Rapid and simple detection of two potato cyst nematode species by real-time multiplex PCR using preserved microarray-based test systems (original) (raw)

2017

Abstract

Accurate diagnostics of plant pathogens is very important to prevent the spread of infection and significant yield losses. This paper presents an easy, rapid and inexpensive system for the complex real-time multiplex PCR diagnostics of two potato cyst nematode species, Globodera rostochiensis and G. pallida, using disposable long-stored stationary PCR microarrays. Laboratory trials have demonstrated an excellent diagnostic efficiency and sensitivity of the developed test systems with the DNA detection limit equal to 1 and 10 pg for G. rostochiensis and G. pallida, respectively. The specificity of the developed primers was successfully confirmed using DNA samples of related nematode species and host plant. A small reaction volume (1.2 μl) and a special technology of lyophilisation and stabilisation of PCR reagents within microreactors significantly reduce the total time of amplification (~30 min) and the number of required manipulations. Ready-to-use microarrays with flexible architecture can be stored up to 6 months at room temperature without any loss of their detection efficiency. The developed test system has a good potential as a rapid field diagnostic method, especially in the case of a low pathogen concentration in plant tissues or soil.

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