Detection of invasive fungi DNA load in human whole blood samples with universal fungi primers and probe

Abstract

Objective 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.Methods The universal fungi primers and the TaqMan-probe were designed on the basis of the multi-copy 5.8S region of the rDNA of the clinically most common invasive fungi.The invasive fungi genomic DNA were extracted with QIAamp?DNA Blood Mini Kit.A 20 μl RQ-PCR amplification system was established,and the simulated blood samples containing various given load of invasive fungi genome and the 71 whole blood samples of the surgical febrile patients were examined.Results The detection limit is 101 copies/μl amplification mixture,namely 105 copies/ml whole blood.The sensitivity and the specificity were 95.5％ and 97.6％,respectively; and the positive predictive value and negative predictive value were 98.7％ and 92.0％,respectively.The correlation coefficient of standard curve was between 0.9931 and 0.9977.The intra-and the inter-assay average coefficients of variation 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)％,and the average coefficients of variation of the relative recovery rate was(14.9 ±4.0)％.No fungi DNA was detected among the 71 blood samples of the surgical febrile patients.Conclusions 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,and is able to discriminate fungi from bacteria.The invasive fungi genome was not detected in this group of surgical patients,which may imply the less possibility of fungi translocation in the surgical febrile patients. Key words: Parenteral nutrition;  Real-time quantitative PCR;  Universal fungi primers and probe;  Gut barrier;  Surgical infection