Improving molecular detection of Candida DNA in whole blood: comparison of seven fungal DNA extraction protocols using real-time PCR.
The limitations of classical diagnostic methods for invasive Candida infections have led to the development of molecular techniques such as real-time PCR to improve diagnosis. However, the detection of low titres of Candida DNA in blood from patients with candidaemia requires the use of extraction methods that efficiently lyse yeast cells and recover small amounts of DNA suitable for amplification. In this study, a Candida-specific real-time PCR assay was used to detect Candida albicans DNA in inoculated whole blood specimens extracted using seven different extraction protocols. The yield and quality of total nucleic acids were estimated using UV absorbance, and specific recovery of C. albicans genomic DNA was estimated quantitatively in comparison with a reference (Qiagen kit/lyticase) method currently in use in our laboratory. The extraction protocols were also compared with respect to sensitivity, cost and time required for completion. The TaqMan PCR assay used to amplify the DNA extracts achieved high levels of specificity, sensitivity and reproducibility. Of the seven extraction protocols evaluated, only the MasterPure yeast DNA extraction reagent kit gave significantly higher total nucleic acid yields than the reference method, although nucleic acid purity was highest using either the reference or YeaStar genomic DNA kit methods. More importantly, the YeaStar method enabled C. albicans DNA to be detected with highest sensitivity over the entire range of copy numbers evaluated, and appears to be an optimal method for extracting Candida DNA from whole blood.
Metwally
L
L
Department of Medical Microbiology, Royal Victoria Hospital, Belfast, Northern Ireland.
Fairley
D J
DJ
Department of Medical Microbiology, Royal Victoria Hospital, Belfast, Northern Ireland.
Coyle
P V
PV
Department of Medical Microbiology, Royal Victoria Hospital, Belfast, Northern Ireland.
Hay
R J
RJ
Queen's University of Belfast, School of Medicine and Dentistry, Belfast, Northern Ireland.
Hedderwick
S
S
Department of Infectious Diseases, Royal Victoria Hospital, Belfast, Northern Ireland.
McCloskey
B
B
Regional Intensive Care Unit, Royal Victoria Hospital, Belfast, Northern Ireland.
O'Neill
H J
HJ
Department of Medical Microbiology, Royal Victoria Hospital, Belfast, Northern Ireland.
Webb
C H
CH
Department of Medical Microbiology, Royal Victoria Hospital, Belfast, Northern Ireland.
Elbaz
W
W
Department of Infectious Diseases, Royal Victoria Hospital, Belfast, Northern Ireland.
McMullan
R
R
Department of Medical Microbiology, Royal Victoria Hospital, Belfast, Northern Ireland.
D002175:Candida / Q000145:classification / Q000235:genetics / Q000302:isolation & purification*; D002176:Candida albicans / Q000145:classification / Q000235:genetics / Q000302:isolation & purification; D002177:Candidiasis / Q000175:diagnosis* / Q000382:microbiology; D004271:DNA, Fungal / Q000032:analysis / Q000097:blood* / Q000302:isolation & purification*; D016469:Fungemia / Q000175:diagnosis* / Q000382:microbiology; D006801:Humans; D016533:Mycological Typing Techniques; D016133:Polymerase Chain Reaction / Q000191:economics / Q000379:methods*; D011933:Reagent Kits, Diagnostic; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D013997:Time Factors
D004271:DNA, Fungal; D011933:Reagent Kits, Diagnostic