Jiang Jianlin, Alderisio Kerri A, Singh Ajaib, Xiao Lihua
Division of Parasitic Disease, National Center for Infectious Diseases, Centers for Disease Control and Prevention, Mail Stop F-12, 4770 Buford Hwy., Atlanta, GA 30341-3717, USA.
Appl Environ Microbiol. 2005 Mar;71(3):1135-41. doi: 10.1128/AEM.71.3.1135-1141.2005.
Extraction of high-quality DNA is a key step in PCR detection of Cryptosporidium and other pathogens in environmental samples. Currently, Cryptosporidium oocysts in water samples have to be purified from water concentrates before DNA is extracted. This study compared the effectiveness of six DNA extraction methods (DNA extraction with the QIAamp DNA minikit after oocyst purification with immunomagnetic separation and direct DNA extraction methods using the FastDNA SPIN kit for soil, QIAamp DNA stool minikit, UltraClean soil kit, or QIAamp DNA minikit and the traditional phenol-chloroform technique) for the detection of Cryptosporidium with oocyst-seeded samples, DNA-spiked samples, and field water samples. The study also evaluated the effects of different PCR facilitators (nonacetylated bovine serum albumin, the T4 gene 32 protein, and polyvinylpyrrolidone) and treatments (the use of GeneReleaser or ultrafiltration) for the relief from or removal of inhibitors of PCR amplification. The results of seeding and spiking studies showed that PCR inhibitors were presented in all DNA solutions extracted by the six methods. However, the effect of PCR inhibitors could be relieved significantly by the addition of 400 ng of bovine serum albumin/mul or 25 ng of T4 gene 32 protein/mul to the PCR mixture. With the inclusion of bovine serum albumin in the PCR mixture, DNA extracted with the FastDNA SPIN kit for soil without oocyst isolation resulted in PCR performance similar to that produced by the QIAamp DNA minikit after oocysts were purified by immunomagnetic separation.
提取高质量DNA是对环境样本中隐孢子虫和其他病原体进行PCR检测的关键步骤。目前，水样中的隐孢子虫卵囊在提取DNA之前必须从水浓缩物中纯化出来。本研究比较了六种DNA提取方法（免疫磁珠分离纯化卵囊后用QIAamp DNA微量提取试剂盒提取DNA，以及使用FastDNA SPIN土壤试剂盒、QIAamp DNA粪便微量提取试剂盒、UltraClean土壤试剂盒或QIAamp DNA微量提取试剂盒和传统酚-氯仿技术的直接DNA提取方法）对含卵囊样本、加样DNA样本和野外水样中隐孢子虫的检测效果。该研究还评估了不同PCR促进剂（非乙酰化牛血清白蛋白、T4基因32蛋白和聚乙烯吡咯烷酮）以及处理方法（使用GeneReleaser或超滤）对缓解或去除PCR扩增抑制剂的效果。接种和加样研究结果表明，六种方法提取的所有DNA溶液中均存在PCR抑制剂。然而，向PCR混合物中添加400 ng牛血清白蛋白/μl或25 ng T4基因32蛋白/μl可显著缓解PCR抑制剂的影响。在PCR混合物中加入牛血清白蛋白后，未分离卵囊直接用FastDNA SPIN土壤试剂盒提取的DNA所产生的PCR效果与免疫磁珠分离纯化卵囊后用QIAamp DNA微量提取试剂盒提取的DNA所产生的PCR效果相似。
