Sequences from the aspergillopepsin PEP gene of Aspergillus fumigatus: evidence on their use in selective PCR identification of Aspergillus species in infected clinical samples.
In immunodeficient patients, Aspergillus species emerge as circumstantial pathogens. Aspergillus fumigatus is a distant first among the pathogenic aspergilli, which cause deep-seated mycoses. Sequences of the pep gene of A. fumigatus as potential PCR primers, which have not been tested before, were used to identify this species and if possible, differentiate it from other, co-identified, clinically important species of the genus. We present results of the three most promising primer pairs, pep-1/pep-22, pep-15/pep-22 and pep-21/pep32. The second pair was of better specificity when tested with DNA extracted from pure cultures of a multitude of aspergilli, whereas the first co-amplified four clinically significant Aspergillus species. The compatibility of the PCR method with the CTAB DNA extraction protocol varied according to the biological fluid tested and the primer pair used. The first two pairs showed moderate adaptability to the different commercial DNA extraction kits, which were tested in whole blood, spiked with Aspergillus fumigatus hyphae and conidia - as were all the biological fluids used. Restriction of the amplification products with MspI produced distinct patterns for different Aspergillus spp. This approach, as a potential diagnostic tool, seems reliable and sensitive due to its flexibility, speed, low cost, ease of application and selectable breadth of detection.
Kambouris
M E
ME
Department of Microbiology, Medical School, University of Athens, Greece.
Reichard
U
U
Legakis
N J
NJ
Velegraki
A
A
D016282:Aspartic Acid Endopeptidases / Q000235:genetics*; D001228:Aspergillosis / Q000175:diagnosis* / Q000382:microbiology; D001230:Aspergillus / Q000145:classification* / Q000235:genetics; D001232:Aspergillus fumigatus / Q000235:genetics* / Q000302:isolation & purification; D017931:DNA Primers; D006801:Humans; D016376:Oligonucleotides, Antisense; D016133:Polymerase Chain Reaction / Q000379:methods*; D012150:Polymorphism, Restriction Fragment Length
D017931:DNA Primers; D016376:Oligonucleotides, Antisense; D016282:Aspartic Acid Endopeptidases; C029094:aspergillopepsin I