Laboratory of Molecular and Cellular Immunology, Institute of Molecular Genetics AS CR, v.v.i., Prague, Czech Republic.
Nat Protoc. 2010 Jun;5(6):1074-80. doi: 10.1038/nprot.2010.68. Epub 2010 May 20.
This protocol describes an improved and optimized PCR-ELISA method for detection and quantification of Leishmania parasites in host tissues. Unlike other DNA-based assays, this method uses digoxigenin- and biotin-labeled primers. This eliminates the need for a separate step of hybridization of the PCR product with labeled probes. The PCR product is detected using sandwich ELISA with antidigoxigenin-detecting antibodies. Primers are complementary to the kinetoplast minicircle conserved region of parasite DNA, allowing the detection of several Leishmania species. For measurement of a wide range of parasite concentrations, +/-25 cycles were optimal. The sensitivity of this technique is 0.3 fg of parasite DNA per reaction in 40-cycle PCR-ELISA, corresponding to 0.004 parasites. DNA preparation by a standard TRI reagent procedure takes about 4 h. When DNA is prepared, a single person can test a large number of samples (at least 150) in a maximum of 7 h. This method might also be suitable for detecting and quantifying other pathogens, especially for detecting small differences in pathogen numbers.
本方案描述了一种改良和优化的 PCR-ELISA 方法，用于检测和定量宿主组织中的利什曼原虫寄生虫。与其他基于 DNA 的检测方法不同，该方法使用地高辛和生物素标记的引物。这消除了 PCR 产物与标记探针杂交的单独步骤的需要。使用抗地高辛检测抗体的夹心 ELISA 检测 PCR 产物。引物与寄生虫 DNA 的动基体微环保守区互补，允许检测几种利什曼原虫物种。为了测量广泛的寄生虫浓度，最佳循环数为 +/-25 个。该技术的灵敏度为 40 个循环 PCR-ELISA 中每个反应 0.3 fg 寄生虫 DNA，相当于 0.004 个寄生虫。通过标准 TRI 试剂程序进行 DNA 制备大约需要 4 小时。准备好 DNA 后，一个人最多可以在 7 小时内测试大量样本（至少 150 个）。该方法也可能适用于检测和定量其他病原体，特别是用于检测病原体数量的微小差异。
