Quantitative RT-PCR assay for high-throughput screening (HTS) of drugs against the growth of Cryptosporidium parvum in vitro.
Quantitative RT-PCR assay for high-throughput screening (HTS) of drugs against the growth of Cryptosporidium parvum in vitro.
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DOI：
10.3389/fmicb.2015.00991
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发表时间：
2015
影响因子：
5.2
通讯作者：
Zhu G
中科院分区：
文献类型：
--
作者：
Zhang H;Zhu G
我们实验室之前开发了一种qRT-PCR方法，通过检测寄生虫18S rRNA的水平来评估药物对小隐孢子虫体外生长的疗效。该方法显示了高达4个数量级的线性动态范围，并且比传统的显微方法节省了大量的劳动。然而，当总RNA需要经过漫长的、多步骤的过程纯化时，传统的qRT-PCR协议不适合高通量分析。最近，几种商用试剂可用于制备可直接用于下游qRT-PCR分析的细胞裂解物（例如，Ambion cell -to- cdna试剂盒和Bio-Rad iScript样品制备试剂）。使用这些试剂，我们能够使qRT-PCR检测适应体外药物的高通量筛选（即96孔和384孔格式分别用于寄生虫培养和qRT-PCR检测）。该qRT-PCR方案能够使用从感染各种数量寄生虫的细胞中分离的样品提供> - 150倍的线性动态范围。新的分析也通过nih推荐的板内、板间和日间均匀性测试进行验证。通过评估paromomycin的抗隐孢子虫功效和对化合物进行小规模筛选，也证实了该检测的稳健性和有效性。
Our laboratory has previously developed a qRT-PCR assay to assess drug efficacy on the growth of Cryptosporidium parvum in vitro by detecting the levels of parasite 18S rRNA. This approach displayed up to four orders of magnitude of linear dynamic range and was much less labor-intensive than the traditional microscopic methods. However, conventional qRT-PCR protocol is not very amendable to high-throughput analysis when total RNA needs to be purified by lengthy, multi-step procedures. Recently, several commercial reagents are available for preparing cell lysates that could be directly used in downstream qRT-PCR analysis (e.g., Ambion Cell-to-cDNA kit and Bio-Rad iScript sample preparation reagent). Using these reagents, we are able to adapt the qRT-PCR assay into high-throughput screening of drugs in vitro (i.e., 96-well and 384-well formats for the cultivation of parasites and qRT-PCR detection, respectively). This qRT-PCR protocol is able to give a >150-fold linear dynamic range using samples isolated from cells infected with various numbers of parasites. The new assay is also validated by the NIH-recommended intra-plate, inter-plate, and inter-day uniformity tests. The robustness and effectiveness of the assay are also confirmed by evaluating the anti-cryptosporidial efficacy of paromomycin and by a small scale screening of compounds.