scholarly journals Impact of primer dimers and self-amplifying hairpins on reverse transcription loop-mediated isothermal amplification detection of viral RNA

The Analyst ◽  
2018 ◽  
Vol 143 (8) ◽  
pp. 1924-1933 ◽  
Author(s):  
Robert J. Meagher ◽  
Aashish Priye ◽  
Yooli K. Light ◽  
Cheng Huang ◽  
Eryu Wang
Keyword(s):  
Reverse Transcription ◽  
Isothermal Amplification ◽  
Viral Rna ◽  
Loop Mediated Isothermal Amplification ◽  
Primer Dimers

Bumping the priming sites for RT-LAMP based on thermodynamic predictions can dramatically reduce non-specific background amplification.

DEVELOPMENT AND ASSESSMENT OF A REAGENT KIT FOR RNA DETECTION OF CRIMEAN-CONGO HEMORRHAGIC FEVER VIRUS WITH USING REVERSE TRANSCRIPTION LOOP-MEDIATED ISOTHERMAL AMPLIFICATION METHOD

2019 ◽  
Vol 64 (9) ◽  
pp. 571-577
Author(s):  
V. A. Ternovoi ◽  
Yu. V. Kononova ◽  
A. V. Zaykovskaya ◽  
E. V. Chub ◽  
A. S. Volynkina ◽  
...  
Keyword(s):  
Reverse Transcription ◽  
High Speed ◽  
Hemorrhagic Fever ◽  
Fever Virus ◽  
Isothermal Amplification ◽  
Viral Rna ◽  
Rna Detection ◽  
Crimean Congo Hemorrhagic Fever ◽  
Loop Mediated Isothermal Amplification ◽  
Hemorrhagic Fever Virus

This study presents the results of laboratory trials of the reagent kit for the rapid detection of RNA of the Crimean-Congo hemorrhagic fever virus (CCHFV) using loop-mediated isothermal amplification with reverse transcription (RT-LAMP). The developed RT-LAMP reagent kit was used to detect the CCHFV and showed a sensitivity of 103 GE/ml of viral RNA, which is sufficient for detection of the CCHFV in the early stage of human infections. The kit showed high specificity and no cross-reactivity with viral panel from the State collection of viruses of the FBRI SRC VB «Vector» (arboviruses and hemorrhagic fever viruses). Laboratory trials of the RT-LAMP kit are showed a high analytical and diagnostic sensitivity and specificity for RNA detection of the CCHFV and high speed of the analysis (60-70 min with sample preparation) compared to real-time PCR. Approbation of the kit field version has showed the possibility of setting the RT-LAMP reaction and viral RNA detection without the using of analytical equipments.

scholarly journals Clinical Evaluation of Self-Collected Saliva by Quantitative Reverse Transcription-PCR (RT-qPCR), Direct RT-qPCR, Reverse Transcription–Loop-Mediated Isothermal Amplification, and a Rapid Antigen Test To Diagnose COVID-19

2020 ◽  
Vol 58 (9) ◽  
Author(s):  
Mayu Nagura-Ikeda ◽  
Kazuo Imai ◽  
Sakiko Tabata ◽  
Kazuyasu Miyoshi ◽  
Nami Murahara ◽  
...  
Keyword(s):  
High Throughput ◽  
Reverse Transcription ◽  
Diagnostic Tests ◽  
Isothermal Amplification ◽  
Viral Rna ◽  
Molecular Diagnostic ◽  
Antigen Test ◽  
Loop Mediated Isothermal Amplification ◽  
Quantitative Reverse Transcription Pcr ◽  
Reverse Transcription Pcr

ABSTRACT The clinical performances of six molecular diagnostic tests and a rapid antigen test for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) were clinically evaluated for the diagnosis of coronavirus disease 2019 (COVID-19) in self-collected saliva. Saliva samples from 103 patients with laboratory-confirmed COVID-19 (15 asymptomatic and 88 symptomatic) were collected on the day of hospital admission. SARS-CoV-2 RNA in saliva was detected using a quantitative reverse transcription-PCR (RT-qPCR) laboratory-developed test (LDT), a cobas SARS-CoV-2 high-throughput system, three direct RT-qPCR kits, and reverse transcription–loop-mediated isothermal amplification (RT-LAMP). The viral antigen was detected by a rapid antigen immunochromatographic assay. Of the 103 samples, viral RNA was detected in 50.5 to 81.6% of the specimens by molecular diagnostic tests, and an antigen was detected in 11.7% of the specimens by the rapid antigen test. Viral RNA was detected at significantly higher percentages (65.6 to 93.4%) in specimens collected within 9 days of symptom onset than in specimens collected after at least 10 days of symptoms (22.2 to 66.7%) and in specimens collected from asymptomatic patients (40.0 to 66.7%). Self-collected saliva is an alternative specimen option for diagnosing COVID-19. The RT-qPCR LDT, a cobas SARS-CoV-2 high-throughput system, direct RT-qPCR kits (except for one commercial kit), and RT-LAMP showed sufficient sensitivities in clinical use to be selectively used in clinical settings and facilities. The rapid antigen test alone is not recommended for an initial COVID-19 diagnosis because of its low sensitivity.

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 Essential properties and pitfalls of colorimetric Reverse Transcription Loop-mediated Isothermal Amplification as a point-of-care test for SARS-CoV-2 diagnosis

2021 ◽  
Vol 27 (1) ◽  
Author(s):  
Bruna de Oliveira Coelho ◽  
Heloisa Bruna Soligo Sanchuki ◽  
Dalila Luciola Zanette ◽  
Jeanine Marie Nardin ◽  
Hugo Manuel Paz Morales ◽  
...  
Keyword(s):  
Viral Load ◽  
Internal Control ◽  
Reverse Transcription ◽  
Color Change ◽  
Point Of Care ◽  
Colorimetric Detection ◽  
False Negative ◽  
Isothermal Amplification ◽  
Clinical Samples ◽  
Loop Mediated Isothermal Amplification

Abstract Background SARS-CoV-2 Reverse Transcription Loop-mediated Isothermal Amplification (RT-LAMP) colorimetric detection is a sensitive and specific point-of-care molecular biology technique used to detect the virus in only 30 min. In this manuscript we have described a few nuances of the technique still not properly described in the literature: the presence of three colors clusters; the correlation of the viral load with the color change; and the importance of using an internal control to avoid false-negative results. Methods To achieve these findings, we performed colorimetric RT-LAMP assays of 466 SARS-CoV-2 RT-qPCR validated clinical samples, with color quantification measured at 434 nm and 560 nm. Results First we determinate a sensitivity of 93.8% and specificity of 90.4%. In addition to the pink (negative) and yellow (positive) produced colors, we report for the first time the presence of an orange color cluster that may lead to wrong diagnosis. We also demonstrated using RT-qPCR and RT-LAMP that low viral loads are related to Ct values > 30, resulting in orange colors. We also demonstrated that the diagnosis of COVID-19 by colorimetric RT-LAMP is efficient until the fifth symptoms day when the viral load is still relatively high. Conclusion This study reports properties and indications for colorimetric RT-LAMP as point-of-care for SARS-CoV-2 diagnostic, reducing false results, interpretations and optimizing molecular diagnostics tests application.

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