Hymas Weston, Stevenson Jeffery, Taggart E W, Hillyard David
Associated Regional and University Pathologists (ARUP) Institute for Clinical and Experimental Pathology, 500 Chipeta Way, Salt Lake City, UT 84108, USA.
J Virol Methods. 2005 Sep;128(1-2):143-50. doi: 10.1016/j.jviromet.2005.05.003.
A quantitative real time PCR assay utilizing an Eclipse minor groove binding hybridization probe was developed to detect and type human herpes 6. A 115 base pair product from the U67 gene was selected for amplification and the assay included a noncompetitive internal control. The probe's melting temperature from the amplified sequence differentiated between HHV6 variants (A and B). In this study, 120 samples (60 spiked and 60 negative) comprising CSF, plasma, and serum were tested at high and medium levels, and near the limit of quantitation. The use of stored standard curves for assay calibration was compared to curves run on each assay, and the stability of liquid frozen versus lyophilized frozen stocks for calibrators and controls was assessed. After 9 months of clinical testing, assay performance was examined to determine the percent positive rate and positive sample reproducibility, as well as to evaluate standard curve stability. We obtained 100% correlation to expected results for positive and negative samples. A stored curve proved easier, more cost effective, and more reliable than running a standard curve on each assay. The use of lyophilized standards contributes substantially to the maintenance of reproducible testing over an extended period of time.
开发了一种利用Eclipse小沟结合杂交探针的定量实时PCR检测方法，用于检测人疱疹病毒6型并进行分型。选择来自U67基因的115个碱基对的产物进行扩增，该检测方法包括一个非竞争性内部对照。探针与扩增序列的熔解温度可区分HHV6变体（A和B）。在本研究中，对120份样本（60份加标样本和60份阴性样本）进行了检测，这些样本包括脑脊液、血浆和血清，检测水平为高、中水平以及接近定量限。将使用储存的标准曲线进行检测校准与每次检测时运行的曲线进行了比较，并评估了校准物和对照品的液体冷冻与冻干冷冻储备的稳定性。经过9个月的临床检测，检查了检测性能，以确定阳性率百分比和阳性样本的重现性，并评估标准曲线的稳定性。我们获得的阳性和阴性样本结果与预期结果的相关性为100%。与每次检测时运行标准曲线相比，使用储存曲线更简便、更具成本效益且更可靠。冻干标准品的使用在很大程度上有助于在较长时间内维持可重复检测。
