Simultaneous Detection of Three Subgroups of Avian Leukosis Virus Using the Nanoparticle-Assisted PCR Assay

Nanoparticle-assisted polymerase chain reaction (nanoPCR) is a novel method for the rapid detection of pathogens. A sensitive and specific multiple nanoPCR assay was developed for simultaneous detection of avian leucosis virus (ALV) subgroups A, B and J. In this study, three pairs of primers were designed, based on the conserved region of the gp85 gene. An exploration of the optimal primer concentration and annealing temperature were carried out, for better performance of the nanoPCR assay. According to the results, the multiple nanoPCR assay amplified 336 pb, 625 bp and 167 bp fragments of ALV-A, -B and -J, respectively, and showed no cross-reactivity with irrelevant pathogens, suggesting the excellent specificity of the assay. The constructed standard DNA templates were used to estimate the limit of detection. As shown by the results, the detection limit of the nanoPCR assay was nearly 10 copies/μL. To further evaluate the detection ability of the assay, 186 clinical samples were detected using the nanoPCR assay, among which, 14 samples were confirmed as ALV positive; the results were further confirmed by sequencing. In conclusion, a highly specific and sensitive nanoPCR assay was successfully developed, which could be a useful tool for clinical diagnosis as well as for the discrimination of ALV-A, -B and -J.