Giemsa-stained thick blood films as a source of DNA for Plasmodium species-specific real-time PCR.
Giemsa-stained thick blood films as a source of DNA for Plasmodium species-specific real-time PCR.
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DOI：
10.1186/1475-2875-9-370
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发表时间：
2010-12-22
期刊：
影响因子：
3
通讯作者：
Jacobs J
中科院分区：
文献类型：
--
作者：
Cnops L;Van Esbroeck M;Bottieau E;Jacobs J
这项研究描述了使用厚血膜 (TBF) 作为 DNA 扩增样本，并使用最近在全血样本上进行了验证的疟原虫物种特异性实时 PCR。 135 个吉姆萨染色的临床 TBF 代表了四种疟原虫物种（具有不同寄生虫密度或仅配子体）的单一感染、混合感染和阴性样本，并可保存长达 12 年。用无菌手术刀刮下一半吉姆萨染色的TBF并收集到磷酸盐缓冲盐水中。 DNA 是用 Qiagen DNA 迷你试剂盒提取的，稍加修改。使用针对四种疟原虫物种的 18S rRNA 实时 PCR 扩增 DNA，使用四种物种特异性引物和探针以及一种属特异性反向引物。将TBF PCR 结果与全血PCR 和显微镜检查结果进行比较。对所有含有恶性疟原虫 (n = 50)、间日疟原虫 (n = 25)、卵形疟原虫 (n = 25) 的 TBF 样本以及除一份三日疟原虫 (n = 10) 样本外的所有样本均获得了单一物种感染的正确鉴定。与全血样本相比，TBF 上的 PCR 观察到较高的 Ct 值，平均差异为 5.93。 TBF 上的 PCR 正确识别了五分之四的混合感染。阴性样本 (n = 20) 均未发出 PCR 信号。 TBF 的 PCR 显示无性寄生虫的检测限为 0.2 个/μl，而全血的检测限为 0.02 个/μl。与全血 PCR (%CV 0.54) 相比，TBF PCR (%CV 1.90) 的运行内变异较高。与显微镜相比，TBF 上的 PCR 在包含单一物种的样本中多产生了三种物种鉴定，并检测到了相同的四种混合感染。吉姆萨染色的 TBF 是疟原虫实时 PCR 分析的可靠 DNA 来源，在没有全血样本的情况下，可以在参考和研究环境中应用。
This study describes the use of thick blood films (TBF) as specimens for DNA amplification with the Plasmodium species-specific real-time PCR that was recently validated on whole blood samples. The panel of 135 Giemsa-stained clinical TBFs represented single infections of the four Plasmodium species with varying parasite densities or only gametocytes, mixed infections, and negative samples and was stored for up to 12 years. Half of the Giemsa-stained TBF was scraped off by a sterile scalpel and collected into phosphate buffered saline. DNA was extracted with the Qiagen DNA mini kit with minor modifications. DNA was amplified with the 18S rRNA real-time PCR targeting the four Plasmodium species with four species-specific primers and probes in combination with one genus-specific reverse primer. Results of the PCR on TBF were compared to those of the PCR on whole blood and to microscopy. Correct identification for single species infections was obtained for all TBF samples with Plasmodium falciparum (n = 50), Plasmodium vivax (n = 25), Plasmodium ovale (n = 25) and in all but one samples with Plasmodium malariae (n = 10). Compared to whole blood samples, higher Ct-values were observed by PCR on TBF with a mean difference of 5.93. Four out of five mixed infections were correctly identified with PCR on TBF. None of the negative samples (n = 20) gave a PCR signal. PCR on TBF showed a detection limit of 0.2 asexual parasites/μl compared to 0.02/μl for whole blood. Intra-run variation was higher for PCR on TBF (%CV 1.90) compared to PCR on whole blood (%CV 0.54). Compared to microscopy, PCR on TBF generated three more species identifications in samples containing a single species and detected the same four mixed-infections. Giemsa-stained TBFs are a reliable source of DNA for Plasmodium real-time PCR analysis, allowing applications in reference and research settings in case whole blood samples are not available.
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影响因子：
3
作者：
Steenkeste N;Incardona S;Chy S;Duval L;Ekala MT;Lim P;Hewitt S;Sochantha T;Socheat D;Rogier C;Mercereau-Puijalon O;Fandeur T;Ariey F
通讯作者：
Ariey F
影响因子：
3
作者：
Bronner, Ulf;Divis, Paul C. S.;Singh, Balbir
通讯作者：
Singh, Balbir
影响因子：
12
作者：
Erdman, Laura K;Kain, Kevin C
通讯作者：
Kain, Kevin C
DOI：
10.4269/ajtmh.1997.57.727
发表时间：
1997-12-01
影响因子：
3.3
作者：
Li, J;Wirtz, RA;McCutchan, TF
通讯作者：
McCutchan, TF
影响因子：
9.4
作者：
Kawamoto, F;Miyake, H;Wataya, W
通讯作者：
Wataya, W