Development Of Nested RT-PCR To Detect Rabies Virus And Its Application In Saliva Of Dogs And Cats In Shanghai

A Nested RT-PCR was developed to detect rabies virus (RV). No cross reaction with five other canine viruses such as CAV, CPIV, CPV, CDV and CCV. The sensitivity of Nested RT-PCR was so high that 0.18fg RNA could be detected. Nested RT-PCR assay was 10000 times more sensitive than that of general RT-PCR. RV positive brains dissolved with PBS and saliva of human being respectively were detected, and with Agarose Electrophoresis we got identical results and 460bp segment was seen under UV. It suggested that saliva containing RV could be detected with Nested RT-PCR.In Guangxi, 20 samples doubted infected rabies virus were detected with Nested RT-PCR and general RT-PCR assay respectively. 15 (75%) out of 20 samples were positive with Nested RT-PCR while no sample was positive with general RT-PCR assay. The results of sequence showed that 15 segments belonged to N gene of rabies virus, and the homologies of the 4 representational strains were 85.1% to 85.5% to ERA, 85.9% to 87% to CVS and 98 % to 99.6 mutually. It suggested that the Nested RT-PCR was sensitive and applied, which reduced RV pseudo-negative extremely.The preservation conditions for saliva samples were grouped for. Saliva, PBS + 2 % FCS and Hanks + 2 % FCS were served as preliminary buffer. The ERA diluted times of the three kinds of buffer were compared under different temperatures and preservation time. Following time passed, the diluted times of ERA which could be detected were reduced. In general, PBS + 2 % FCS was the best buffer, saliva is the second. Although the ability of buffer Hanks + 2 % FCS was higher than the two others in theory, the ability for preserving RV was the lowest. When the time of preserving was in 2 days, the samples should be preserved in 4℃ because freezing and melting would inactivate viruses. When the time of preserving was more than 2 days, they should be frozen mixing with antibiotics. When the...