Establishment and application of duplex RT-PCR detection for Newcastle disease virus and avian pneumovirus

ObjectiveA method has been established for simultaneous detection of Newcastle disease virus(NDV) and avian pneumovirus(APV) by duplex reverse transcriptase polymerase chain reaction(RT-PCR), which could provide tech-nical support for control of NDV and APV. MethodTwo pair of specific primers based on F gene sequence of NDV(Gen-Bank accession number JX840454) and F gene sequence of APV(GenBank accession number AF187154) were designed. A duplex RT-PCR was set to detect NDV and APV with the designed primers. Amplification condition was optimized. Prac-ticability of the detection was verified by specificity test, sensitivity test and clinical sample validation. ResultSpecified amplification results by optimized duplex RT-PCR showed that objective bands of tested NDV strains (Strain Lasota, Strain F48E9 and Strain Ⅰ) and APV/MN-10 strain were 247 and 424 bp in length but there were no special bands were detected at 247 and 424 bp in other control strains. The minimum detection limit was 10 pg RNA for NDV and APV. In clinical samples detection using duplex RT-PCR, out of 132 sick chickens collected in Guangxi, there were 26 NDV positive samples and 2 APV positive samples. The result was in accordance with that by serological method. Two NDV positive samples were randomly selected. PCR products of the two NDV positive samples and 2 APV positive samples were se-quenced. Homology analysis showed that PCR products of two NDV samples were 100% homologous with F gene sequence of NDV(GenBand accession number JX840454), and PCR products of APV samples were 100% homologous with F gene se-quence of APV(GenBand accession number AF187154). ConclusionDuplex RT-PCR for NDV and APV is a detection method with high specificity, high sensibility and rapid detection. It can be applied to rapid clinical identification and serve as a technical support for control of Newcastle disease viruse and avian pneumovirus.