Immunocapture-reverse transcriptase loop-mediated isothermal amplification assay for detection of plant RNA viruses

Authors: HAJERI, SUBHAS - Central California Tristeza Eradication Agency; Yokomi, Raymond - Ray

Submitted to: Methods in Molecular Biology
Publication Type: Book / Chapter
Publication Acceptance Date: 10/3/2020
Publication Date: 12/15/2021
Citation: Hajeri S., Yokomi R. 2021. Immunocapture-reverse transcriptase loop-mediated isothermal amplification assay for detection of plant RNA viruses. In: Wang, A., Li, Y., editors. Plant Virology. Methods in Molecular Biology, vol 2400. New York, NY: Humana. p. 245-252. https://doi.org/10.1007/978-1-0716-1835-6_23.
DOI: https://doi.org/10.1007/978-1-0716-1835-6_23

Interpretive Summary:

Technical Abstract: Loop-mediated isothermal amplification (LAMP) is a sensitive method that can rapidly amplify a specific nucleic acid target with high specificity. The LAMP reaction process has no denaturation step, instead DNA amplification occurs by strand displacement activity of the Bacillus stearothermophilus (Bst) DNA polymerase under isothermal conditions. It utilizes three sets of forward and reverse oligonucleotide primers specific to six distinct sequences on the target gene. These primers are used to generate amplification products that contain single-stranded loops, thereby allowing primers to bind to these sequences without the need for repeated cycles of thermal denaturation. For diagnosis of pathogens with RNA genomes, LAMP has been merged with reverse transcription (RT) step to create RT-LAMP. To further reduce the cost of diagnosis and increase the throughput, immunocapture (IC) step was added to develop IC-RT-LAMP assay. Hence this chapter focuses on utilizing IC-RT-LAMP assay to specifically identify severe strain of a plant virus from field samples.