Development of qPCR assays for quantitative detection of Heterodera avenae, H. filipjevi and H. latipons

Fateh Toumi, Lieven Waeyenberge, Nicole Viaene, Abdelfattah A. Dababat, Julie M. Nicol, Francis C. Ogbonnaya, Maurice Moens

    Onderzoeksoutput: Hoofdstuk in Boek/Rapport/CongresprocedureC3: Congres abstract

    Uittreksel

    Twelve Heterodera species are considered of major economic significance in cereals; Heterodera avenae, H. filipjevi and H. latipons are the most important ones. Precise identification and quantification of these nematodes are necessary to develop effective integrated pest control. We report the results of a qPCR assay that we developed for the quick detection and quantification of the three species. Three qPCR primer sets comprising two primers and a probe, were designed and optimized. All developed assays were able to detect a single second-stage juvenile (J2). Their specificity was confirmed by the lack of amplification of 13 other Heterodera species. A qPCR using DNA extracted from 106, 114 and 114 J2 + eggs of H. avenae, H. filipjevi and H. latipons resulted in steady Ct-values (Ct = 22.33 ± 0.1, Ct = 21.83 ± 0.05 and Ct= 18.6 ± 0.12, respectively). Dilution series of DNA extracted from known numbers of J2 + eggs of the three species were made. The assays resulted in a standard curve showing a highly significant linearity between the Ct-values and the dilution rates (R² = 0.99; slope = -3.05, R² = 0.99; slope = -3.4 and R² = 0.99; slope = -3.5 for H. avenae, H. filipjevi and H. latipons respectively). The three qPCR assays provide a sensitive and valid tool for the rapid detection and quantification of the three species whether they occur alone or in mixtures with other species. Unfortunately, the assay for the detection and quantification of H. filipjevi was not successful for all H. filipjevi populations. This is probably due to polymorphism. Further investigation is needed.
    TaalEngels
    Titelthe 6th International Congress of Nematology : 6th ICN
    Datummei-2014
    StatusGepubliceerd - mei-2014

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