2005 Fiscal Year Final Research Report Summary
Pathogenic aspects of the secondary structure at the 5'-untranstlated regions specific to pastivirus
| Project/Area Number |
15580253
|
|---|
| Research Category |
Grant-in-Aid for Scientific Research (C)
|
| Allocation Type | Single-year Grants |
|---|
| Section | 一般 |
|---|
| Research Field |
Basic veterinary science/Basic zootechnical science
|
|---|
| Research Institution | Iwate University (2004-2005)
The University of Tokyo (2003) |
|---|
Principal Investigator |
HARASAWA Ryo Iwate University, Fac.of Agric., Prof., 農学部, 教授 (70159101)
|
|---|
| Project Period (FY) |
2003 – 2005
|
| Keywords | Pestivirus / virulence / 5'-UTR / Palindrome |
|---|
| Research Abstract |
Pestivirus strains isolated from diseased cattle and cell cultures were subjected to (1)nucleotide sequencing following the nested RT-PCR to amplify the 5'-untranstlated region of pestivirus genomes, (2)viral titration by using real-time PCR. Viral RNA samples were isolated by the single-step guanidinium isothiocyanate-phenol-chloroform method. The RNA solution was subsequently subjected to complementary deoxyribonucleic acids synthesis by reverse transcription. The oligonucleotide primers amplified the expected targets in real-time RT-PCR analysis using the SmartCycler. The universal primers amplified a 254 bp fragment which was evident on agarose gel electrophoresis. The conventional RT-PCR used for the specific detection of pestivirus species requires visualization step by agarose gel electrophoresis. While we describe here a rapid method to detect pestiviral RNA by real-time RT-PCR using SYBR Green I. Therefore, a simple and rapid detection and identification procedure for pestivirus strains was developed in this study.
|
