Abe Manabu, Klett Corinne, Wieland Eberhard, Gille Sascha, Landt Olfert
Central Institute for Clinical Chemistry and Laboratory Medicine, Klinikum Stuttgart Katharinenhospital, Stuttgart, Germany.
Folia Med (Plovdiv). 2008 Jul-Sep;50(3):5-13.
Current HIV-1 viral-load assays are too expensive and time-consuming for small sample quantity or resource-limited setting. In addition, some commercial assays have shown shortcomings in quantifying rare genotypes. We developed an internally controlled, two-step, reverse transcription-initiated real-time PCR protocol on the LightCycler instrument achieving a favourable detection limit with an extended quantification range, detecting all HIV-1 subtypes suitable for laboratories with low sample throughput. The detection limit was found to be 100 copies/ml, the dynamic range up to 500,000,000 copies/ml. Intra and inter assay imprecision were 1.6% and 2.0% (n=10), respectively. The assay was calibrated against WHO Standard 97/656. The HIV-1 RNA values obtained by real-time PCR assay were highly correlated with those obtained by the Cobas Amplicor HIV-1 Monitor (r = 0.954; p < 0.001). This rapid (3 h) and cost effective assay is suitable for both HIV-1 detection and disease monitoring with the ability to detect and quantify all HIV-1 subtypes including O1 and O2.
对于小样本量或资源有限的环境，目前的HIV-1病毒载量检测方法过于昂贵且耗时。此外，一些商业检测方法在定量罕见基因型方面存在缺陷。我们在LightCycler仪器上开发了一种内部对照的两步逆转录启动实时PCR方案，该方案具有良好的检测限和扩展的定量范围，可检测所有HIV-1亚型，适用于低样本通量的实验室。检测限为100拷贝/毫升，动态范围高达500,000,000拷贝/毫升。批内和批间不精密度分别为1.6%和2.0%（n=10）。该检测方法以WHO标准97/656进行校准。通过实时PCR检测获得的HIV-1 RNA值与通过Cobas Amplicor HIV-1 Monitor获得的值高度相关（r = 0.954；p < 0.001）。这种快速（3小时）且经济高效的检测方法适用于HIV-1检测和疾病监测，能够检测和定量所有HIV-1亚型，包括O1和O2。
