Reproducibility of HPV 16 and HPV 18 viral load quantitation using TaqMan real-time PCR assays
Reproducibility of HPV 16 and HPV 18 viral load quantitation using TaqMan real-time PCR assays
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DOI：
10.1016/s0166-0934(03)00186-1
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发表时间：
2003-09-01
影响因子：
3.1
通讯作者：
Shah, KV
中科院分区：
文献类型：
--
作者：
Gravitt, PE;Peyton, C;Shah, KV
重现性研究旨在评估靶向HPV 16、HPV 18和人甘油醛-3-磷酸脱氢酶（GAPDH）假基因的三种实时PCR检测试剂的检测内、日间和实验室间变异性。分别通过GAPDH和HPV 16以及GAPDH和HPV 18检测试剂对15份HPV 16和15份HPV 18宫颈拭子样本进行一式三份扩增。所有样本均在同一实验室中以未稀释和1：10稀释度在不同的2天进行扩增，并且相同的样本在不同的实验室中进行扩增。HPV 16和HPV 18标准化病毒载量报告为每20 000个GAPDH拷贝的HPV基因组数量。HPV 16和18检测的分析特异性分别为100%和97%。HPV 16、HPV 18和GAPDH的组内相关系数（ICC）分别为0.99、0.97和0.98，表明实验误差引起的变异性非常低。病毒载量的十倍差异可以在六个数量级的动态范围内容易地区分（约100）。5-5 x 10（6）份）。在更高的靶浓度（> 5000拷贝）下，辨别能力增加。两个实验室之间标准化HPV 16和18病毒载量的相关性较高（分别为斯皮尔曼rho（rho）= 0.96和0.87）。这些具有GAPDH标准化的HPV 16和HPV 18定量PCR检测在宽线性动态范围内可重复定量，允许在流行病学研究中应用以测量病毒载量。(C)2003 Elsevier B. V.保留所有权利。
A reproducibility study was designed to assess within-assay, between-day, and interlaboratory variability of three real-time PCR assays targeting HPV 16, HPV 18, and the human glyceraldehyde-3-phosphate dehydrogenase (GAPDH) pseudogenes. Fifteen HPV 16 and fifteen HPV 18 cervical swab samples were amplified in triplicate by GAPDH and HPV 16 and by GAPDH and HPV 18 assays, respectively. All samples were amplified undiluted and at a 1: 10 dilution on 2 separate days in the same laboratory, and the same samples were amplified in a separate laboratory. HPV 16 and HPV 18 normalized viral load is reported as the number of HPV genomes per 20 000 GAPDH copies. The analytic specificity of the HPV 16 and 18 assays was 100 and 97%, respectively. The intraclass correlation coefficients (ICC) were 0.99, 0.97, and 0.98 for HPV 16, HPV 18, and GAPDH, respectively, indicating that the variability due to experimental error was very low. Ten-fold differences in viral load could be readily discriminated across a six order of magnitude dynamic range (ca. 5-5 x 10(6) Copies). Power of discrimination was increased at higher target concentrations ( > 5000 copies). The correlation of normalized HPV 16 and 18 viral load was high between the two laboratories (Spearman rho (rho) = 0.96 and 0.87, respectively). These HPV 16 and HPV 18 quantitative PCR assays with GAPDH normalization are reproducibly quantitative over a broad linear dynamic range allowing for application in epidemiologic studies for measurement of viral load. (C) 2003 Elsevier B.V. All rights reserved.