Pathogenic aspects of the secondary structure at the 5'-untranstlated regions specific to pastivirus

Pastivirus 特有的 5-非翻译区二级结构的致病方面

• 批准号: 15580253
• 负责人: HARASAWA Ryo
• 金额: $ 2.05万
• 依托单位: Iwate University (2004-2005)The University of Tokyo (2003)
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 2003
• 资助国家: 日本
• 起止时间: 2003 至 2005
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-15580253/
• 关键词: Pestivirus; virulence; 5'-UTR; Palindrome; 牛ウイルス性下痢ウイルス; 豚コレラウイルス; 5'ボーダー病ウイルス; 回文様塩基置換

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.

对从病牛和细胞培养物中分离的瘟病毒株进行（1）巢式RT-PCR后的核苷酸测序，以扩增瘟病毒基因组的5 '-非转录区，（2）使用实时PCR进行病毒滴定。采用异硫氰酸胍-苯酚-氯仿一步法分离病毒RNA。随后通过逆转录对RNA溶液进行互补脱氧核糖核酸合成。在使用SmartCycler的实时RT-PCR分析中，寡核苷酸引物扩增了预期的靶标。通用引物扩增出一个254 bp的片段，在琼脂糖凝胶电泳上是明显的。用于瘟病毒种特异性检测的常规RT-PCR需要通过琼脂糖凝胶电泳的可视化步骤。我们在此描述了一种使用SYBR绿色I通过实时RT-PCR快速检测瘟病毒RNA的方法。因此，本研究建立了一种简便、快速的瘟病毒株检测和鉴定方法。

项目成果

Giangaspero, M., Harasawa, R.: "Genetic variety of Bovine viral diarrhea virus 2 strains isolated from sheep."Journal of Veterinary Medical Science. 66・3. 323-326 (2004)

Giangaspero, M., Harasawa, R.：“从绵羊中分离出的牛病毒性腹泻病毒 2 株的遗传多样性”。兽医医学杂志 66・3 (2004)。

Rapid detection and differentiation of the major mycoplasma contarniants in cell cultures.

快速检测和分化细胞培养物中的主要支原体污染物。

• 发表时间: 2005
• 期刊: Microbiol. Immunol. 49
• 作者: Harasawa;R. et al.
• 通讯作者: R. et al.

New classification of animal viruses[2005].

动物病毒新分类[2005]。

• 发表时间: 2005
• 期刊: J.Vet.Med. 58
• 作者: Fuse;T.;Kanai;Y;et al.;Harasawa R.
• 通讯作者: Harasawa R.

動物ウイルスの新しい分類[2005]

动物病毒新分类[2005]

• 发表时间: 2005
• 期刊: 獣医畜産新報 58
• 作者: 原澤 亮

Rapid detection and differentiation of the major mycoplasma contaminants in cell cultures.

快速检测和区分细胞培养物中的主要支原体污染物。

• 发表时间: 2005
• 期刊: Microbiol.Immunol. 49
• 作者: Harasawa;R. et al.
• 通讯作者: R. et al.