National Research Council, US Environmental Protection Agency, Ada, OK 748201, USA.
Appl Environ Microbiol. 2011 Sep;77(18):6476-85. doi: 10.1128/AEM.00677-11. Epub 2011 Jul 29.
Extraction of high-quality mRNA from Cryptosporidium parvum is a key step in PCR detection of viable oocysts in environmental samples. Current methods for monitoring oocysts are limited to water samples; therefore, the goal of this study was to develop a rapid and sensitive procedure for Cryptosporidium detection in soil samples. The efficiencies of five RNA extraction methods were compared (mRNA extraction with the Dynabeads mRNA Direct kit after chemical and physical sample treatments, and total RNA extraction methods using the FastRNA Pro Soil-Direct, PowerSoil Total RNA, E.Z.N.A. soil RNA, and Norgen soil RNA purification kits) for the direct detection of Cryptosporidium with oocyst-spiked sandy, loamy, and clay soils by using TaqMan reverse transcription-PCR. The study also evaluated the presence of inhibitors by synthesis and incorporation of an internal positive control (IPC) RNA into reverse transcription amplifications, used different facilitators (bovine serum albumin, yeast RNA, salmon DNA, skim milk powder, casein, polyvinylpyrrolidone, sodium hexametaphosphate, and Salmonella enterica serovar Typhi) to mitigate RNA binding on soil components, and applied various treatments (β-mercaptoethanol and bead beating) to inactivate RNase and ensure the complete lysis of oocysts. The results of spiking studies showed that Salmonella cells most efficiently relieved binding of RNA. With the inclusion of Salmonella during extraction, the most efficient mRNA method was Dynabeads, with a detection limit of 6 × 10(2) oocysts g(-1) of sandy soil. The most efficient total RNA method was PowerSoil, with detection limits of 1.5 × 10(2), 1.5 × 10(3), and 1.5 × 10(4) C. parvum oocysts g(-1) soil for sandy, loamy, and clay samples, respectively.
从微小隐孢子虫中提取高质量的 mRNA 是 PCR 检测环境样本中活卵囊的关键步骤。目前监测卵囊的方法仅限于水样；因此，本研究的目的是开发一种快速灵敏的方法来检测土壤样品中的隐孢子虫。比较了五种 RNA 提取方法的效率（在化学和物理样品处理后用 Dynabeads mRNA Direct 试剂盒提取 mRNA，以及使用 FastRNA Pro Soil-Direct、PowerSoil Total RNA、E.Z.N.A. soil RNA 和 Norgen soil RNA 纯化试剂盒提取总 RNA），用于用 TaqMan 逆转录-PCR 直接检测用卵囊接种的沙质、壤质和粘质土壤中的隐孢子虫。该研究还通过将内部阳性对照 (IPC) RNA 掺入逆转录扩增中来评估抑制剂的存在，使用不同的促进剂（牛血清白蛋白、酵母 RNA、鲑鱼 DNA、脱脂奶粉、酪蛋白、聚乙烯吡咯烷酮、六偏磷酸钠和伤寒沙门氏菌血清型 Typhi）来减轻 RNA 与土壤成分的结合，并应用各种处理（β-巯基乙醇和珠磨）来灭活 RNA 酶并确保卵囊完全裂解。加标研究的结果表明，沙门氏菌细胞最有效地缓解了 RNA 的结合。在提取过程中加入沙门氏菌时，最有效的 mRNA 方法是 Dynabeads，其检测限为 6×10(2)个卵囊 g(-1)沙质土壤。最有效的总 RNA 方法是 PowerSoil，其检测限分别为 1.5×10(2)、1.5×10(3)和 1.5×10(4) C. parvum 卵囊 g(-1)沙质、壤质和粘质土壤。
