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Optimization of a magnetic capture RT-LAMP assay for fast and real-time detection of potato virus Y and differentiation of N and O serotypes.

ABSTRACT: Potato virus Y (PVY) infection has been a global challenge for potato production and the leading cause of downgrading and rejection of seed crops for certification. Accurate and timely diagnosis is a key for effective disease control. Here, we have optimized a reverse transcription loop-mediated amplification (RT-LAMP) assay to differentiate the PVY O and N serotypes. The RT-LAMP assay is based on isothermal autocyclic strand displacement during DNA synthesis. The high specificity of this method relies heavily on the primer sets designed for the amplification of the targeted regions. We designed specific primer sets targeting a region within the coat protein gene that contains nucleotide signatures typical for O and N coat protein types, and these primers differ in their annealing temperature. Combining this assay with total RNA extraction by magnetic capture, we have established a highly sensitive, simplified and shortened RT-LAMP procedure as an alternative to conventional nucleic acid assays for diagnosis. This optimized procedure for virus detection may be used as a preliminary test for identifying the viral serotype prior to investing time and effort in multiplex RT-PCR tests when a specific strain is needed.

SUBMITTER: Treder K

PROVIDER: S-EPMC5799334 | biostudies-literature | 2018 Feb

REPOSITORIES: biostudies-literature

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Optimization of a magnetic capture RT-LAMP assay for fast and real-time detection of potato virus Y and differentiation of N and O serotypes.

Treder Krzysztof K Chołuj Joanna J Zacharzewska Bogumiła B Babujee Lavanya L Mielczarek Mateusz M Burzyński Adam A Rakotondrafara Aurélie M AM

Archives of virology 20171108 2

Potato virus Y (PVY) infection has been a global challenge for potato production and the leading cause of downgrading and rejection of seed crops for certification. Accurate and timely diagnosis is a key for effective disease control. Here, we have optimized a reverse transcription loop-mediated amplification (RT-LAMP) assay to differentiate the PVY O and N serotypes. The RT-LAMP assay is based on isothermal autocyclic strand displacement during DNA synthesis. The high specificity of this method ...[more]

PMID: 29119360

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Project description:The Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) pandemic demanded rapid diagnosis to isolate new COVID-19 cases and prevent disease transmission. Quantitative Real-Time Polymerase Chain Reaction (qRT-PCR) rapidly became the gold standard for diagnosis. However, due to the high cost and delay of the results, other types of diagnosis were implemented, such as COVID-19 Ag Rapid Tests and Reverse Transcription Technique followed by Loop-Mediated isothermal Amplification (RT-LAMP). In this work, we validated the use of RT-LAMP in saliva samples rather than nasopharyngeal swabs, as the collection is more comfortable. First, we selected 5 primer sets based on the limit of detection for SARS-CoV-2 RNA, then validated their sensitivity and specificity in patient samples. A total of 117 samples were analyzed by fluorometric RT-LAMP and compared with qRT-PCR results. Our results show that the use of a high-sensitive primer ORF1-a, together with a low-sensitive primer set Gene E (time to threshold of 22.9 and 36.4 minutes, respectively, using 200 copies of viral RNA), achieved sensitivity in purified RNA from saliva samples of 95.2% (95% CI 76.1‒99.8) with 90.5% specificity (95% CI 69.6‒98.8) (n = 42).As RNA purification increases the turnaround time, we tested the outcome of RT-LAMP utilizing raw saliva samples without purification. The test achieved a sensitivity of 81.8% (95% CI 59.7‒94.8) and a specificity of 90.9% (95% CI 70.8‒98.8). As a result, the accuracy of 92.9% (95% CI 80.5‒98.5) in purified RNA-saliva samples was lowered to an acceptable level of 86.4% (95% CI 72.6‒94.8) in raw saliva. Although mass vaccination has been implemented, new strains and low vaccination progress helped to spread COVID-19. This study shows that it is feasible to track new COVID-19 cases in a large population with the use of raw saliva as sample in RT-LAMP assay which yields accurate results and offers a less invasive test.

Dataset Information

Optimization of a magnetic capture RT-LAMP assay for fast and real-time detection of potato virus Y and differentiation of N and O serotypes.

Publications

Optimization of a magnetic capture RT-LAMP assay for fast and real-time detection of potato virus Y and differentiation of N and O serotypes.

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