An oligonucleotide microarray for multiplex real-time PCR identification of HIV-1HBV and HCV
An oligonucleotide microarray for multiplex real-time PCR identification of HIV-1HBV and HCV
复制标题
DOI：
10.2144/000112628
复制
发表时间：
2008-02-01
期刊：
影响因子：
2.7
通讯作者：
Mikhailovich, Vladimir M.
中科院分区：
文献类型：
--
作者：
Khodakov, Dmitry A.;Zakharova, Natalia V.;Mikhailovich, Vladimir M.
我们描述了一种新的基于微阵列的方法，用于同时鉴定和定量供体血浆标本中的人类免疫缺陷病毒1型（HIV-1）和肝炎B和C病毒（HBV和HCV）。该方法基于在微阵列水凝胶垫内进行的多重实时RT-PCR。双链扩增产物使用非特异性SYBR绿色I染料同时检测，因为反应在带有5 '-固定化特异性引物的单独垫中进行。基于132份分析的血液标本，该检测的灵敏度和特异性均为100%（HBV、HCV和HIV-1血清学阳性标本分别为56、26和8份; HIV-1和HCV均阳性标本22份，所有三种病毒均阳性标本2份;病原体阴性标本18份）。定量分析的动态范围涵盖了每项测定100至10（6）个基因组当量的六阶区间。每次检测的95%检出限为HIV-1 14 gEq、HBV 10 gEq（1.7 IU）和HCV 15 gEq（7.5 IU）。所提出的方法被认为是通用的，可以适用于同时识别和定量的许多遗传目标。
We describe a novel microarray-based approach for simultaneous identification and quantification of human immunodeficiency virus type 1 (HIV-1) and hepatitis B and C viruses (HBV and HCV) in donor plasma specimens. The method is based on multiplex real-time RT-PCR performed within the microarray hydrogel pads. Double-stranded amplification products are simultaneously detected using nonspecific SYBR Green I dye due to the reaction run in separate pads bearing 5 '-immobilized specific primers. Both the sensitivity and specificity of the assay, based on 132 blood specimens analyzed, were 100% (56, 26, and 8 specimens were seropositive to HBV HCV and HIV-1, respectively; 22 were positive to both HIV-1 and HCV and 2 positive to all three viruses; 18 samples were pathogen-negative). The dynamic range of the quantitative analysis covered a six-order interval ranging from 100 to 10(6) genome equivalents per assay. The 95% detection limits were 14 gEq for HIV-1, 10 gEq (1.7 IU) for HBV and 15 gEq (7.5 IU for HCV per assay. The proposed approach is considered to be versatile and could be adapted for simultaneous identification and quantification of numerous genetic targets.