Rapid pathotyping of Newcastle disease virus from allantoic fluid and organs of experimentally infected chickens using two novel probes.

Abstract

A system including RNA isolation, primers and two novel oligonucleotide probes (NC22 and VC22) was established to rapidly pathotype Newcastle disease virus (NDV) from infected allantoic fluid; The sequence of the probes is: VC22, 5'-AAGGAGGCAGAAACGCTTTATA-3'; NC22, 5'-GGGGAGACAGG GGCGCCTTATA-3'. Application efficacy of the probes was verified by differentiating NDV that derived from experimentally infected organs. NC22 and VC22 both were labeled with digoxigenin (DIG) and were successful in differentiating NDV strains from the infected allantoic fluid and organs of experimentally infected SPF chickens. The RT-PCR products of NDV isolates and strains were dotted onto nylon membrane and then hybridized with two specific probes separately. The VC22 probe is specific to virulent strains, and the NC22 probe is specific to avirulent strains. The results of hybridization coincide with viral intracerebral pathologenicity index (ICPI). The specificity of two probes and sensitivity of NC22 probe were evaluated. NC22 could detect down to 10(-8) dilution from 10(7.5) EID(50)/ml or 800 fg of viral RNA. The system could be completed within 1 day to conduct experiment from clinical sample to the result of assay, and its potential practical application in clinical assay was discussed basing on specificity, sensitivity and rapidness.