Specific amplification of Aspergillus fumigatus DNA by polymerase chain reaction

Abstract

We have developed a specific and sensitive method to detect the human pathogen Aspergillus fumigatus by polymerase chain reaction (PCR) with an objective of detecting the organism in peripheral blood and urine which can be obtained by non-invasive procedures. A pair of oligonucleotide primers for PCR were designed based on the published partial protein sequences of an 18 KD IgE-binding protein of A. fumigatus Asp f1 and the ribotoxins mitogillin and restrictocin of A. restrictus, and alpha-sarcin of A. giganteus. The primers were specific in amplifying an expected 315 bp region of the homologous genes in A. fumigatus and A. restrictus but not in A. giganteus. Also, there was no amplification of human DNA or DNA of A. flavus, A. niger, A. fischeri, Penicillium sp., Candida albicans and Pneumocystis carinii. The sensitivity of the PCR detection of A. fumigatus DNA is about 20 pg on an ethidium bromide gel and 0.6 pg by Southern analysis using a 32P-labelled internal oligonucleotide. In preliminary analysis of 13 urine specimens of patients suspected of invasive aspergillosis (IA), two were PCR positive, one of whom died of IA with brain lesion. Further analyses of both urine and blood specimens of IA are in progress to determine the comparative utility of PCR over the conventional antigen tests.