Molecular Typing of Cyst‐Forming Nematodes <i><scp>G</scp>lobodera pallida</i> and <i><scp>G</scp>. rostochiensis</i>, Using Real‐Time <scp>PCR</scp> and Evaluation of Five Methods for Template Preparation

Abstract

Abstract Globodera pallida and G . rostochiensis are two cyst‐forming nematodes known to infest potato crops, causing severe economic losses worldwide. In this study, a real‐time T aq M an PCR assay was developed and optimized for the simultaneous detection of G . pallida and G . rostochiensis . The assay's analytical and diagnostic sensitivity and specificity were evaluated using reference isolates. Four different DNA extraction methods and one rapid crude template‐preparation procedure were compared in terms of extraction purity, efficiency for PCR applications, utility and cost. Extraction methods A and B included two commercially available kits that utilize silica columns and magnetic beads, respectively. Method C was based on DNA isolation using C helex resin, and method D was a standard chemistry in‐house protocol. Procedure E included the direct use of crude mixture composed of disrupted cysts in T ris– EDTA buffer. The multiplex T aq M an PCR assay successfully discriminated the two nematode species from all reference cyst samples and its recorded diagnostic sensitivity ( D se) and specificity ( D sp) was 100%. On the contrary, in conventional ( C o) PCR tests, the overall D sp and D se were lower and estimated at 94 and 87% for G . pallida , and 97 and 88% for G . rostochiensis , respectively. Spectrophotometric results showed that DNA extraction methods A , B and C yielded the purest DNA and gave the lowest mean C t values as well as the most consistent results in C o PCR . Alternative crude preparation method E resulted in statistically similar and C t values consistent with those obtained with methods A to C when tested by T aq M an PCR . The developed assay, using crude template‐preparation E , allows the simple, accurate and cost‐effective testing of a large number of cyst samples and can be applied in surveys and certification schemes.