Development of a reliable assay protocol for identification of diseases (RAPID)-bioactive amplification with probing (BAP) for detection of Newcastle disease virus.

Due to appearance of new genotypes of Newcastle disease virus (NDV) with no cross-protection and with vaccine strains, some outbreaks have been reported in Taiwan that caused significant damage to the poultry industry. A reliable assay protocol, (RAPID)-bioactive amplification with probing (BAP), for detection of NDV that uses a nested PCR and magnetic bead-based probe to increase sensitivity and specificity, was developed. Primers and probes were designed based on the conserved region of the F protein-encoding gene sequences of all NDV Taiwan isolates. The optimal annealing temperature for nested reverse transcription-polymerase chain reaction (RT-PCR) to amplify the gene was 61 degrees C and optimal hybridization occurred when buffer 1x SSC and 0.5% SDS were used at 50 degrees C. The sensitivity of RAPID-BAP was 1 copy/microl for standard plasmids and 10 copy/mul for transcribed F protein-encoding gene of NDV with comparable linearity (R(2)=0.984 versus R(2)=0.99). This sensitivity was superior to that of other techniques currently used. The assay was also highly specific because the negative controls, including classical swine fever virus, avian influenza virus, avian reovirus, and infectious bursa disease virus could not be detected. Thirty-four field samples were tested using conventional RT-PCR, nested RT-PCR, real-time quantitative RT-PCR, and RAPID-BAP assay and the positive rates were 24%, 30%, 41%, and 53%, respectively. The developed assay allows for rapid, correct, and sensitive detection of NDV and fulfils all of the key requirements for clinical applicability. It could reliably rule out false negative results from antibody-based assays and also facilitate a rapid diagnosis in the early phase of the disease for emergency quarantine that may help prevent large-scale outbreaks.