Rapid detection of Banna virus by reverse transcription-loop-mediated isothermal amplification (RT-LAMP)

Abstract

Objectives: Banna virus (BAV) is classified in the genus Seadornavirus within the Reoviridae family and considered to be an emerging pathogen. We aimed to develop a rapid and simple molecular detection approach for all BAV subgroups in isothermal conditions. Method: A set of six specific primers was designed to target the segment 12 of BAV, and the reverse transcription-loop mediated isothermal amplification (RT-LAMP) assay was developed and compared with conventional RT-PCR method. Results: The amplification of the RT-LAMP assay can be obtained within 40 min at 65 C. The results from specificity showed that only target BAVs RNA including genotypes A, B and C were amplified and the assay demonstrated a sensitivity of 3.6 Â 10 À2 PFU/mL, which was higher than conventional RT-PCR measurement. A good reliability for the assay was presented in the further evaluation for BAVs RNA from serial diluted BAV-spiked serum and 47 pools of field mosquito samples. Conclusions: Our findings present a rapid, sensitive and specific RT-LAMP assay that can be applied for BAV detection in clinical or field samples in the future.

Rapid detection of Broad bean wilt virus 2 and Turnip mosaic virus in Pseudostellaria heterophylla by reverse transcription loop‐mediated isothermal amplification assay

“…Every technology has its disadvantages and limitations, and RT‐LAMP is no exception. One of the limitations of RT‐LAMP is as follows: after LAMP amplification, if we opened the centrifuge tube lids, this easily leads to product volatilization and aerosol pollution (Rachid et al, 2019; Xia et al, 2018), which may lead to many false positives in future RT‐LAMP assays using the same set of primers. Aiming to isolate the reaction product from the outside environment, we added two drops (approximately 20 μl) of sterile, colourless mineral oil after adding the reaction liquid to the tubes and then added 2 μl SYBR Green I dye to the lid of the centrifuge tubes.…”

“…Every technology has its disadvantages and limitations, and RT-LAMP is no exception. One of the limitations of RT-LAMP is as follows: after LAMP amplification, if we opened the centrifuge tube lids, this easily leads to product volatilization and aerosol pollution (Rachid et al, 2019;Xia et al, 2018), which may lead to many false positives in future RT-LAMP assays using the same In this study, we established a rapid, simple, intuitive, accurate and sensitive method for the detection of TuMV and BBWV2 in P. heterophylla and improved some easy-to-produce contamination of the experimental steps. This is of great significance to the promotion of RT-LAMP rapid detection method.…”

Molecular Approaches for Low-Cost Point-of-Care Pathogen Detection in Agriculture and Forestry

“…Several examples of portable devices exploiting fluorescence detectors for POC plant pathogen analysis can be found. Such devices combine fluorescence detectors with several amplification methods, such as qPCR (Koo et al, 2013;DeShields et al, 2018;Fotiou et al, 2019), LAMP (Larrea-Sarmiento et al, 2018;Tahzima et al, 2019;Xia et al, 2019), RCA (Zhang et al, 2019), and RPA (Lei et al, 2019). A possible alternative to the classical fluorescence-based detection methods is the so-called surface-enhanced Raman scattering (SERS), a technique that exploits the signal enhancement given by metal nanoparticles surfaces upon laser excitation (Kneipp et al, 1997).…”

Advances in isothermal nucleic acid amplification methods for hepatitis B virus detection

“…Moreover, the LAMP reaction system requires at least four primers, resulting in its excellent specificity. Since its invention, it has been successfully used to detect various pathogenic microorganisms, including bacteria, 60–62 viruses, 63–65 parasites 66–68 and so on.…”