One-step reverse transcription loop-mediated isothermal amplification: a simple, sensitive and rapid assay for detection of potato virus X in potato leaves and tubers

Raigond, Baswaraj; Verma, Ashwani Kumar; Roach, Shivani; Kochhar, Tarvinder; Shilpa, .; Jeevalatha, A.; Kumar, Ravinder; Sharma, Shubham; Chakrabarti, S. K.

doi:10.1007/s42360-019-00147-4

Cited by 10 publications

(8 citation statements)

References 24 publications

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“…These results were comparable with those of LAMP testing on other RNA viruses. For examples, LAMP was 100 times more sensitive than rt-PCR on the detection of Citrus leaf blotch virus [21] and Little cherry virus 1 [22]; on Potato virus X [23] and Sugarcane streak mosaic virus [24], LAMP was 10 times more sensitive than rt-PCR. Compared to rt-qPCR, LAMP was similarly sensitive for detection of Onion yellow dwarf virus [25] and Potato leafroll virus [26].…”

Section: Discussionmentioning

confidence: 99%

Development and evaluation of a loop-mediated isothermal amplification (LAMP) assay for the detection ofTomato brown rugose fruit virus(ToBRFV)

Sarkes

Feindel

et al. 2020

Preprint

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Tomato brown rugose fruit virus (ToBRFV) is a member of Tobamovirus infecting tomato and pepper. Within North America, both the United States and Mexico consider ToBRFV to be a regulated pest. In Canada, the presence of ToBRFV has been reported, but an efficient diagnostic system has not yet been established. Here, we describe the development and assessment of a loop-mediated isothermal amplification (LAMP)-based assay to detect ToBRFV. The LAMP test was efficient and robust, and results could be obtained within 35 min with an available RNA sample. Amplification was possible when either water bath or oven were used to maintain the temperature at isothermal conditions (65°C), and results could be read by visual observation of colour change. Detection limit of the LAMP was eight target RNA molecules. Under the experimental conditions tested, LAMP was as sensitive as qPCR and 100 times more sensitive than the currently used rt-PCR. We recommend this sensitive, efficient LAMP protocol to be used for routine lab testing of ToBRFV.

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Section: Discussionmentioning

confidence: 99%

Development and evaluation of a loop-mediated isothermal amplification (LAMP) assay for the detection ofTomato brown rugose fruit virus(ToBRFV)

Sarkes

Feindel

et al. 2020

Preprint

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show abstract

“…In previous studies, LAMP was 100 times more sensitive than RT-PCR on the detection of Citrus leaf blotch virus [23] and Little cherry virus 1 [24], while on Potato virus X [25] and Sugarcane streak mosaic virus [26], LAMP was 10 times more sensitive than RT-PCR. Compared to RT-qPCR, LAMP was similarly sensitive for detection of Onion yellow dwarf virus [27] and Potato leafroll virus [28].…”

Section: Sensitivity Of the Lampmentioning

confidence: 94%

“…Positive signals were generated from gBlock ToBRFV when its molecule number was more than or equal to 6 per 25-μL reaction (Fig 2A). When gBlock ΔF1c and gBlock ΔB2 were used as the templates, positive signals could be generated when their molecule numbers were more than or equal to 600 per 25-μL reaction; there was no positive signal when the molecule numbers were less than or equal to 60 per 25-μL reaction (Fig 2B). These results confirmed that the LAMP primers were ToBRFV-specific.…”

Section: Lamp On the Gblockmentioning

confidence: 99%

Development and evaluation of a loop-mediated isothermal amplification (LAMP) assay for the detection of Tomato brown rugose fruit virus (ToBRFV)

