AUTHOR=Wang Di , Jiao Xinya , Jia Haijiang , Cheng Shumei , Jin Xi , Wang Youhua , Gao Yunhua , Su Xiaofeng 

TITLE=Detection and quantification of Verticillium dahliae and V. longisporum by droplet digital PCR versus quantitative real-time PCR

JOURNAL=Frontiers in Cellular and Infection Microbiology

VOLUME=Volume 12 - 2022

YEAR=2022

URL=https://www.frontiersin.org/journals/cellular-and-infection-microbiology/articles/10.3389/fcimb.2022.995705

DOI=10.3389/fcimb.2022.995705

ISSN=2235-2988

ABSTRACT=Vascular wilt, caused by Verticillium dahliae and V. Longisporum, drastically restricts agricultural development, particularly by affecting the yield and quality of host plants. Although the application of quantitative real-time Polymerase Chain Reaction (qPCR) has largely improved the diagnosis of these two destructive agents compared with the traditional time-consuming isolation method, solutions are required to address the relatively poor detection sensitivity and high measurement bias for traceable matrix-rich samples. In this study, we developed a droplet digital PCR (ddPCR) assay for the accurate and sensitive detection and quantification of V. dahliae and V. longisporum. We compared the analytical and diagnostic performance in detail of ddPCR and the corresponding qPCR assay against the genomic DNA (gDNA) of the two fungi from cultured and field samples. We evaluated species specificity, linearity, analytical sensitivity, and measurement viability. The results showed that ddPCR has substantial advantages over qPCR, with enhanced diagnostic sensitivity, decreased quantification bias, and less susceptibility to inhibitors. Although ddPCR had comparable sensitivity (even slightly higher LoD95% values) for testing cultured gDNA of V. dahliae and V. longisporum, its positive detection rates were much higher than those of qPCR when identifying field samples. This detection discrepancy was potentially caused by the inhibition of residual matrix in the extracts. Taken together, digital PCR is a more sensitive and accurate quantitative detection method for testing traceable amounts of pathogen samples than the qPCR system, and can facilitate initial management to limit or prevent the further spread of disease.