Studies On The Detection And Differentiation Of Virulent And Avirulent Strains Of Newcastle Disease Virus In Chickens Using Reverse Transcription-Nested Polymerase Chain Reaction

In order to diagnose Newcastle Disease (ND) rapidly and accurately, three pairs of primers (P1+P2, P3+P4 and P3+P5) were designed and synthesized respectively, according to the genome structure of Newcastle disease virus (NDV) and the sequence differences at F0 cleavage sites between virulent and avirulent strains, and the reverse transcription-nested polymerase chain reaction (RT-nested PCR) technique was developed for detecting NDV and differentiating NDV virulent and avirulent strains. The RNAs extracted from allantoic fluid of SPF chicken embryos inoculated with standard virulent NDV strain F4gEg, avirulent La Sota strain, local virulent isolate ( HJ strain) and control viruses (i.e. IBV, IBDV) respectively and the samples of tissue from chickens infected with the above-mentioned NDV strains experimentally and naturally were tested by this RT-nested PCR technique. The results were as follows: ?l andP2 are general primers for NDV strains. The RNAs extracted from two standard strains of NDV and HJstrain were amplified using primer pair Pj+Pj, and a single band was observed with the expected size of about 1. 7kb, in addition, as a control, no specific products were observed when RNAs extracted from IBV, IBDV, normal allanotic fluid of SPF chicken embryos and samples of non-vaccinated healthy chickens' tissue were performed on. P3 is the shared primer for nested PCR. Taking the products of RT-PCR as model, the nested PCR using primer pair P3+P4 resulted in a fragment of 254bp only with RNA from virulent NDV, whereas the nested PCR using primer pair P3+P5 resulted in a 254bp fragment only with RNA of avirulent NDV. The lowest content of lOfg RNA of NDV could be detected by this technique. These results demonstrated that this RT-nested PCR technique was a specific, sensitive, rapid and convenient method for diagnosis of ND. It could be used not only for detection of NDV strains derived from the inoculated chicken embryo and the samples of tissue from morbid or dead chicken, but also for differentiation of virulent and avirulent NDV strains. It is suggested that this molecular biological method is potential for diagnosis and epidemiological investigation of ND and differentiation of NDV.