Real-Time PCR-Based Pathotyping of Newcastle Disease Virus by Use of TaqMan Minor Groove Binder Probes

摘要

A real-time reverse-transcription PCR was developed to detect and pathotype Newcastle disease viruses (NDV) in clinical samples. Degenerate oligonucleotide primers and TaqMan probes with nonfluorescent minor groove binder (MGB) quencher amplified and hybridized to a region in the fusion protein (F) gene that corresponds to the cleavage site of the F0 precursor, which is a key determinant of NDV pathogenicity. The application of degenerate primers and TaqMan MGB probes provided high specificity to the assay, as was shown by the successful and rapid pathotype determination of 39 NDV strains representing all the known genotypes (I to VIII) and pathotypes (lentogens/mesogens/velogens). The PCR assays specific for lentogenic and velogenic/mesogenic strains had high analytical sensitivity, detecting approximately 10 and 20 copies of the target molecule per reaction, respectively. The detection limit was also determined in terms of 50% egg infective dose (EID₅₀) by using dilution series of virus stock solutions to be approximately 101.0 and 10?1.3 EID₅₀/ml for lentogens and velogens/mesogens, respectively. Organ, swab, and stool specimens from experimentally infected animals were tested to prove the clinical suitability of the method. The results of this study suggest that the described real-time PCR assay has the potential to be used for the rapid detection/pathotyping of NDV isolates and qualitative/quantitative measurement of the virus load.