Evaluation of DNA extraction and PCR methods for detection of Enterocytozoon bienuesi in stool specimens
Evaluation of DNA extraction and PCR methods for detection of Enterocytozoon bienuesi in stool specimens
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DOI：
10.1128/jcm.42.8.3490-3494.2004
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发表时间：
2004-08-01
影响因子：
9.4
通讯作者：
Leelayoova, S
中科院分区：
文献类型：
--
作者：
Subrungruang, I;Mungthin, M;Leelayoova, S
使用已知浓度的比氏肠细胞虫孢子样本，对三种 DNA 提取方法（即 FTA 滤纸、QIAamp 粪便迷你试剂盒和传统苯酚-氯仿法）的灵敏度进行了评估。 FTA滤纸和QIAamp粪便迷你试剂盒是最灵敏的方法，可以检测到浓度为800个孢子/ml的标本中的E. bieneusi。我们还比较了先前描述的五种 PCR 方法，这些方法使用五种不同的引物对来检测 E. bieneusi，并表明 MSP3-MSP4B 和 EBIEF1-EBIER1 是最敏感的引物。尽管两组引物表现出相同的灵敏度，但使用MSP3-MSP4B引物可以直接通过测序提供基因型信息。还进行了盲法诊断测试，以比较 PCR 和光学显微镜方法检测粪便样本中的 E. bieneusi。使用FTA滤纸提取DNA并结合使用引物对MSP3-MSP4B的PCR方法检测粪便标本中的E. bieneusi显示出100%的灵敏度和100%的特异性，而光镜法的灵敏度为86.7%，特异性为100%。
An evaluation of the sensitivities of three DNA extraction methods, i.e., FTA filter paper, a QIAamp stool mini kit, and a conventional phenol-chloroform method, by using specimens with known concentrations of Enterocytozoon bieneusi spores was performed. FTA filter paper and the QIAamp stool mini kit were the most sensitive methods, which could detect E. bieneusi in specimens with a concentration of 800 spores/ml. We also compared five previously described PCR methods that use five different primer pairs for the detection of E. bieneusi and showed that MSP3-MSP4B and EBIEF1-EBIER1 were the most sensitive primers. Although both sets of primers showed the same sensitivity, using the MSP3-MSP4B primers can directly provide genotypic information by sequencing. A blinded diagnostic test to compare PCR and light microscopy methods for the detection of E. bieneusi in stool specimens was also conducted. The use of FTA filter paper for DNA extraction together with the PCR method using the primer pair MSP3-MSP4B showed 100% sensitivity and 100% specificity for the detection of E. bieneusi in stool specimens, while the light microscopy method gave a sensitivity of 86.7% and a specificity of 100%.