Khodakov Dmitry A, Zakharova Natalia V, Gryadunov Dmitry A, Filatov Felix P, Zasedatelev Alexander S, Mikhailovich Vladimir M
Engelhardt Institute of Molecular Biology, Russian Academy of Sciences, Russia.
Biotechniques. 2008 Feb;44(2):241-6, 248. doi: 10.2144/000112628.
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 106 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.
我们描述了一种基于微阵列的新方法，用于同时鉴定和定量供体血浆样本中的1型人类免疫缺陷病毒（HIV-1）以及乙型和丙型肝炎病毒（HBV和HCV）。该方法基于在微阵列水凝胶垫内进行的多重实时逆转录聚合酶链反应（RT-PCR）。由于反应在带有5'固定化特异性引物的单独垫中进行，双链扩增产物使用非特异性SYBR Green I染料同时检测。基于对132份血液样本的分析，该检测方法的灵敏度和特异性均为100%（分别有56、26和8份样本对HBV、HCV和HIV-1呈血清学阳性；22份样本对HIV-1和HCV均呈阳性，2份样本对所有三种病毒均呈阳性；18份样本病原体呈阴性）。定量分析的动态范围涵盖了每个检测从100到106基因组当量的六个数量级区间。每次检测的95%检测限为HIV-1 14基因组当量、HBV 10基因组当量（1.7国际单位）和HCV 15基因组当量（7.5国际单位）。所提出的方法被认为具有通用性，可适用于同时鉴定和定量众多遗传靶点。
