A real-time PCR primer/probe set for reliable identification and quantification of Heterodera glycines

Summary The soybean cyst nematode, Heterodera glycines , is a major pest in soybean cultivation. Identifying and quantifying nematodes can be achieved through probe-based real-time PCR, and two sets of primers/probes have been developed to target H. glycines . However, we found that one set showed low species specificity, and the other showed significant variations in Ct values among H. glycines populations, potentially leading to inaccurate quantification. Therefore, for more reliable identification and quantification, we developed a novel primer/probe set based on the H. glycines mitochondrial cytochrome c oxidase subunit I gene. The novel primer/probe set amplified DNA of all tested H. glycines populations but not that of the other tested species. In addition, there were no significant differences in Ct values among the tested H. glycines populations, indicating accurate quantification across populations using a fixed standard curve. The detection sensitivity of the novel primer/probe set was higher than or comparable to that of the existing primer/probe sets. The novel primer/probe set thus facilitates reliable identification and quantification of H. glycines , supporting decision-making for the appropriate control of H. glycines .