Detection of virus genomic sequence in perilymph of deaf patient by using the polymerase chain reaction

聚合酶链式反应检测聋哑人外淋巴液中病毒基因组序列

• 批准号: 10671592
• 负责人: TAMURA Manabu
• 金额: $ 1.98万
• 依托单位: Osaka University Graduate School of Medicine
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 1998
• 资助国家: 日本
• 起止时间: 1998 至 1999
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-10671592/
• 关键词: virus infection of the inner ear; measles virus; RT-PCR; Deafness; ウイルス; 内耳炎

The etiology of the profound sensory hearing loss is mostly unknown. Virus infection of the inner ear infection is one of the important cause of acquired deafness. The aim of our study was to detect measles virus genome in the perilymph of the affected patients using RT-PCR system. Ten μ l of the perilymph was obtained during the surgery of cochlear implant and frozen until the performance of RT-PCR.Total RNA was extracted by the guanidinium thiocyanate-phenol-chloroform method. The extracted RNA was submitted to reverse transcription (RT) With random primers. The cDNA was then amplified by nested PCR using primers specific for nucleoprotein (NP) of measles virus. Identity of PCR product was confirmed by direct sequence method. NP gene of measles virus was detected four out of 5 samples by RT-PCR.Seven nucleotide substitutions were present in 4 samples compared with Edomonston strain and CAM strain. Deduced amino acid sequences showed 2 mutations in 4 samples compared with Edomonston strain and CAM strain. The perilymph obtaqined from the patients of otosclerosis was also analyzed by this RT-PCR method. Three nucleotide substitutions were present in 3 samples compared with Edomonston strain and CAM strain. Deduced amino acid sequences showed 1 mutations in 3 samples compared with Edomonston strain and CAM strain.

深度感觉性听力损失的病因大多未知。内耳病毒感染是后天性耳聋的重要原因之一。本研究旨在应用RT-PCR技术检测麻疹患者外淋巴液中麻疹病毒基因组。人工耳蜗植入术中取外淋巴液10 μ l，冻存至RT-PCR检测，用硫氰酸胍-酚-氯仿法提取总RNA。提取的RNA用随机引物进行逆转录（RT）。用麻疹病毒核蛋白（NP）特异性引物，通过巢式PCR扩增其cDNA。PCR产物经直接测序法鉴定。用RT-PCR方法检测了5份麻疹病毒标本中4份标本的NP基因，与Edomonston株和CAM株比较，有4份标本存在7个核苷酸替换。推导的氨基酸序列与Edomonston株和CAM株比较，有4例样品有2处突变。并对耳硬化症患者的外淋巴液进行了RT-PCR分析。与Edomonston株和CAM株相比，3个样品中存在3个核苷酸替换。推导的氨基酸序列与Edomonston株和CAM株比较，有3个样品有1处突变。