Purpose

The major problem of the PCR method for the search of protozoan cysts/oocysts in environmental samples is the presence of inhibitors. DNA extraction methods capable of removing inhibitory substances of environmental origin and recovering the DNA are decisive for the efficiency of PCR. This study aimed to compare the efficiency of different DNA extraction methods for the search by Cryptosporidium oocysts in water samples by molecular methods.

Methods

DNA extraction from water samples was performed using four different methods. Two methods use a chaotropic buffer to extract DNA and promote the selective binding of DNA to a silica membrane (GuSCN-silica and GFX Kit). The other method is based on the lysis and digestion of the samples in buffer and proteinase K, adsorption of impurities by an “InhibitEX” insertion matrix and purification of the DNA by a silica column (QIAamp Kit). The fourth method uses ionic and non-ionic detergents and proteinase K, to solubilize and separate the DNA from proteins, and a paramagnetic resin for DNA purification in the presence of high concentrations of guanidine ions (MAGNEX DNA Kit). Nested-PCR was performed, and the Cryptosporidium SSU rDNA gene amplified.

Results

The results demonstrated that MAGNEX and GFX commercial kits showed higher sensitivity, with detection of up to 10⁰ oocysts/mL and 10⁴ oocysts/mL respectively.

Conclusion

In conclusion, this study confirmed that for low-DNA environmental samples, extraction methods should include an efficient oocyst wall breaking step, and showed that the best Cryptosporidium DNA extraction methods are those that use paramagnetic resins.

中文翻译：

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对隐孢子虫不同卵囊 DNA 提取方法的评价。环境样本研究。

目的

在环境样品中寻找原生动物包囊/卵囊的 PCR 方法的主要问题是抑制剂的存在。能够去除环境来源的抑制物质并回收 DNA 的 DNA 提取方法对 PCR 的效率具有决定性意义。本研究旨在比较不同DNA提取方法对水样中隐孢子虫卵囊的分子检测效率。

方法

使用四种不同的方法从水样中提取 DNA。两种方法使用离液缓冲液提取 DNA 并促进 DNA 选择性结合到硅胶膜上（GuSCN-silica 和 GFX 试剂盒）。另一种方法基于样品在缓冲液和蛋白酶 K 中的裂解和消化，通过“InhibitEX”插入基质吸附杂质以及通过硅胶柱（QIAamp Kit）纯化 DNA。第四种方法使用离子和非离子去污剂和蛋白酶 K，从蛋白质中溶解和分离 DNA，以及在高浓度胍离子（MAGNEX DNA 试剂盒）存在下用于 DNA 纯化的顺磁性树脂。进行巢式 PCR，并扩增了隐孢子虫SSU rDNA 基因。

结果

结果表明，MAGNEX 和 GFX 商业试剂盒显示出更高的灵敏度，分别检测到高达 10 ⁰卵囊/mL 和 10 ⁴卵囊/mL。

结论

总之，本研究证实，对于低 DNA 环境样品，提取方法应包括有效的卵囊破壁步骤，并表明最好的隐孢子虫DNA 提取方法是使用顺磁性树脂的方法。