Abstract: The aim of present study was to develop serotyping real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR) methods for serotype identification of Palyam virus (PALV) from clinical samples or insect vector. According to segment 2 (Seg-2) sequences of Chinese epidemic PALV strains, PALV serotype-specific qRT-PCR methods were established and their specificity, sensitivity and repeatability were evaluated. The reliability of the established qRT-PCR methods was assessed by 28 Chinese PALV strains and 90 PALV-positive blood samples, then the established methods were applied to identify the serotypes of PALV from the collected Culicoides samples. The established PALV serotyping qRT-PCR methods showed highly specificity and sensitivity, with the minimum copy number of detectable viral nucleic acid ranging from 22 to 28 copies·μL^-1. The qRT-PCR identification results of 28 PALV strains are consistent with the results of virus sequencing. Furthermore, serotype qRT-PCR identification results of 90 PALV-positive blood samples from infected sentinel animals were consistent with serotyping results of the isolated PALVs. The established method can accurately identify the serotype of PALV collected from Culicoides samples. With features of strong specificity, high sensitivity and positive repeatability, the PALV serotype-specific qRT-PCR methods established in this study could be used for rapid and accurate diagnosis of the serotypes of PALV in vectors and animals.

Key words: Palyam virus, serotype identification, real-time quantitative reverse transcription polymerase chain reaction, detection method