MULTI-RESOLUTION NESTED PRIMER PCR ASSAY FOR RAPID DETECTION AND IDENTIFICATION OF FUNGI IN HUMAN BLOOD
Invasive fungal infection is an increasingly life-threatening condition in immunocompromised hosts. Standard diagnostic techniques to detect pathologic fungi are time consuming and lack the sensitivity required for early intervention. We developed a rapid and sensitive PCR-based assay to detect fungi present in whole blood samples. Three independent nested PCR primer pairs were designed to specifically amplify different regions of the fungal 18S ribosomal RNA gene: set I targets Aspergillus specific sequences; set II targets Candida specific sequences; and set III is fungal specific, targeting sequences conserved in essentially all fungi. Each group of primers amplifies different regions of the 18S gene such that cross-contamination between the groups cannot occur. The reverse fungal specific primer is tailed with M13 universal sequence to allow nucleotide sequencing of the PCR product for species identification using the Basic Local Alignment Search Tool (Altschul et al., J. Mol Bio, 1990, 215: 403). Species determination may be important as a guide in therapeutic decisions since fluconazole-resistant strains of Candida albicans and Candida tropicalis have been reported. Furthermore, sequencing offers the potential to identifiy fungal organisms not previously thought to be pathogenic. Specificity of the primers was established by optimizing PCR conditions using DNA from five species of Aspergillus, four species of Candida, Histoplasma capsulatum, Saccharomyces cerevisiae, and Homo sapien. PCR sensitivity was determined by serial dilution of fungal DNA into human DNA. As little as 1 fg of fungal DNA was detectable in a background of 250 ng of human DNA. Reconstitution studies were performed using serial dilutions of fungal cells in whole blood. Approximately, 10 fungal cells were routinely detectable in 500 ul of blood. A prospective study is currently being conducted to ascertain the ability of this assay to detect fungal infection in bone marrow transplant (BMT) patients. Positive PCR assays have been correlated with confirmed fungal infection in samples obtained post-BMT. This multi-resolution nested primer PCR assay potentially offers a rapid and sensitive method for detection of pathologic fungi in the immunocompromised host.
- ASH 40th (1999)
Full conference title:
40th Annual Meeting of the American Society of Hematology.