[1]乔 斌,李 艳,汪 明,等.新型TaqMan-MGB探针实时荧光定量PCR检测HCV-RNA方法学的建立[J].现代检验医学杂志,2017,32(05):32-35.[doi:10.3969/j.issn.1671-7414.2017.05.009]
　QIAO Bin,LI Yan,WANG Ming,et al.Development of a New TaqMan-MGB Probe-Based Real-Time qPCR Method for the Detection of HCV-RNA[J].Journal of Modern Laboratory Medicine,2017,32(05):32-35.[doi:10.3969/j.issn.1671-7414.2017.05.009]
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新型TaqMan-MGB探针实时荧光定量PCR检测HCV-RNA方法学的建立()
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《现代检验医学杂志》[ISSN:/CN:]

卷:
第32卷
期数:
2017年05期
页码:
32-35
栏目:
论著
出版日期:
2017-11-02

文章信息/Info

Title:
Development of a New TaqMan-MGB Probe-Based Real-Time qPCR Method for the Detection of HCV-RNA
文章编号:
1671-7414(2017)05-032-04
作者:
乔 斌; 李 艳; 汪 明; 熊 格
武汉大学人民医院检验科,武汉 430060
Author(s):
QIAO Bin; LI Yan; WANG Ming; XIONG Ge
Department of Clinical Laboratory,Renmin Hospital of Wuhan University,Wuhan 430060,China
关键词:
TaqMan-MGB探针;  实时荧光定量PCR;  丙型肝炎病毒核酸;  假病毒颗粒
分类号:
Q503; R512.63
DOI:
10.3969/j.issn.1671-7414.2017.05.009
文献标志码:
A
摘要:
目的 建立一种快速、准确定量HCV-RNA的实时荧光定量PCR检测方法。方法 Clustal X软件对HCV核酸序列进行比对分析,在HCV-RNA的保守区域5'UTR设计引物和探针,棋盘滴定法对实时荧光定量PCR体系进行性能优化,制作假病毒颗粒作为定量标准品对新建方法进行性能评价,并与临床常用HCV-RNA定量检测试剂盒进行比对分析,探讨其应用价值。结果 建立HCV核酸定量高灵敏度方法,该方法可以检测1a,1b,2a,3a,3b,6a,6b等多个HCV基因型,检测HCV-RNA的灵敏度为50 IU/ml,精密度CV<5%,检测结果可溯源至卫生部临床检验中心丙型肝炎病毒核酸(HCV-RNA)血清标准物质GBW09151a。新建方法与凯杰HCV-RNA荧光定量检测试剂盒同时检测40例临床样本,阳性一致率为100%,阴性一致率为56%; 自建方法检测阳性的14个样本中,凯杰试剂0个阳性,表明自建方法的检测灵敏度要高于凯杰试剂。结论 建立的HCV核酸定量新方法灵敏度高、特异性好、精密度高,可应用于临床。
Abstract:
Abstract:Objective Developing a rapid andaccurate real-time qPCR method for the detection of HCV-RNA.Methods HCV nucleotide sequence was analysed in Clustal software and primers and probewere designed in the conserved region of 5'UTR.The reaction system optimization of real-time qPCR method was used chessboard titration,pseudoviral particles were used as quantitative standard to assess the performance.New methods was compared with clinical commonly used kit of HCV-RNA and discuss the application value.Results The sensitivity of new real-time qPCRmethod was 50 IU/ml,coefficient variation was less than 5%.The quantitative results of this method could be traceable to national standards of GBW09151a.40samples were determined by new methods and clinical commonly used kit of HCV-RNA,the positive concordance rate was 100%,the negative concordance rate was 56%.14 samples were positive by new method,but negative by Qiagen kit,illustrating that the sensitivity of new method was superior to Qiagen kit.Conclusion New TaqMan-MGB probe-based real-time qPCR method is a specific,sensitive,simple,rapid and exactly used to detection of HCV-RNA.

参考文献/References:

[1] World Health Organization Guidelines Approved by the Guidelines Review Committee.Guidelines for the screening,care and treatment of persons with hepatitis C infection[M].Geneva:World Health Organization,2014(172):343-346.
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[3] American Association for the Study of Liver Diseases.Recommendations forTesting,Managing,and Treating Hepatitis C[OL].http://www.hcvguidelines.org,2014.
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备注/Memo

备注/Memo:
作者简介:乔 斌(1988-),男,硕士研究生,技师,主要从事临床分子诊断,E-mail:qiaobin223@126.com。 通讯作者:李 艳,E-mail:liyan@whu.edu.cn。
更新日期/Last Update: 1900-01-01