title: Development of a novel quantitative real-time assay using duplex mutation             primers for rapid detection of Candida species.
abstract: We developed a novel quantitative real-time PCR for quantitating Candida DNA based on the duplex mutation primer principle, in which a signal is generated by melting a duplex mutation primer during renaturation. The duplex mutation primers are much more specific than double-stranded DNA dyes such as SYBR-Green I and, unlike other probes, do not require the double-labeled synthesis of fluorophore and a quencher on the same molecule. A total of 176 clinical blood specimens were obtained from patients hospitalized in our hospital with clinically proven or suspected systemic Candida infection. The presence of DNA from pathogens in the Candida species was detected using real-time PCR targeting of an internal transcribed spacer region of a fungal gene. The assay exhibited a low limit of detection (10 CFU/ml of blood), an excellent reproducibility and specificity. Twenty-eight positive samples exhibited a wide range of Candida species loads, extending from 13 to 90,528 CFU/ml of blood. The sensitivity and specificity of the present assay were 100 and 97.4%, respectively, compared with the results of blood culture. Our data suggest that this assay may be appropriate for use in clinical laboratories as a simple, low-cost and rapid screening test for the most frequently encountered Candida species.
authors.affiliation: Faculty of Tropical Medicine and Laboratory Medicine, Hainan Medical College, Haikou, Hainan 570102, PR China. xiaqianfeng@sina.com
authors.forename: Qian-Feng
authors.initials: QF
authors.lastname: Xia
authors.forename: Jin-Bo
authors.initials: JB
authors.lastname: Liu
authors.forename: Ping
authors.initials: P
authors.lastname: Liu
authors.forename: Xi
authors.initials: X
authors.lastname: Qin
authors.forename: Shi-Yun
authors.initials: SY
authors.lastname: Qian
authors.forename: Zhi-Guang
authors.initials: ZG
authors.lastname: Tu
mesh_terms: D001483:Base Sequence; D052160:Benzothiazoles; D002175:Candida / Q000235:genetics* / Q000302:isolation & purification; D002177:Candidiasis / Q000175:diagnosis / Q000382:microbiology*; D017931:DNA Primers / Q000737:chemistry / Q000235:genetics; D004271:DNA, Fungal / Q000032:analysis; D003959:Diamines; D006801:Humans; D008969:Molecular Sequence Data; D009154:Mutation / Q000235:genetics; D009930:Organic Chemicals / Q000737:chemistry; D011804:Quinolines; D060888:Real-Time Polymerase Chain Reaction; D012680:Sensitivity and Specificity; D016415:Sequence Alignment
chemical_list: D052160:Benzothiazoles; D017931:DNA Primers; D004271:DNA, Fungal; D003959:Diamines; D009930:Organic Chemicals; D011804:Quinolines; C098022:SYBR Green I