Multiplex real-time polymerase chain reaction for identifying potato cyst nematodes, Globodera pallida and Globodera rostochiensis, and the tobacco cyst nematode, Globodera tabacum

• Published in: Canadian journal of plant pathology 2008-12, Vol.30 (4), p.554-564
• Main Authors: Madani, M., Ward, L.J., De Boer, S.H.
• Format: Article
• Language: English
• Subjects: Biological and medical sciences; Fundamental and applied biological sciences. Psychology; Phytopathology. Animal pests. Plant and forest protection; Protozoa. Invertebrates
• ISSN: 0706-0661; 1715-2992
• DOI: 10.1080/07060660809507555

Because the potato cyst nematodes (PCNs), Globodera rostochiensis and Globodera pallida, and tobacco cyst nematode (TCN), Globodera tabacum, may occur in the same geographic regions, their accurate identification is essential for implementing appropriate regulatory and crop loss mitigating strategies. Molecular methods for differentiating PCNs based on the internal genomic transcribed spacer (ITS) sequences have been developed but the potential of the many DNA amplification-based test formats have not yet been fully explored and most published work has not included TCN. In our study, the two PCN species and TCN could be distinguished by melting curve analysis of ITS amplicons generated in an EvaGreen-based real-time polymerase chain reaction (PCR) test. However, a multiplex real-time Taqman PCR test, which is also based on ITS sequences, was developed in this study with primers and probes modified with locked nucleic acids and proved to be superior for identification of one or more Globodera spp. in samples containing DNA from cysts and (or) second-stage juveniles. The test was specific for the PCN and TCN species with efficiencies of 0.91, 1.02, and 0.89 for G. rostochiensis, G. pallida, and G. tabacum tabacum, respectively. The multiplex, real-time PCR test was useful for distinguishing PCNs and TCN from other nematodes as well as their identification to species level in a single assay, correctly identifying each species in a DNA template from mixed populations. Key words: potato cyst nematode, golden cyst nematode, pale cyst nematode, tobacco cyst nematode, real-time PCR, melting curve analysis, locked nucleic acid, molecular identification. Étant donné que les nématodes à kystes de la pomme de terre (NKP) Globodera pallida et G. rostochiensis ainsi que le nématode à kystes du tabac (NKT) G. tabacum peuvent être présents dans une même zone géographique, il est essentiel de pouvoir les identifier avec précision afin d'appliquer les mesures adéquates relatives aux stratégies d'atténuation en cas de pertes de cultures. Des méthodes moléculaires permettant de différencier les NKP, basées sur les séquences de l'espaceur interne transcrit (ITS), ont été développées, mais le potentiel des nombreux tests d'amplification de l'ADN n'a pas encore été complètement exploré, et la plupart des travaux qui ont été publiés sur le sujet n'ont pas encore été faits sur les NKT. Dans notre étude, les deux espèces de NKP et les NKT pouvaient être caractérisés par l'analyse d