A Highly Sensitive Real-Time Reverse Transcription Polymerase Chain Reaction for Detecting Nucleocapsid Protein Gene of Both Classes I and Ii of Newcastle Disease Virus

Abstract

Newcastle disease virus (NDV) is a worldwide viral agent that infects over 200 species of birds and is responsible for outbreaks of ND. Although a series of real-time reverse transcription polymerase chain reaction (rRT-PCR) assays have been developed for detecting different genes of NDV, diagnostic sensitivity and efficiency still can be improved. This study describes a nucleocapsid protein gene rRT-PCR screening assay based on TaqMan technology for the detection of divergent NDV strains. All 23 representative NDV strains of classes I and II in the tested panel were detected using the NP-gene rRT-PCR assay, whereas eight class I and two class II NDV isolates cannot be detected by the USDA-validated matrix-gene assay. The detection limit of the NP-gene assay was approximately 10[Formula: see text] EID[Formula: see text]/mL. The new assay also demonstrated a high degree of specificity with no false-positive results of 35 non-NDV viruses. A total of 146 clinical specimens were also tested and the NP-gene assay gave high relative sensitivity (100%) and specificity (96.61%) when compared with virus isolation. This NP-gene rRT-PCR assay offers a sensitive, specific and rapid assay for detecting both class I and II NDV and can be used alongside with the existing diagnostic assays for this notifiable disease agent.