Studies Of Polymerase Chain Reaction Assay For Detection Of Canine Parvovirus

Canine parovirus (CPV) is the causative agent of acute hemorrhagic enteritis and myocarditis in dogs. The disease is highly contagious and spread rapidly via infected faeces. CPV in faecal samples has been detected by several methods including virus isolation (VI) in cell cultures, haemagglutination test (HA), electron microscopy (EM). EM and VI, although highly specific and sensitive, are too time consuming and expensive for routine use. The HA test lacks reliability without a confirmatory haemagglutination inhibition test .In this study, PCR assay and In situ~PCR have been used as a highly sensitive and specific method to detect of CPV in cell culture, faecal samples .The main content includes: 1. Feline kidney F81 cells was used for virus infecting. 2. The CPV DNA was extracted by phenol chloroform from ini~cted cell . Then the CPV DNA was amplified by polymerase chain reaction using two pairs of primers. With optimized reaction conditions, 1051 bp and 226bp were generated. The results are stable in lots of experiments. The products were not found in negative samples. 3. CPV was also detected from faecal samples. It appeared the second pair of primers, which generated 226bp products, was more sensitive and reproductive. The known positive samples was detected with PCR and HA. It showed that PCR method was better than HA test in sensitivity (P<0.05). 4. 20 field faecal samples were detected in HA and PCR with the second pair of primers, 13 samples were positive by PCR method and 8 samples were positive by HA test. 5 samples which were negative by 4 HA test were found to be positive by PCR. Cells was infected these 5 samples and pathological changes were same as the changes caused by standard CPV. 5. Feline kidney Fsi cells were cultured in slide and infected CPV. The cells infected CPV in different stages were detected by direct in situ PCR, which use biolin-labelled primers. After amplication the amplified signals was detected by anti-biotin antibody alkaline linked phosphatase and NTP/BCIP. Some signals were found in cytoplasm, and some in nucleus. And these infected cells were also detected with immunohistochemical method. Utilizing In situ PCR method, a lot of signals were found from cells, which were propagated virus from 12 hours to 72 hours. Utilizing immuohistochemical method, no signals were found from cells propagated virus for 12 hours, and sporadic signals from cells propagated virus for 48 hours. The signals detected by these two methods were compared by statistic method. The result indicated that the In situ PCR not only has high sensitivity, but also can locate the position of virus.