Torsten Seemann ◽  
...  
Keyword(s):  
Sensitivity And Specificity ◽  
Reverse Transcription ◽  
Rapid Detection ◽  
Single Step ◽  
Isothermal Amplification ◽  
N Gene ◽  
Assay Sensitivity ◽  
Single Tube ◽  
Clinical Specimens ◽  
Loop Mediated Isothermal Amplification

2.AbstractIntroductionThe SARS-CoV-2 pandemic of 2020 has resulted in unparalleled requirements for RNA extraction kits and enzymes required for virus detection, leading to global shortages. This has necessitated the exploration of alternative diagnostic options to alleviate supply chain issues.AimTo establish and validate a reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay for the detection of SARS-CoV-2 from nasopharyngeal swabs.MethodologyWe used a commercial RT-LAMP mastermix from OptiGene Ltd in combination with a primer set designed to detect the CDC N1 region of the SARS-CoV-2 nucleocapsid (N) gene. A single-tube, single-step fluorescence assay was implemented whereby as little as 1 μL of universal transport medium (UTM) directly from a nasopharyngeal swab could be used as template, bypassing the requirement for RNA purification. Amplification and detection could be conducted in any thermocycler capable of holding 65°C for 30 minutes and measure fluorescence in the FAM channel at one-minute intervals.ResultsAssay evaluation by assessment of 157 clinical specimens previously screened by E-gene RT-qPCR revealed assay sensitivity and specificity of 87% and 100%, respectively. Results were fast, with an average time-to-positive (Tp) for 93 clinical samples of 14 minutes (SD ±7 minutes). Using dilutions of SARS-CoV-2 virus spiked into UTM, we also evaluated assay performance against FDA guidelines for implementation of emergency-use diagnostics and established a limit-of-detection of 54 Tissue Culture Infectious Dose 50 per ml (TCID50 mL−1), with satisfactory assay sensitivity and specificity. A comparison of 20 clinical specimens between four laboratories showed excellent interlaboratory concordance; performing equally well on three different, commonly used thermocyclers, pointing to the robustness of the assay.ConclusionWith a simplified workflow, N1-STOP-LAMP is a powerful, scalable option for specific and rapid detection of SARS-CoV-2 and an additional resource in the diagnostic armamentarium against COVID-19.3.Data summaryThe authors confirm all supporting data, code and protocols have been provided within the article or through supplementary data files.

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 Colorimetric Reverse Transcription–Loop-Mediated Isothermal Amplification Assay for Rapid Detection of SARS-CoV-2

2021 ◽  
Author(s):  
Meng Yee Lai ◽  
Soo Nee Tang ◽  
Yee Ling Lau
Keyword(s):  
Sensitivity And Specificity ◽  
Reverse Transcription ◽  
Virus Detection ◽  
Lamp Assay ◽  
Isothermal Amplification ◽  
Clinical Sensitivity ◽  
Loop Mediated Isothermal Amplification ◽  
Resource Limited ◽  
Detection Technology ◽  
Simple Detection

Coronavirus disease 2019 (COVID-19), which is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has been spreading rapidly all over the world. In the absence of effective treatments or a vaccine, there is an urgent need to develop a more rapid and simple detection technology of COVID-19. We describe a WarmStart colorimetric reverse transcription–loop-mediated isothermal amplification (RT-LAMP) assay for the detection of SARS-CoV-2. The detection limit for this assay was 1 copy/µL SARS-CoV-2. To test the clinical sensitivity and specificity of the assay, 37 positive and 20 negative samples were used. The WarmStart colorimetric RT-LAMP had 100% sensitivity and specificity. End products were detected by direct observation, thereby eliminating the need for post-amplification processing steps. WarmStart colorimetric RT-LAMP provides an opportunity to facilitate virus detection in resource-limited settings without a sophisticated diagnostic infrastructure.

A Rapid and Sensitive Reverse Transcription-Loop-Mediated Isothermal Amplification (RT-LAMP) Assay for the Detection of Indian Citrus Ringspot Virus

Plant Disease ◽  
2020 ◽  
Author(s):  
Amol Kokane ◽  
Sunil Kokane ◽  
Ashish Warghane ◽  
Mrugendra G Gubyad ◽  
Ashwani Kumar Sharma ◽  
...  
Keyword(s):  
Reverse Transcription ◽  
Large Scale ◽  
Color Change ◽  
Lamp Assay ◽  
Single Step ◽  
Isothermal Amplification ◽  
Ringspot Virus ◽  
Lamp Method ◽  
Loop Mediated Isothermal Amplification ◽  
Field Samples

Indian citrus ringspot virus (ICRSV) is a devastating pathogen that has a particularly deleterious effect on the ‘Kinnow mandarin’, a commercial citrus crop cultivated in the north-west of India. ICRSV belongs to the Mandarivirus genus within the family of Alphaflexiviridae and has a positive sense single-stranded RNA (ssRNA) genome consisting of six open reading frames (ORFs). Severe cases of ICRSV result in a significant reduction in both the yield and quality of crops. Consequently, there is an urgent need to develop methods to detect ICRSV in an accurate and timely manner. Current methods involve a two-step reverse transcriptase-polymerase chain reaction (RT-PCR) that is time-consuming. Here, we describe a novel, one-step, reverse transcription-loop-mediated isothermal amplification (RT-LAMP) method for the sensitive and rapid detection of ICRSV. The RT-LAMP assay was standardized by designing and testing four different primers that targeted the coat protein gene of ICRSV. Amplification results were visualized by a color change after addition of SYBR Green I. The standardized RT-LAMP assay was highly specific and successfully detected all 35 ICRSV isolates tested from the Punjab and Haryana states of India. Furthermore, there was no cross-reaction with 17 isolates of five other citrus pathogens that are common in India. ICRSV-RT-LAMP assay developed in the present study is a simple, rapid, sensitive, and specific, technique. Moreover, the assay consists of only a single step and is more cost-effective than existing methods. This represents the first application of RT-LAMP for the detection of ICRSV. Our RT-LAMP assay is a powerful tool for the detection of ICRSV and will be particularly useful for large scale indexing of field samples in diagnostic laboratories, nurseries, and for quarantine applications.

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