Open AccessCas12a-Based Diagnostics for Potato Purple Top Disease Complex Associated with Infection by ‘Candidatus Phytoplasma trifolii’-Related Strains
Author(s) -
Matthew S. Wheatley,
Qin Wang,
Wei Wei,
Kristi D. Bottner-Parker,
Yan Zhao,
Yig Yang
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-09-21-2119-re
Subject(s) - biology , phytoplasma , 16s ribosomal rna , loop mediated isothermal amplification , intergenic region , ribosomal dna , ribosomal intergenic spacer analysis , virology , internal transcribed spacer , ribosomal rna , bacteria , botany , dna , polymerase chain reaction , restriction fragment length polymorphism , genetics , genome , gene , phylogenetics
‘Candidatus Phytoplasma trifolii’ is a cell wall-less phytopathogenic bacterium that infects many agriculturally important plant species such as alfalfa, clover, eggplant, pepper, potato, and tomato. The phytoplasma is responsible for repeated outbreaks of potato purple top (PPT) and potato witches’ broom (PWB) that occurred along the Pacific Coast of the United States since 2002, inflicting significant economic losses. To effectively manage these phytoplasmal diseases, it is important to develop diagnostic tools for specific, sensitive and rapid detection of the pathogens. Here we report the development of a DNA endonuclease targeted CRISPR trans reporter (DETECTR) assay that couples isothermal amplification and Cas12a trans-cleavage of fluorescent oligonucleotide reporter for highly sensitive and specific detection of ‘Candidatus Phytoplasma trifolii’-related strains responsible for PPT and PWB. The DETECTR assay was capable of specifically detecting the 16S–23S ribosomal DNA (rDNA) intergenic transcribed spacer (ITS) sequences from PPT- and PWB-diseased samples at the attomolar sensitivity level. Furthermore, the DETECTR strategy allows flexibility to capture assay outputs with fluorescent microplate reader or lateral flow assay for potentially high-throughput and/or field-deployable disease diagnostics.