Application of quantitative real-time reverse transcription-PCR in assessing drug efficacy against the intracellular pathogen Cryptosporidium parvum in vitro
Application of quantitative real-time reverse transcription-PCR in assessing drug efficacy against the intracellular pathogen Cryptosporidium parvum in vitro
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DOI：
10.1128/aac.49.11.4437-4442.2005
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发表时间：
2005-11-01
影响因子：
4.9
通讯作者：
Zhu, G
中科院分区：
文献类型：
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作者：
Cai, XM;Woods, KM;Zhu, G
我们报道了一种定量实时逆转录-聚合酶链式反应(qRT-PCR)检测方法，用于评估药物对细胞内微小隐孢子虫的疗效。QRT-PCR方法检测两种寄生虫的18S rRNA转录本，即寄生虫(C，P，，，)和宿主细胞(C-T[H18S])的18S rRNA的周期阈值，并评估寄生虫和宿主rRNA水平之间的相对表达(即，Delta C-T=C-T[P18S]-C-T[H18S])，以尽量减少实验和操作上的错误。本研究选择定量聚合酶链式反应(qRT-PCR)与定量聚合酶链式反应(QPCR)相比，(I)感染寄生虫的对数(log[PI)与rRNA的归一化相对水平(Delta Delta CT)之间的关系是线性的，动态范围为四倍；(Ii)活体寄生虫的RNA水平比DNA的水平更能代表活体寄生虫的水平，因为在体外条件下，死亡寄生虫的RNA(99%，类似于3h)的衰变速度快于DNA(99%，类似于24~48h)。通过检测硝唑尼特和帕罗霉素对两株微小隐孢子虫(爱荷华株和KSU-1株)在体外培养的HCT-8细胞生长的影响，验证了qRT-PCR方法的可靠性。两种化合物均表现出剂量依赖的抑制作用。硝唑尼特和帕罗霉素的MIC值分别为0.3~0.45mU·g/ml和89.7~119.0 mU·g/ml，与文献报道值相当。利用QRT-PCR方法，我们还观察到吡唑在体外对微小隐孢子虫的发育有抑制作用(MLC(50)=15.8 mM)，这表明最近发现的隐孢子虫醇脱氢酶可能成为新的药物靶点。
We report here on a quantitative real-time reverse transcription-PCR (qRT-PCR) assay for assessing drug efficacy against the intracellular pathogen Cryptosporidium parvum. The qRT-PCR assay detects 18S rRNA transcripts from both parasites, that is, the cycle threshold for 18S rRNA from parasites (C,,P,,,,) and host cells (C-T[H18S]), and evaluates the relative expression between parasite and host rRNA levels (i.e., Delta C-T = C-T[P18S] - C-T[H18S]) to minimize experimental and operational errors. The choice of qRT-PCR over quantitative PCR (qPCR) in this study is based on the observations that (i) the relationship between the logarithm of infected parasites (log[PI) and the normalized relative level of rRNA (Delta Delta CT) is linear, with a fourfold dynamic range, by qRT-PCR but sigmoidal (nonlinear) by qPCR; and (ii) the level of RNA represents that of live parasites better than that of DNA, because the decay of RNA (99% in similar to 3 h) in dead parasites is faster than that of DNA (99% in similar to 24 to 48 h) under in vitro conditions. The reliability of the qRT-PCR method was validated by testing the efficacies of nitazoxanide and paromomycin on the development of two strains of C. parvum (IOWA and KSU-1) in HCT-8 cells in vitro. Both compounds displayed dose-dependent inhibitions. The observed MIC,, values for nitazoxanide and paromomycin were 0.30 to 0.45 mu g/ml and 89.7 to 119.0 mu g/ml, respectively, comparable to the values reported previously. Using the qRT-PCR assay, we have also observed that pyrazole could inhibit C. parvum development in vitro (MlC(50) = 15.8 mM), suggesting that the recently discovered Cryptosporidium alcohol dehydrogenases may be explored as new drug targets.