Sarkes

Feindel

et al. 2020

PLoS ONE

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Tomato brown rugose fruit virus (ToBRFV) is a member of Tobamovirus infecting tomato and pepper. Within North America, both the United States and Mexico consider ToBRFV to be a regulated pest. In Canada, the presence of ToBRFV has been reported, but an efficient diagnostic system has not yet been established. Here, we describe the development and assessment of a loop-mediated isothermal amplification (LAMP)-based assay to detect ToBRFV. The LAMP test was efficient and robust, and results could be obtained within 35 min with an available RNA sample. Amplification was possible when either water bath or oven were used to maintain the temperature at isothermal conditions (65˚C), and results could be read by visual observation of colour change. Detection limit of the LAMP was eight target RNA molecules. Under the experimental conditions tested, LAMP was as sensitive as qPCR and 100 times more sensitive than the currently used RT-PCR. We recommend this sensitive, efficient LAMP protocol to be used for routine lab testing of ToBRFV.

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“…After the short RT incubation, the rest of the primers (F3, FIP, LF and LB) and Bst polymerase are added, and the LAMP reaction is performed at a constant temperature (65 C, 60 min.). It is important to note that our modified protocol involving a separate RT step with only B3 and BIP primers offers superior sensitivity to the conventional 2-step RT-LAMP process where RT incubation is performed with either random hexamers or all of the LAMP primers [22][23][24] .…”

Section: Improved Rt-lamp Assaymentioning

confidence: 99%

“…Finally, we tested our RT-LAMP reactions detecting SARS-CoV-2 virus in 50 VTM and 34 saliva clinical samples. It is important to note that our protocol gives superior sensitivity to the conventional 2-step RT-LAMP protocol where reverse transcription (RT) is separately performed with all primers followed by an amplification step [22][23][24] . We believe, with our improved LOD and simple RNA extraction-free protocol, our approach will allow rapid scaling of testing and detection of cases which might have been otherwise missed due to low viral loads.…”

mentioning

confidence: 99%

RT-LAMP assay for ultra-sensitive detection of SARS-CoV-2 in saliva and VTM clinical samples

Ganguli

Mostafa

Berger

et al. 2020

Preprint

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The COVID-19 pandemic has underscored the shortcomings in the deployment of state-of-the-art diagnostic platforms. Although several PCR-based techniques have been rapidly developed to meet the growing testing needs, such techniques often need samples collected through a swab, the use of RNA extraction kits, and expensive thermocyclers in order to successfully perform the test. Isothermal amplification-based approaches have also been recently demonstrated for rapid SARS-CoV-2 detection by minimizing sample preparation while also reducing the instrumentation and reaction complexity. There are limited reports of saliva as the sample source and some of these indicate inferior sensitivity when comparing RT-LAMP with PCR-based techniques. In this paper, we demonstrate an improved sensitivity assay to test saliva using a 2-step RT-LAMP assay, where a short 10-minute RT step is performed with only B3 and BIP primers before the final reaction. We show that while the 1-step RT-LAMP demonstrate satisfactory results, the optimized 2-step approach allows for single molecule sensitivity per reaction and performs significantly better than the 1-step RT-LAMP and conventional 2-step RT-LAMP approaches with all primers included in the RT Step. Importantly, we demonstrate RNA extraction-free RT-LAMP based assays for detection of SARS-CoV-2 from VTM and saliva clinical samples.

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One-step reverse transcription loop-mediated isothermal amplification: a simple, sensitive and rapid assay for detection of potato virus X in potato leaves and tubers

Cited by 10 publications

References 24 publications

Development and evaluation of a loop-mediated isothermal amplification (LAMP) assay for the detection ofTomato brown rugose fruit virus(ToBRFV)

Development and evaluation of a loop-mediated isothermal amplification (LAMP) assay for the detection ofTomato brown rugose fruit virus(ToBRFV)

Development and evaluation of a loop-mediated isothermal amplification (LAMP) assay for the detection of Tomato brown rugose fruit virus (ToBRFV)

RT-LAMP assay for ultra-sensitive detection of SARS-CoV-2 in saliva and VTM clinical samples

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