干血斑样本用于HIV-1基因序列测定和亚型分析的研究

张麒; 潘品良; 邓红; 王临虹; 蒋岩

Abstract

张麒,潘品良,邓红,王临虹,蒋岩.干血斑样本用于HIV-1基因序列测定和亚型分析的研究[J].Chinese journal of Epidemiology,2006,27(9):798-802

干血斑样本用于HIV-1基因序列测定和亚型分析的研究

Study on the use of dried blood spgt in sequencing and subtyping HIV-1 DNA genome

Received:December 22, 2005

DOI：

KeyWord: 艾滋病病毒亚型分析干血斑

English Key Word: Human immunodeficiency virus Subtype analysis Dried blood spot

FundProject:国家“十五”科技攻关计划课题(2004BA719A03)

Author Name	Affiliation	E-mail
ZHANG Qi	National Center for Woman and Children Health,Chinese Center,for Disease Control and Prevention,Beijing 100013, China
PAN Pin-liang	National Center for Woman and Children Health,Chinese Center,for Disease Control and Prevention,Beijing 100013, China
DENG Hong	中国疾病预防控制中心性病艾滋病预防控制中心
WANG Lin-hong	中国疾病预防控制中心性病艾滋病预防控制中心
JIANG Yan	新疆维吾尔族自治区疾病预防控制中心	Jangyan03@263.net

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Abstract:

目的将干血斑样本用于HIV-1基因序列测定和亚型分析。方法采集20例静脉吸毒HIV-1感染者的EDTA抗凝静脉全血2 ml,80μl制备干血斑样本。QIAamp Blood Mini试剂盒,10%Chelex 100树脂分别提取全血和干血斑中细胞DNA。HIV-1 env基因Gp41区2对外侧和内侧引物,按照巢式PCR方法,扩增Gp41区,PCR产物用内侧正反向引物测序。MEGA 3．0等软件完成序列比对及亚型分析。结果18对符合要求的干血斑和全血样本进入研究。16对经序列分析归为中国流行的BC重组亚型,样本间的基因平均离散率为5．79%,与中国流行的BC重组亚型的基因离散率为4．91%,与C亚型代表序列基因离散率为8．89%,与其他几个国际亚型代表株的基因离散率在15%～20%之间。另2对样本分别归属于泰国B'亚型和欧美B亚型。结论干血斑样本与全血样本HIV-1 DNA PCR产物亚型分析一致,氨基酸序列比对同义和非同义替换比例、位置和类型基本一致,其易采集,便于运输,保存条件和生物危险性低,费用低廉的优点适合作为实验条件较差的偏远地区HIV-1分子流行病学研究的一种样本采集手段。

English Abstract:

Objective To use dried blood spot (DBS) in studying the sequence and subtype analysis of HIV-1 genome. Methods 2 ml whole blood containing EDTA anticoagulant from 20 HIV-1 infected patients were collected, then 80 p1 blood was used to propare DBS. QIAamp Blood Mini kit and 10% Chelex.100 resin extracted DNA genome from whole blood and DIiS as well as nested PCR amplified specifically HIV-1 Gp41 region from the two kinds of DNA extraction. Software MAGE 3.0 was used to study the sequence and subtype of the PCR products. Results Eligible 18 paired samples were analysed to show that 16 of them belonged to C54A, 97CNGX-7I', 98CN006 subtypes. The other two samples might belong to B. CN._.RL42 and B. US. 90WEAU160.Conclusion Data showed that there wem parallel results between the whole blood and DBS samples including' subtype analysis,position of mutation and types of amino acid sequencing. Since D$S itself facilitated the collection, transportation and storage, it could be used as a measure to collect blood sample in resource limited area and to develop molecular epidemiologic research as well as early diagnosis on infant exposed to HIV.

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