Novel Isothermal Reverse Transcription Polymerase Spiral Reaction (RT-PSR) Assay for the Detection of Newcastle Disease Virus in Avian Species

Newcastle disease in avian species is caused by Newcastle disease virus (NDV). It is responsible for large economic losses to the poultry industry. The early diagnosis of NDV infection is important for epidemiologic control and clinical management. The present study aimed to develop reverse transcription polymerase spiral reaction (RT-PSR) assay that accurately detects the NDV at isothermal temperature. The RT-PSR assay was developed using specifically designed primers targeting the conserved fusion gene of NDV. The assay could be performed within 2 h at an isothermal temperature of 65 °C. The developed assay is highly sensitive and capable to detect as low as 1.08 fg of template. It was found to be 1000-fold more sensitive than conventional RT-PCR and was comparable to real-time RT-PCR assay. The developed assay was found to be specific as it did not detect closely related infectious bronchitis virus and Merck disease virus. Further, the developed assay was evaluated with clinical samples and it has shown > 99% concordance with RT-PCR methods. To the best of our knowledge this is the first report of isothermal detection of NDV without the need of high-end equipment like thermal cycler. Moreover, this assay has the potential to detect NDV under field conditions. In conclusions, the results of this study indicated that the RT-PSR assay is a simple, rapid, sensitive, and specific assay for the detection of NDV that may improve diagnostic potential in clinical laboratories or can be used at diagnostic laboratories with the least infrastructure or even at field condition.