A real-time, quantitative PCR method using hydrolysis probes for the monitoring of Plasmodium falciparum load in experimentally infected human volunteers.
A real-time, quantitative PCR method using hydrolysis probes for the monitoring of Plasmodium falciparum load in experimentally infected human volunteers.
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DOI：
10.1186/1475-2875-10-48
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发表时间：
2011-02-28
期刊：
影响因子：
3
通讯作者：
Sloots TP
中科院分区：
文献类型：
--
作者：
Rockett RJ;Tozer SJ;Peatey C;Bialasiewicz S;Whiley DM;Nissen MD;Trenholme K;Mc Carthy JS;Sloots TP
通过 PCR 准确定量恶性疟原虫寄生虫数量是监测感染者和随后接受抗疟疾药物治疗的受试者生长动力学的重要工具。采用引物和针对 18S rRNA 基因的水解探针的实时定量 PCR (rt-qPCR) 方法进行了调整和优化，以估计血液样本中的寄生虫含量。样本包括实验室制备的不同寄生虫浓度的血液样本（每 500 μl 浓缩红细胞 (500pRBC) 6.4 × 105 至 6.4 个寄生虫）以及从实验感染的人类受试者在 10 天内的 19 个时间点收集的血液样本。将样品制备和提取、检测化学、测定重现性和检测限与之前发布的疟疾疫苗临床试验中使用的 SYBR Green rt-qPCR 进行比较。 rt-qPCR 水解探针测定和 SYBR Green rt-qPCR 的检测限均为每 500pRBC 6.4 × 101 个寄生虫。然而，SYBR Green rt-qPCR 检测中的非特异性扩增导致对每 500pRBC 6.4 × 102 个寄生虫水平以下的寄生虫负荷估计不准确，并导致阴性样品中寄生虫的假阳性检测。 rt-qPCR 水解探针测定具有特异性，可对寄生虫血症进行可靠的定量，低至每 500pRBC 6.4 × 101 个寄生虫。值得注意的是，从实验感染受试者收集的 19 个连续样本中，有 12 个样本的拷贝数等于或低于每 500pRBC 6.4 × 102 拷贝。这些结果表明，对于人类血液样本中恶性疟原虫的定量，水解探针 rt-qPCR 测定优于 SYBR Green rt-qPCR。水解探针 rt-qPCR 目前在昆士兰儿科传染病实验室 (QPID) 使用，用于监测实验感染的临床试验受试者的寄生虫血症。
The accurate quantification of Plasmodium falciparum parasite numbers by PCR is an important tool for monitoring growth kinetics in subjects infected and subsequently treated with anti-malarial agents. A real-time quantitative PCR (rt-qPCR) method using primers and a hydrolysis probe that targets the 18S rRNA gene was adapted and optimized to estimate parasite load in blood samples. Samples included laboratory prepared blood samples of varying parasite concentrations (6.4 × 105 to 6.4 parasites per 500 μl of packed red blood cells (500pRBC)) and blood samples collected from an experimentally infected human subject collected at 19 time points over 10 days. Sample preparation and extraction, detection chemistry, assay reproducibility, and limit of detection were compared to a previously published SYBR Green rt-qPCR used in a malaria vaccine clinical trial. Both the rt-qPCR hydrolysis probe assay and SYBR Green rt-qPCR provided a limit of detection of 6.4 × 101 parasites per 500pRBC. However non-specific amplification in the SYBR Green rt-qPCR assay led to either inaccurate estimation of parasite load at levels below 6.4 × 102 parasites per 500pRBC and to false-positive detection of parasites in negative samples. The rt-qPCR hydrolysis probe assay was specific and provided reliable quantification of parasitaemia down to 6.4 × 101 parasites per 500pRBC. Notably, 12 of the 19 consecutive samples collected from the experimentally infected subject were at or below 6.4 × 102 copies per 500pRBC. These results show that the hydrolysis probe rt-qPCR assay is superior to the SYBR Green rt-qPCR for the quantification of P. falciparum in human blood samples. The hydrolysis probe rt-qPCR is now in use in the Queensland paediatric infectious diseases laboratory (QPID) to monitor parasitaemia in experimentally-infected clinical trial subjects.
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影响因子：
3.6
作者：
Bialasiewicz, S.;Whiley, D. M.;Sloots, T. P.
通讯作者：
Sloots, T. P.
DOI：
10.4269/ajtmh.2005.73.191
发表时间：
2005-07-01
影响因子：
3.3
作者：
Andrews, L;Andersen, RF;Gilbert, SC
通讯作者：
Gilbert, SC
DOI：
10.1016/j.diagmicrobio.2003.10.014
发表时间：
2004-05-01
影响因子：
2.9
作者：
Whiley, DM;LeCornec, GM;Sloots, TP
通讯作者：
Sloots, TP
影响因子：
1.5
作者：
Dixon, Matthew W. A.;Peatey, Christopher L.;Trenholme, Katharine R.
通讯作者：
Trenholme, Katharine R.
影响因子：
1.5
作者：
Hermsen, CC;Telgt, DSC;Sauerwein, RW
通讯作者：
Sauerwein, RW