One-step RT-qPCR with an internal control system for the detection of turkey rotaviruses in faecal samples

Turkey rotaviruses are one of the major pathogens responsible for the poult enteritis syndrome (PES). In this study a one step real-time reverse transcription polymerase chain reaction (RT-qPCR) assay targeting the rotaviral non-structural protein 4 (NSP4) was developed. The NSP4 is a highly conserved gene inside the turkey rotavirus genome and contains an internal control system to monitor any potential RT-qPCR inhibitors. The detection limit of the optimized NSP4-RT-qPCR assay ranged from 8.15 to 8.15 x 10(5) copy numbers. In total 149 faecal samples were collected from eight different flocks of commercial turkey farms. Faecal samples from hens and toms were collected separately at 2-week intervals from the 2nd week of age through the 16th and 20th week of age (age of slaughter for female and male, respectively) and tested. One farm reared only hens. The samples were tested previously using conventional RT-PCR targeting the same gene. When the conventional RT-PCR was compared with the developed NSP4-RT-qPCR, the results revealed that 11% of the samples of the conventional RT-PCR were false negative. The results indicate that this NSP4-RT-qPCR is highly sensitive for the detection of turkey rotaviruses in faeces. In addition, it could be suitable for the development of high-throughput screening. (C) 2011 Elsevier B.V. All rights reserved.