吉姆萨厚血膜用于疟原虫种特异性实时 PCR 的 DNA 来源。
Giemsa-stained thick blood films as a source of DNA for Plasmodium species-specific real-time PCR.
机构信息
Department of Clinical Sciences, Institute of Tropical Medicine, Antwerp, Belgium.
出版信息
Malar J. 2010 Dec 22;9:370. doi: 10.1186/1475-2875-9-370.
BACKGROUND
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.
METHODS
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.
RESULTS
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.
CONCLUSIONS
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.
背景
本研究描述了使用厚血膜（TBF）作为模板进行 DNA 扩增的方法，该方法最近已在全血样本上得到验证，采用的是针对疟原虫种属特异性的实时 PCR。
方法
该研究共纳入了 135 份经吉姆萨染色的临床 TBF 标本，这些 TBF 标本代表了四种疟原虫的单一感染，包括不同密度的寄生虫或仅含配子体、混合感染以及阴性样本，这些 TBF 标本的储存时间最长可达 12 年。用无菌手术刀刮取一半吉姆萨染色的 TBF，收集到磷酸盐缓冲液中。使用 Qiagen DNA mini 试剂盒提取 DNA，提取方法稍作修改。采用针对四种疟原虫的 18S rRNA 实时 PCR 对 TBF 中的 DNA 进行扩增，该 PCR 使用了四对种属特异性引物和探针以及一对属特异性反转引物。将 TBF 上的 PCR 结果与全血上的 PCR 结果和显微镜检查结果进行比较。
结果
对于所有的 TBF 样本（恶性疟原虫 n = 50，间日疟原虫 n = 25，卵形疟原虫 n = 25，三日疟原虫 n = 10），采用实时 PCR 检测 18S rRNA 可以正确鉴定单一物种的感染。与全血样本相比，TBF 上的 PCR 观察到的 Ct 值更高，平均差异为 5.93。采用 TBF 上的 PCR 可以正确鉴定出 5 个混合感染中的 4 个。20 个阴性样本（n = 20）均未产生 PCR 信号。与全血相比，TBF 上的 PCR 检测下限为 0.2 个无性体/μl，而全血的检测下限为 0.02/μl。TBF 上的 PCR 内重复性（%CV 1.90）高于全血上的 PCR （%CV 0.54）。与显微镜检查相比，TBF 上的 PCR 在仅含单一物种的样本中可多鉴定出 3 种物种，并且可以检测到同样的 4 种混合感染。
结论
吉姆萨染色的 TBF 是疟原虫实时 PCR 分析的可靠 DNA 来源，在无法获得全血样本的情况下，可应用于参考和研究环境中。
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