干血浆和血斑储存条件对 HIV-1 RNA 定量及耐药基因分型 PCR 扩增的影响。

Effect of storage conditions of dried plasma and blood spots on HIV-1 RNA quantification and PCR amplification for drug resistance genotyping.

机构信息

UMR 145, Institut de Recherche pour le Développement (IRD) and University of Montpellier 1, Montpellier, France.

出版信息

J Antimicrob Chemother. 2010 Aug;65(8):1562-6. doi: 10.1093/jac/dkq205. Epub 2010 Jun 11.

DOI:10.1093/jac/dkq205

PMID:20542904

Abstract

OBJECTIVES

Dried blood spots (DBS) and dried plasma spots (DPS) are easy to collect and store, and have been successfully tested as an alternative to plasma for performing virological analyses. Adequate storage conditions still need to be established and cell-associated proviral DNA in DBS can contribute to the amplified products. We evaluated these two parameters.

METHODS

Residual samples from 34 HIV-1-infected patients [mean viral load (VL) = 3.93 log(10) copies/mL] were used to prepare DPS and DBS, then stored at 20 degrees C and 37 degrees C. HIV-1 nucleic acids were extracted, with or without DNase treatments, to perform HIV-1 VL quantification and nested RT-PCR to amplify the reverse transcriptase gene (798 bp).

RESULTS

For DBS stored for 3 months at 20 degrees C, VL could be measured for all samples and results were comparable to plasma VL. At 37 degrees C, a slight decrease was observed after 2 and 3 months (0.16 and 0.37 log(10) copies/mL mean difference, respectively). For DPS, a significant decrease in VL (0.70 and 1.07 log(10) copies/mL after 1 and 2 months, respectively) was seen at 37 degrees C, but not at 20 degrees C. PCR amplifications from DPS were only successful for 50% of samples with an initial VL >10 000 copies/mL after 1 month at 20 degrees C. From DBS, PCR amplifications are possible until 3 months for samples with plasma VL >5000 copies/mL. VL and PCR results for DBS treated with DNase are close to results obtained for DPS.

CONCLUSIONS

Virological monitoring is still feasible for DBS after 3 months of storage at 37 degrees C when VL is >5000 copies/mL, but DNA contributes largely to the final results.

摘要

目的

干血斑（DBS）和干血浆斑（DPS）易于采集和储存，并且已成功测试作为替代血浆进行病毒学分析的方法。仍需要建立足够的储存条件，并且 DBS 中的细胞相关前病毒 DNA 可能会导致扩增产物增加。我们评估了这两个参数。

方法

使用 34 名 HIV-1 感染患者的剩余样本（平均病毒载量（VL）= 3.93 log(10) 拷贝/ml）来制备 DPS 和 DBS，然后在 20°C 和 37°C 下储存。提取 HIV-1 核酸，进行或不进行 DNase 处理，以进行 HIV-1 VL 定量和巢式 RT-PCR 以扩增逆转录酶基因（798 bp）。

结果

对于在 20°C 下储存 3 个月的 DBS，所有样本均可以测量 VL，并且结果与血浆 VL 相当。在 37°C 下，2 个月和 3 个月后观察到 VL 略有下降（分别为 0.16 和 0.37 log(10) 拷贝/ml 的平均差异）。对于 DPS，在 37°C 下，1 个月和 2 个月后 VL 分别显著下降（0.70 和 1.07 log(10) 拷贝/ml），而在 20°C 下则没有。在 20°C 下储存 1 个月后，初始 VL >10 000 拷贝/ml 的 50%样本的 DPS 中 PCR 扩增仅成功。对于血浆 VL >5000 拷贝/ml 的样本，DBS 的 PCR 扩增可在 3 个月内进行。经过 DNase 处理的 DBS 的 VL 和 PCR 结果与 DPS 的结果接近。