Demonstration of hepatitis C virus RNA with in situ hybridization employing a locked nucleic Acid probe in humanized liver of infected chimeric mice and in needle-biopsied human liver.
Demonstration of hepatitis C virus RNA with in situ hybridization employing a locked nucleic Acid probe in humanized liver of infected chimeric mice and in needle-biopsied human liver.
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DOI：
10.1155/2013/249535
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发表时间：
2013
影响因子：
1.8
通讯作者：
Tsutsumi Y
中科院分区：
文献类型：
--
作者：
Shiogama K;Inada K;Kohara M;Teramoto H;Mizutani Y;Onouchi T;Tsutsumi Y
背景资料。高灵敏度的原位杂交(ISH)已被要求在福尔马林固定、石蜡包埋(FFPE)的肝脏切片中显示丙型肝炎病毒(HCV)RNA。方法：研究方法。采用锁定核酸(LNA-)修饰的寡核苷酸探针和非生物素催化信号放大系统(CSAII)的ISH技术检测肝组织中的丙型肝炎病毒RNA。提取FFPE组织总RNA，采用套式逆转录聚合酶链式反应(RT-PCR)进行丙型肝炎病毒基因分型。目标组织包括丙型肝炎病毒感染的嵌合小鼠人源化肝脏的FFPE组织切片(丙型肝炎病毒1a、1b和2a和未感染的)和来自丙型肝炎病毒感染患者的针吸活组织切片。结果。用ISH技术在两种嵌合体小鼠和11例丙型肝炎病毒感染者中的9例(82%)的肝组织中检测到了丙型肝炎病毒RNA。丙型肝炎病毒信号对核糖核酸酶敏感。在11个肝脏中，有8个(73%)证实了基因分型，其中1b型6个，2a型2个。在慢性乙肝、脂肪肝、自身免疫性肝炎和肝细胞癌中未检测到丙型肝炎病毒RNA。结论。用LNA修饰的寡核苷酸探针和CSAII进行原位杂交，可用于检测常规制备的FFPE肝组织中的丙型肝炎病毒RNA。
Background. In situ hybridization (ISH) with high sensitivity has been requested to demonstrate hepatitis C virus (HCV) RNA in formalin-fixed, paraffin-embedded (FFPE) sections of the liver. Methods. ISH employing a locked-nucleic-acid- (LNA-)modified oligonucleotide probe and biotin-free catalyzed signal amplification system (CSAII) was applied to HCV-RNA detection in the liver tissue. Nested reverse-transcription polymerase chain reaction (RT-PCR) was performed for HCV genotyping using total RNA extracted from FFPE sections. The target tissues included FFPE tissue sections of humanized livers in HCV-infected chimeric mice (HCV genotypes 1a, 1b, and 2a and noninfected) and of needle-biopsied livers from HCV-infected patients. Results. HCV-RNA was demonstrated with the ISH technique in HCV-infected liver tissues from both chimeric mice and 9 (82%) of 11 patients with HCV infection. The HCV signals were sensitive to RNase. Nested RT-PCR confirmed the genotype in 8 (73%) of 11 livers (type 1b: 6 lesions and type 2a: 2 lesions). HCV-RNA was not identified in chronic hepatitis B lesions, fatty liver, autoimmune hepatitis, and hepatocellular carcinoma. Conclusion. ISH using the LNA-modified oligonucleotide probe and CSAII was applicable to detecting HCV-RNA in routinely prepared FFPE liver specimens.
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