Development of a real-time PCR assay to detect the fusion gene of the D26 strain of a commercial avian avulavirus 1

Abstract

Newcastle disease (ND), caused by avian avulavirus 1 (AA1), an enveloped, negative sense, single stranded RNA virus belonging to the Paramyxoviridae family. ND is found world-wide and leads to severe economic losses from mortality and condemnation of carcasses. Virulent ND causes clinical signs such as respiratory distress, central nervous signs, drop in egg production, weakness, gastro-intestinal symptoms and death. The disease is listed by the World Organisation for Animal Health (OIE) and outbreaks require reporting to the OIE. The OIE requires a definitive diagnosis of virulent AA1 to enable effective control of an outbreak by strict control measures and trade restrictions. Currently the real-time reverse transcription polymerase chain reaction (RRT-PCR) assay used to diagnose ND does not differentiate between field and vaccine strain. The aim of this study was to develop and optimise a real time RT-PCR assay that detects chickens vaccinated with Vectormune® HVT NDV vaccine based on the F gene of the D26 strain. NDV F gene sequences were downloaded from Genbank® and aligned. A region unique to the D26 strain, between nucleotides 69 to 131 (using accession number M24692 for numbering) was identified and a TaqMan® MGB™ assay was developed. Primer and probe concentrations were optimised at 200 nM. Nucleic acid was purified using a MagMax™ Pathogen RNA/DNA extraction kit and a MagMax™ Express Magnetic Particle Processor (ThermoFisher Scientific). TaqMan Fast Advanced Master Mix PCR reagents were used to amplify the AA1 F gene with one StepOnePlus Real-time PCR system. The PCR efficiency was calculated to be 81.8% with 0.9942 coefficient correlation (R2). The 95% limit of detection was 10-1.31 plaque forming units per reaction. The assay was specific and did not detect any other AA1 isolates tested. Twenty-four spleen impression smear field samples from chickens (12 Vectormune® HVT NDV vaccine samples and 12 vaccinated with ND virus conventional vaccine) preserved on Whatman® FTA cards, were collected between day 21 and 28 post vaccination. The assay detected only the D26 vaccine strain and was negative when tested on other field samples. The developed real time PCR was sensitive, reliable and repeatable and will also be able to produce results rapidly as compared to other conventional methods.