Detection of Aspergillus fumigatus and a Mutation That Confers Reduced Susceptibility to Itraconazole and Posaconazole by Real-Time PCR and Pyrosequencing

Abstract

A real-time PCR and pyrosequencing method was developed to detect Aspergillus fumigatus in whole blood by amplifying the cyp51A gene and sequencing the codon for glycine 54. Mutations in this codon can result in amino acid substitutions that confer reduced susceptibility to itraconazole and posaconazole.

Population structure and diversity of the pathogenic fungus Aspergillus fumigatus isolated from different sources and geographic origins

“…These facts could have repercussions for the diagnosis and treatment of patients with aspergillosis, since it has become more common to find great variation within this species. Changes due to mutations or recombinations can occur and do reflect the Aspergillus resistance to anti-mycotic treatments (Díaz-Guerra et al 2000, Trama et al 2005.…”

Stress, Drugs, and Evolution: the Role of Cellular Signaling in Fungal Drug Resistance

“…Molecular beacon technology seems well suited to meet these needs and rapidly identify mutations in cyp51A. Molecular beacons have demonstrated the sensitivity to detect single-base-pair mutations (10), and other studies have used real-time PCR to detect mutations in cyp51A at codon 54 (218).…”

Pyrosequencing-An Alternative to Traditional Sanger Sequencing

“…Pyrosequencing is well suited for de novo sequencing and resequencing (Ronaghi, 2001). Currently, pyrosequencing method is broadly being used in many applications such as Single Nucleotide Polymorphism (SNP) genotyping (Ahmadian et al, 2000a;Nordstrom et al, 2000;Milan et al, 2000), identification of bacteria (Gharizadeh, 2003;Grahn et al, 2003;Jonasson et al, 2002), fungal (Gharizadeh et al, 2005;Trama et al, 2005) and viral typing (Gharizadeh et al, 2001;. Moreover, the method has demonstrated the ability to determine difficult secondary structures (Ronaghi, 2001) and perform mutation detection (Ahmadian et al, 2000b;Garcia et al, 2000), DNA methylation analysis (Neve et al, 2002;Uhlmann et al, 2002), multiplex sequencing (Gharizadeh et al, 2003a;2006) (Gharizadeh et al, 2003b;Gharizadeh et al, 2006), tag sequencing of cDNA library (Nordstrom et al, 2001) and clone checking (Nourizad et al, 2003).…”