title: Methods for rapid and effective PCR-based detection of 'Candidatus Liberibacter solanacearum' from the insect vector Bactericera cockerelli: streamlining the DNA extraction/purification process.
abstract: This study provides a protocol for rapid DNA isolation from psyllid vectors (Bactericera cockerelli and Diaphorina citri) that can be used directly with DNA-based methods for the detection of 'Candidatus (Ca.) Liberibacter solanacearum,' the bacterial causal agent of potato zebra chip disease and eventually for 'Ca. Liberibacter asiaticus' the causal agent of huanglongbing disease in citrus. The fast DNA extraction protocol was designed to work with conventional polymerase chain reaction (cPCR) DNA amplification as well as Loop mediated PCR DNA amplification. Direct cPCR of the psyllid 28S rDNA gene from samples prepared using the fast DNA extraction method was as reliable as from samples prepared using standard DNA purification (> 97% from live insects) as tested in B. cockerelli. However, samples prepared using the fast DNA extraction method had to be diluted 1:100 in sterile water for reliable amplification, presumably to dilute PCR inhibitors in the crude extract. Similarly, both cPCR and loop mediated PCR DNA amplification detected 'Ca. Liberibacter' in psyllids infected with either the zebra chip or huanglongbing pathogen equally well from diluted samples prepared using the fast DNA extraction method or from samples prepared using a DNA purification step. In addition to being reliable, the time required to complete the fast DNA extraction for 10 samples was on average approximately 5 min and required no special reagents or laboratory equipment. Thus, the fast DNA extraction method shows strong promise as a rapid, reliable, and expedient method when coupled with PCR-based analyses for detection of 'Ca. Liberibacter' pathogens in psyllids.
authors.affiliation: Department of Horticultural Sciences, HSF Building, Room 202, Texas A&M University, College Station, TX 77843, USA.
authors.forename: Julien
authors.initials: J
authors.lastname: Lévy
authors.forename: Joseph
authors.initials: J
authors.lastname: Hancock
authors.forename: Aravind
authors.initials: A
authors.lastname: Ravindran
authors.forename: Dennis
authors.initials: D
authors.lastname: Gross
authors.forename: Cecilia
authors.initials: C
authors.lastname: Tamborindeguy
authors.forename: Elizabeth
authors.initials: E
authors.lastname: Pierson
mesh_terms: D000818:Animals; D004269:DNA, Bacterial / Q000235:genetics / Q000302:isolation & purification*; D004275:DNA, Ribosomal / Q000235:genetics / Q000302:isolation & purification; D006430:Hemiptera / Q000382:microbiology*; D007303:Insect Vectors / Q000382:microbiology; D021141:Nucleic Acid Amplification Techniques; D010935:Plant Diseases / Q000382:microbiology; D018515:Plant Leaves / Q000382:microbiology; D016133:Polymerase Chain Reaction / Q000191:economics / Q000379:methods*; D015203:Reproducibility of Results; D012230:Rhizobiaceae / Q000235:genetics / Q000302:isolation & purification*; D013045:Species Specificity; D013997:Time Factors
chemical_list: D004269:DNA, Bacterial; D004275:DNA, Ribosomal