| The critically ill surgical patients who are under long-term parenteral nutrition support often experience the damage of gut barrier and the translocation of intestinal microorganisms into the circulation system leading to systemic infection and fever. The impropriate administration of antibiotics brings no therapeutic effects but worsen the damage of the gut barrier. So it’s necessary to establish certain fast methods to discriminate whether there are bacteria or fungus inside the human blood. This study aimed to establish a real-time quantitative PCR (RQ-PCR) assay for fast detection of invasive fungi DNA in human whole blood samples with universal fungi primers and probe.In this section, the universal fungi primers and the TaqMan-probe were designed on the basis of the multi-copy5.8S region of the rDNA of the clinically most common invasive fungi. The invasive fungi genomic DNA were extracted with QIAamp(?) DNA Blood Mini Kit. A20μl RQ-PCR amplification system was established, and the simulated blood samples containing various given load of invasive fungi genome and the71whole blood samples of the surgical febrile patients were examined. The detection limit is1.0×101copies/μl amplification mixture, namely1.0×105copies/ml whole blood. The sensitivity and the specificity were95.5%and97.6%respectively; and the positive predictive value and negative predictive value were98.7%and92.0%respectively. The correlation coefficient of standard curve was between0.9931and0.9977. The intra-and the inter-assay average coefficients of variation (CVs) were (10.4±4.0)%and (27.9±2.0)%, respectively. The average relative recovery rate of fungi genomic DNA in blood samples was (91.0±7.6)%, the average CV of the relative recovery rate was (14.9±4.0)%. No fungi DNA were detected among the71blood samples of the surgical febrile patients. The RQ-PCR assay for fast quantitative detection of invasive fungal DNA in human whole blood samples with the universal fungi primers and the TaqMan-probe was of high sensitivity, specificity, accuracy and precision, which could discriminate fungi from bacteria. The invasive fungi genome was not detected in this group of surgical patients, which might imply the less possibility of fungi translocation in the surgical febrile patients. |
