Validation of high-throughput real time polymerase chain reaction assays for simultaneous detection of invasive citrus pathogens

•Total nucleic acid from tissue print, manual and high throughput extraction was compared for citrus pathogen detection.•Singleplex RT-qPCR assays to detect Hop stunt viroid and Citrus exocortis viroid were developed.•A multiplex RT-qPCR assay was developed to detect CLas, generic CTV and virulent CTV genotypes (VT and T3) (Mix A).•A multiplex RT-qPCR assays was developed to detect HSVd, CEVd and plant mRNA (internal control nad5) (Mix B).•High throughput multiplex RT-qPCR assays accurately detected the target pathogens in a panel of field samples. A number of important citrus pathogens are spread by graft propagation, arthropod vector transmission and inadvertent import and dissemination of infected plants. For these reasons, citrus disease management and clean stock programs require pathogen detection systems which are economical and sensitive to maintain a healthy industry. To this end, multiplex quantitative real-time PCR (qPCR) assays were developed allowing high-throughput and simultaneous detection of some major invasive citrus pathogens. Automated high-throughput extraction comparing several bead-based commercial extraction kits were tested and compared with tissue print and manual extraction to obtain nucleic acids from healthy and pathogen-infected citrus trees from greenhouse in planta collections and field. Total nucleic acids were used as templates for pathogen detection. Multiplex reverse transcription-qPCR (RT-qPCR) assays were developed for simultaneous detection of six targets including a virus, two viroids, a bacterium associated with huanglongbing and a citrus RNA internal control. Specifically, two one-step TaqMan-based multiplex RT-qPCR assays were developed and tested with target templates to determine sensitivity and detection efficiency. The first assay included primers and probes for ‘Candidatus Liberibacter asiaticus’ (CLas) and Citrus tristeza virus (CTV) broad spectrum detection and genotype differentiation (VT- and T3-like genotypes). The second assay contained primers and probes for Hop stunt viroid (HSVd), Citrus exocortis viroid (CEVd) and the mitochondrial NADH dehydrogenase (nad5) mRNA as an internal citrus host control. Primers and TaqMan probes for the viroids were designed in this work; whereas those for the other pathogens were from reports of others. Based on quantitation cycle values, automated high-throughput extraction of samples proved to be as suitable as manual extraction. The multiplex RT-qPCR assays detected b