Evaluation of DNA extraction and PCR methods for detection of Enterocytozoon bienuesi in stool specimens

Abstract

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%.

FIG. 1.

PCR analysis using the MSP3-MSP4B primer pair specific to the SSU rRNA gene of E. bieneusi. Lane M, molecular markers of 100-bp DNA ladder; lane 1, negative control; lanes 2 to 6, different concentrations of spores (100,000, 20,000, 4,000, 800, and 160 spores/ml, respectively); lane 7, positive control.