Nested-quantitative PCR approach with improved sensitivity for the detection of low titer levels of Candidatus Liberibacter asiaticus in the Asian citrus psyllid, Diaphorina citri Kuwayama

• Published in: Journal of microbiological methods Vol. 102; pp. 15 - 22
• Main Authors: Coy, M.R., Hoffmann, M., Kingdom Gibbard, H.N., Kuhns, E.H., Pelz-Stelinski, K.S., Stelinski, L.L.
• Format: Journal Article
• Language: English
• Published: Netherlands Elsevier B.V 01.07.2014
• Subjects: Animals; Citrus; Citrus - microbiology; Diaphorina citri; DNA, Bacterial - genetics; DNA, Ribosomal - genetics; False-negatives; Hemiptera - microbiology; Huanglongbing; Humans; Insecta - microbiology; Kuwayama; Low-abundance template; Microbial detection methods; Plant pathogen; Plasmids; Polymerase Chain Reaction - methods; Real-Time Polymerase Chain Reaction - methods; Rhizobiaceae - genetics; Rhizobiaceae - isolation & purification; RNA, Ribosomal, 16S - genetics; Sensitivity and Specificity; Template secondary structure; Template secondary structure; Huanglongbing; Microbial detection methods; Plant pathogen; False-negatives; Low-abundance template
• ISSN: 0167-7012; 1872-8359
• DOI: 10.1016/j.mimet.2014.04.007

Candidatus Liberibacter asiaticus (CLas) is a phloem-limited bacterium transmitted by the Asian citrus psyllid, Diaphorina citri, and the presumptive causal agent of citrus greening disease. The current method of detection for CLas within plant and insect samples is by a presence/absence qPCR assay using the CLas 16S rDNA gene target. Although qPCR is highly sensitive, low bacterial titers or suboptimal qPCR conditions can result in false-negatives. Using a nested qPCR assay, we determined the false-negative rate of the 16S presence/absence qPCR assay was greater than 50%. Studies to determine the performance parameters of the qPCR assays for CLas 16S and Wingless (Wg), the D. citri endogenous gene, using plasmid and psyllid DNA, revealed suboptimal and variable performance of the 16S assay in psyllid samples. Average efficiencies and sensitivity limits of the plasmid assays were 99.0% and 2.7 copies of template for Wg, respectively, and 98.5% and 2.2-22.1 copies for 16S, respectively. Variability in efficiency was significantly greater in psyllid samples for both gene targets compared to the corresponding plasmid assays, and efficiencies as low as 76% were obtained for 16S. A secondary structure analysis revealed the formation of two stem-loop structures that block the forward and probe binding sites in the 16S template, which could hinder amplification. In summary, our results suggest that suboptimal qPCR efficiency is not uncommon for the 16S presence/absence qPCR assay, which combined with lowCLas titers in some samples, could contribute significantly to the under-reporting of CLas infection in psyllid and plant samples. •A nested-qPCR assay to detect Candidatus Liberibacter asiaticus is described.•The false-negative rate using the current qPCR method is greater than 50%.•Performance of the qPCR gene targets used in the current method is reported.•The 16S target used in the current method is suboptimal and highly variable.•Low abundance and secondary structure of 16S probably contribute to poor performance.