Abstract: The study aimed to detect the presence of microsatellite instability (MSI) in Marek's disease (MD) tumors by high-resolution melting (HRM) method. Clinical samples were collected and diagnosed as Marek's disease by clinical signs, pathological anatomy, histopathology, PCR and sequencing. Muscle, liver, spleen, blood and tumor samples were collected from five affected chickens. DNA was extracted from the samples, and HRM assay was performed by using 15 pairs of primers for amplifying microsatellite DNA sequences. The difference melting curves of the normalization processing were obtained. The differences in the melting curves among samples were compared and analyzed for the presence of MSI in 15 microsatellite markers. The various clinical symptoms and pathological findings of the affected chickens were consistent with MD. The sequence of the viral meq gene had high homology with the virulent strain of MDV prevalent in China in recent years. HRM analysis showed that melting curves of PCR production were significantly different in muscle, liver, spleen, blood, and tumor samples. That is MSI phenomenon and MSI frequencies were different in chickens and microsatellites markers. There were 14,7,5,3 and one microsatellite markers presented MSI in five chickens respectively. The markers MCW0200 and MCW0220 presented MSI in all chickens' samples, however the markers ADL0158 had no MSI phenomenon in all chickens' samples. These microsatellite PCR amplification fragments differ in size were further demonstrated by nondenaturing polyacrylamide gel electrophoresis and sequencing analysis. There was an increase and decrease of microsatellite repeat units, and insertions or deletions of gene fragments in the sequences. The results suggest that the MSI phenomenon in MD tumors can be detected by HRM analysis, and HRM was a sensitive, accurate, simple and high-throughput method.

Key words: high-resolution melting, Marek's disease, microsatellite instability, tumor