Detection of HGV-RNA by Hybridizati0n with Fluorescein．Labeled Probe

NING De-Gang; LIU Hai-Yan; LEI Shi-Mei; QIN Wei-Wei; XIANG En-Hong; LI Tian-Xian

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April 1999

NING De-Gang, LIU Hai-Yan, LEI Shi-Mei, QIN Wei-Wei, XIANG En-Hong and LI Tian-Xian. Detection of HGV-RNA by Hybridizati0n with Fluorescein．Labeled Probe[J]. Virologica Sinica, 1999, 14(2): 147-151.

Citation: NING De-Gang, LIU Hai-Yan, LEI Shi-Mei, QIN Wei-Wei, XIANG En-Hong, LI Tian-Xian. Detection of HGV-RNA by Hybridizati0n with Fluorescein．Labeled Probe .VIROLOGICA SINICA, 1999, 14(2) : 147-151.

荧光素标记的庚型肝炎病毒基因探针的制备及应用

摘要

参考国内外已完成测序的庚型肝炎病毒（ＧＢＶＣ／ＨＧＶ）基因序列，选取毒株间系列较保守的基因片段，并合成与之互补的核苷酸序列（Ａｎｔｉｓｅｎｓｅ），用末端转移酶将荧光素Ｎ６ｄｄＡＴＰ标记该片段，制成庚肝病毒基因探针。在严格控制温度的条件下，与固定于硝酸纤维膜（ＮＣ膜）上血清斑点杂交，洗膜后与抗荧光素碱性磷酸酶（ＡＰ）结合，加底物后化学发光自显影判断结果。该方法检测与套式逆转录聚合酶链反应（ＮｅｓｔｅｄＲＴＰＣＲ）检测结果的阳性符合率为８８．２％，阴性符合率为１００％；并且与其他相关病毒基因无交叉反应，具有较好的特异性与灵敏性。其检测结果比ＥＩＡ法检测庚肝病毒抗体更具临床意义。
- 庚型肝炎病毒
- , 逆转录-聚合酶链反应
- , 荧光素标记基因探针
- , 分子杂交

Detection of HGV-RNA by Hybridizati0n with Fluorescein．Labeled Probe

1.
南宁市卫生防痤站，南宁530011

Abstract

The sequences of HGV gene that had been reported were checked and analyzed. After comparison of HGV seqences from different virus strains, a gene fragment of 31 nucleotides (7121-7152 nt) served as a probe was labeled by fluorescein N 6 dd ATP with terminal transferase. The specificity of HGV probe was tested by dot blot hybridization with HCV RNA related virus RNA, C.T DNA, HGV RNA, and Southern blot hybridization with RT PCR product of HGV. Positive results obstained only from the HGV RNA of positive sera that was confirmed by nested RT PCR, all others were negative. The sensitivity of fluorescein labeled probe was examined. The result showed that the fluorescein labeled probe was able to check1.56 pg of target genome. The conformity of HGV RNA detection using RT PCR and HGV gene probe was 97.9% . The experiment suggested that fluorescein labeled probe can be used in clinic detection and epidemic study.
- HGV
- , RTPCR
- , Fluorescdnlabeled probe
- , Hybridizatio

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通讯作者: 陈斌, bchen63@163.com

1.
沈阳化工大学材料科学与工程学院沈阳 110142

Detection of HGV-RNA by Hybridizati0n with Fluorescein．Labeled Probe

1. 南宁市卫生防痤站，南宁530011

Abstract: The sequences of HGV gene that had been reported were checked and analyzed. After comparison of HGV seqences from different virus strains, a gene fragment of 31 nucleotides (7121-7152 nt) served as a probe was labeled by fluorescein N 6 dd ATP with terminal transferase. The specificity of HGV probe was tested by dot blot hybridization with HCV RNA related virus RNA, C.T DNA, HGV RNA, and Southern blot hybridization with RT PCR product of HGV. Positive results obstained only from the HGV RNA of positive sera that was confirmed by nested RT PCR, all others were negative. The sensitivity of fluorescein labeled probe was examined. The result showed that the fluorescein labeled probe was able to check1.56 pg of target genome. The conformity of HGV RNA detection using RT PCR and HGV gene probe was 97.9% . The experiment suggested that fluorescein labeled probe can be used in clinic detection and epidemic study.