Open AccessA diagnostic real-time PCR assay for the rapid identification of the tomato-potato psyllid, Bactericera cockerelli (Šulc, 1909) and development of a psyllid barcoding database
Author(s) -
Jason Sumner-Kalkun,
M. Jennifer Sjölund,
Yvonne M. Arnsdorf,
Mairi Carnegie,
Fiona Highet,
David Ouvrard,
A. F. C. Greenslade,
James R. Bell,
Roland Sigvald,
D. M. Kenyon
Publication year - 2020
Publication title -
plos one
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.99
H-Index - 332
ISSN - 1932-6203
DOI - 10.1371/journal.pone.0230741
Subject(s) - biology , taqman , pest analysis , genomic dna , dna extraction , outbreak , specific identification , polymerase chain reaction , vector (molecular biology) , dna barcoding , dna , botany , virology , genetics , zoology , gene , recombinant dna
The accurate and rapid identification of insect pests is an important step in the prevention and control of outbreaks in areas that are otherwise pest free. The potato-tomato psyllid Bactericera cockerelli (Šulc, 1909) is the main vector of ‘ Candidatus Liberibacter solanacearum’ on potato and tomato crops in North America and New Zealand; and is considered a threat for introduction in Europe and other pest-free regions. This study describes the design and validation of the first species - specific TaqMan probe-based real-time PCR assay, targeting the ITS2 gene region of B . cockerelli . The assay detected B . cockerelli genomic DNA from adults, immatures, and eggs, with 100% accuracy. This assay also detected DNA from cloned plasmids containing the ITS2 region of B . cockerelli with 100% accuracy. The assay showed 0% false positives when tested on genomic and cloned DNA from 73 other psyllid species collected from across Europe, New Zealand, Mexico and the USA. This included 8 other species in the Bactericera genus and the main vectors of ‘ Candidatus Liberibacter solanacearum’ worldwide. The limit of detection for this assay at optimum conditions was 0.1ng DNA (~200 copies) of ITS2 DNA which equates to around a 1:10000 dilution of DNA from one single adult specimen. This assay is the first real-time PCR based method for accurate, robust, sensitive and specific identification of B . cockerelli from all life stages. It can be used as a surveillance and monitoring tool to further study this important crop pest and to aid the prevention of outbreaks, or to prevent their spread after establishment in new areas.