Development of two real-time quantitative TaqMan PCR assays to detect circulating Aspergillus fumigatus DNA in serum.
Several PCR assays have been developed for detecting Aspergillus fumigatus DNA in blood of patients with invasive aspergillosis. However, the best blood fraction to be assayed has not been defined and the multicopy genes used as the DNA targets for amplification not characterized. Firstly, we developed a real-time PCR assays based on the TaqMan technology targeted to a single copy gene. To compare serum, white cell pellet, and plasma for effectiveness as blood assay fractions, we spiked whole blood with A. fumigatus DNA and processed these fractions similarly. The difference between white cell pellet and serum was not significant. In contrast, the yield from plasma was 10 times lower than from serum. Then, we compared serum processed immediately or after 24 h at room temperature and observed a lower yield after 24 h. Secondly, a real-time PCR assay targeted to a mitochondrial gene was also developed. The copy number was estimated between 9 and 10 mitochondrial genes per single copy gene. Therefore, we recommend serum, stored and frozen as soon as possible, to be used for detecting circulating A. fumigatus DNA for diagnosis. Moreover, the mitochondrial multicopy gene was characterized in order to compare results from different patients.
Costa
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C
Laboratoire de Parasitologie-Mycologie, Hôpital Henri Mondor-APHP and Université Paris XII, 51 avenue du Général DeLattre de Tassigny, 94010, Cedex, Créteil, France.
Vidaud
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D
Olivi
M
M
Bart-Delabesse
E
E
Vidaud
M
M
Bretagne
S
S
D001228:Aspergillosis / Q000097:blood* / Q000175:diagnosis; D001232:Aspergillus fumigatus / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification; D017931:DNA Primers / Q000737:chemistry; D004271:DNA, Fungal / Q000097:blood* / Q000737:chemistry / Q000235:genetics; D004272:DNA, Mitochondrial / Q000737:chemistry / Q000235:genetics; D018628:Gene Dosage; D006801:Humans; D007962:Leukocytes / Q000737:chemistry; D016133:Polymerase Chain Reaction / Q000379:methods*; D019914:Taq Polymerase / Q000378:metabolism
D017931:DNA Primers; D004271:DNA, Fungal; D004272:DNA, Mitochondrial; D019914:Taq Polymerase