scholarly journals Reverse Transcription-Loop-mediated Isothermal Amplification (RT-LAMP) Assay for Zika Virus and Housekeeping Genes in Urine, Serum, and Mosquito Samples

10.3791/58436 ◽  
2018 ◽  
Author(s):  
Sarah N. Bartolone ◽  
Maya O. Tree ◽  
Michael J. Conway ◽  
Michael B. Chancellor ◽  
Laura E. Lamb
Keyword(s):  
Reverse Transcription ◽  
Zika Virus ◽  
Lamp Assay ◽  
Housekeeping Genes ◽  
Isothermal Amplification ◽  
Loop Mediated Isothermal Amplification ◽  
Urine Serum

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.

Reverse transcription-loop mediated isothermal amplification (RT-LAMP) assay for detection of AhR receptor responsive xenobiotics

2021 ◽  
pp. 1-13
Author(s):  
Deeksha Sharma ◽  
Payal Rani ◽  
Suneel Kumar Onteru ◽  
Partha Roy ◽  
Rakesh Kumar Tyagi ◽  
...  
Keyword(s):  
Reverse Transcription ◽  
Lamp Assay ◽  
Isothermal Amplification ◽  
Loop Mediated Isothermal Amplification

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.

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 Detection and discrimination of multiple strains of Zika virus by reverse transcription-loop-mediated isothermal amplification

2020 ◽  
Vol 48 (1) ◽  
Author(s):  
Hiroka Aonuma ◽  
Itoe Iizuka-Shiota ◽  
Tokio Hoshina ◽  
Shigeru Tajima ◽  
Fumihiro Kato ◽  
...  
Keyword(s):  
Virus Strain ◽  
Reverse Transcription ◽  
Zika Virus ◽  
Virus Disease ◽  
Isothermal Amplification ◽  
Lamp Method ◽  
Simple Method ◽  
Loop Mediated Isothermal Amplification ◽  
Specificity And Sensitivity ◽  
Virus Strains

Abstract Background Monitoring both invasion of Zika virus disease into free countries and circulation in endemic countries is essential to avoid a global pandemic. However, the difficulty lies in detecting Zika virus due to the large variety of mutations in its genomic sequence. To develop a rapid and simple method with high accuracy, reverse transcription-loop-mediated isothermal amplification (RT-LAMP) was adopted for the detection of Zika virus strains derived from several countries. Results Common primers for RT-LAMP were designed based on the genomic sequences of two standard Zika strains: African lineage, MR-766, and Asian lineage, PRVABC59. RT-LAMP reactions using a screened primer set, targeting the NS3 region, detected both Zika virus strains. The minimum detectable quantity was 3 × 10−2 ng of virus RNA. Measurable lag of reaction times among strains was observed. The RT-LAMP method amplified the target virus sequence from the urine and serum of a patient with a travel history in the Caribbean Islands and also provided a prediction about which lineage of Zika virus strain was present. Conclusions The RT-LAMP method using a well-optimized primer set demonstrated high specificity and sensitivity for the detection of Zika virus strains with a variety in genomic RNA sequences. In combination with the simplicity of LAMP reaction in isothermal conditions, the optimized primer set established in this study may facilitate rapid and accurate diagnosis of Zika fever patients with virus strain information.

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