Update and validation of the 16S rDNA qPCR assay for the detection of three ‘Candidatus’ Liberibacter species following current MIQE guidelines and workflow

Osman et al. (2022). PhytoFrontiers™
Ca. Liberibacter asiaticus Liberibacter
General Medicine
An updated real-time multiplex quantitative polymerase chain reaction (qPCR) assay was designed and validated for the simultaneous detection of three ‘Candidatus Liberibacter species’ (CLsp), Ca. Liberibacter asiaticus (CLas), africanus (CLaf) and americanus (CLam), associated with the Huanglongbing (HLB) disease of citrus. The multiplex assay was designed based on the previously published qPCR assay by Li et al., 2006, taking into consideration all available CLsp 16S rRNA gene sequences in the GenBank and the MIQE guidelines and workflow for qPCR optimization, which became available after 2006. When using the updated multiplex CLsp qPCR assay compared to singleplex qPCR, no significant increase in Cq values was detected. The specificity and sensitivity of the updated qPCR assay was optimal and measuring the intra and inter assay variations confirmed the reproducibility and repeatability of the assay. The assay was also successfully used with a large number of diverse samples, at independent laboratories in four countries, thus demonstrating its transferability, applicability, practicability, and robustness as different qPCR reaction conditions or instruments had a minor effect on Cq values. This updated multiplex CLsp qPCR assay can be used in a variety of citrus surveys, germplasm, or nursery stock programs that require different pathogen detection tools for their successful operation. Keywords: Citrus greening disease, COX internal DNA control; validation; citrus germplasm; budwood; citrus nursery, citrus survey, regulatory diagnostics, Citrus Clonal Protection Program (CCPP), National Clean Plant Network (NCPN)
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