Evaluation of reverse transcription-loop-mediated isothermal amplification for rapid detection of SARS-CoV-2

Quino, Willi; Flores-León, Diana; Caro-Castro, Junior; Hurtado, Carmen V.; Silva, Iris; Gavilán, Ronnie G.

doi:10.1038/s41598-021-03623-y

Cited by 8 publications

(5 citation statements)

References 39 publications

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“…In contrast, the previously established TiPV qPCR assay showed a detection limit at 2.56 × 10 1 copies/μL (Yamkasem et al, 2022), while the TiPV LAMP in this study showed the detection limit at 5.25 × 10 3 copies/ μL. Other LAMP assays have revealed detection limits between 10 and 100 copies for the detection of viruses (Baek et al, 2020;Lu et al, 2020;Park et al, 2020;Quino et al, 2021). Although LAMP assays are reliable and sensitive in detecting TiPV-infected fish, the qPCR assay had 100-times higher sensitivity than the LAMP assay.…”

Section: Sensitivity Of the Tipv Lamp Assaycontrasting

confidence: 97%

Specific and rapid detection of tilapia parvovirus using loop‐mediated isothermal amplification (LAMP) method

Phusantisampan

Yamkasem

Tattiyapong

et al. 2022

Journal of Fish Diseases

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Section: Sensitivity Of the Tipv Lamp Assaycontrasting

confidence: 97%

Specific and rapid detection of tilapia parvovirus using loop‐mediated isothermal amplification (LAMP) method

Phusantisampan

Yamkasem

Tattiyapong

et al. 2022

Journal of Fish Diseases

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“…While the study presented here is limited to synthetic RNA control templates, the choice of optimal primer set and the sensitivity benefit of primer set combination is a fundamental starting point for any diagnostic assay. The benefit of combining RT-LAMP primer sets for increased sensitivity as we described in [10] has since been demonstrated with extracted RNA and directly from clinical samples [21][22][23][24] and thus we believe the analysis of many additional potential primer sets described here is of benefit to additional assay development. SARS-CoV-2 unfortunately remains a significant public health concern with continuing needs for diagnostic and surveillance testing, increasingly in field, point-of-care, and even home settings removed for traditional clinical laboratory infrastructure where RT-LAMP is particularly valuable.…”

Section: Plos Onementioning

confidence: 86%

Improving RT-LAMP detection of SARS-CoV-2 RNA through primer set selection and combination

Zhang

Tanner²

2022

PLoS ONE

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Reverse transcription loop-mediated isothermal amplification (RT-LAMP) has emerged as a viable molecular diagnostic method to expand the breadth and reach of nucleic acid testing, particularly for SARS-CoV-2 detection and surveillance. While rapidly growing in prominence, RT-LAMP remains a relatively new method compared to the standard RT-qPCR, and contribution to our body of knowledge on designing LAMP primer sets and assays can have significant impact on its utility and adoption. Here we select and evaluate 18 LAMP primer sets for SARS-CoV-2 previously identified as sensitive ones under various conditions, comparing their speed and sensitivity with two LAMP formulations each with 2 reaction temperatures. We find that both LAMP formulations have some effects on the speed and detection sensitivity and identify several primer sets with similar high sensitivity for different SARS-CoV-2 gene targets. Significantly we observe a consistent sensitivity enhancement by combining primer sets for different targets, confirming and building on earlier work to create a simple, general approach to building better and more sensitive RT-LAMP assays.

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“…In contrast, real-time fluorometric detection offers greater sensitivity, efficiency, precision, and the possibility of quantifying viral loads [ 18 , 19 ]. Several studies have reported RT-LAMP assays for the detection of SARS-CoV-2 [ 20 – 25 ]. However, most of these use commercial reaction mixtures and enzymes subjected to availability that can limit diagnostic tests during the pandemic [ 26 ].…”

Section: Introductionmentioning

confidence: 99%

Starting from scratch: Step-by-step development of diagnostic tests for SARS-CoV-2 detection by RT-LAMP

Tapia-Sidas¹,

Vargas-Hernández²,

Ramírez-Pool³

et al. 2023

PLoS ONE

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The pandemic caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has affected millions of people worldwide. Public health strategies to reduce viral transmission are based on widespread diagnostic testing to detect and isolate contagious patients. Several reverse transcription (RT)-PCR tests, along with other SARS-CoV-2 diagnostic assays, are available to attempt to cover the global demand. Loop-mediated isothermal amplification (LAMP) based methods have been established as rapid, accurate, point of care diagnostic tests for viral infections; hence, they represent an excellent alternative for SARS-CoV-2 detection. The aim of this study was to develop and describe molecular detection systems for SARS-CoV-2 based on RT-LAMP. Recombinant DNA polymerase from Bacillus stearothermophilus and thermostable engineered reverse transcriptase from Moloney Murine Leukemia Virus were expressed using a prokaryotic system and purified by fast protein liquid chromatography. These enzymes were used to set up fluorometric real time and colorimetric end-point RT-LAMP assays. Several reaction conditions were optimized such as reaction temperature, Tris-HCl concentration, and pH of the diagnostic tests. The key enzymes for RT-LAMP were purified and their enzymatic activity was determined. Standardized reaction conditions for both RT-LAMP assays were 65°C and a Tris-HCl-free buffer at pH 8.8. Colorimetric end-point RT-LAMP assay was successfully used for viral detection from clinical saliva samples with 100% sensitivity and 100% specificity compared to the results obtained by RT-qPCR based diagnostic protocols with Ct values until 30. The developed RT-LAMP diagnostic tests based on purified recombinant enzymes allowed a sensitive and specific detection of the nucleocapsid gene of SARS-CoV-2.

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Evaluation of reverse transcription-loop-mediated isothermal amplification for rapid detection of SARS-CoV-2

Cited by 8 publications

References 39 publications

Specific and rapid detection of tilapia parvovirus using loop‐mediated isothermal amplification (LAMP) method

Specific and rapid detection of tilapia parvovirus using loop‐mediated isothermal amplification (LAMP) method

Improving RT-LAMP detection of SARS-CoV-2 RNA through primer set selection and combination

Starting from scratch: Step-by-step development of diagnostic tests for SARS-CoV-2 detection by RT-LAMP

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