Open AccessGenome-Informed Design of a LAMP Assay for the Specific Detection of the Strain of ‘Candidatus Phytoplasma asteris’ Phytoplasma Occurring in Grapevines in South Africa
Author(s) -
Špela Alič,
Marina Dermastia,
Johan T. Burger,
M. Dickinson,
Gerhard Pietersen,
Gert Pietersen,
T. Dreo
Publication year - 2022
Publication title -
plant disease
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.663
H-Index - 108
eISSN - 1943-7692
pISSN - 0191-2917
DOI - 10.1094/pdis-10-21-2312-re
Subject(s) - phytoplasma , biology , aster yellows , genome , strain (injury) , genetics , computational biology , virology , gene , polymerase chain reaction , restriction fragment length polymorphism , anatomy
Grapevine yellows is one of the most damaging phytoplasma-associated diseases worldwide. It is linked to several phytoplasma species, which can vary regionally due to phytoplasma and insect-vector diversity. Specific, rapid and reliable detection of the grapevine yellows pathogen has an important role in phytoplasma control. The purpose of this study was to develop and validate a specific LAMP assay for detection of a distinct strain of grapevine ‘Candidatus Phytoplasma asteri’ that is present in South Africa, through implementation of a genome-informed test design approach. Several freely available, user-friendly, web-based tools were coupled to design the specific LAMP assays. The criteria for selection of the assays were set for each step of the process, which resulted in four experimentally operative LAMP assays that targeted ftsH/hflB gene region, specific to the aster yellows phytoplasma strain from South Africa. A real-time PCR was developed, targeting the same genetic region, to provide extensive validation of the LAMP assay. The validated molecular assays are highly specific to the targeted aster yellows phytoplasma strain from South African, with good sensitivity and reproducibility. We show a genome-informed molecular test design and an efficient validation approach for molecular tests if reference and sample materials are sparse and hard to obtain.