Development of Candida-Specific Real-Time PCR Assays for the Detection and Identification of Eight Medically Important Candida Species

Abstract

Culture-based identification methods have been the gold standard for the diagnosis of fungal infection. Currently, molecular technologies such as real-time PCR assays with short turnaround time can provide desirable alternatives for the rapid detection of Candida microbes. However, most of the published PCR primer sets are not Candida specific and likely to amplify DNA from common environmental contaminants, such as Aspergillus microbes. In this study, we designed pan-Candida primer sets based on the ribosomal DNA-coding regions conserved within Candida but distinct from those of Aspergillus and Penicillium. We demonstrate that the final two selected pan-Candida primer sets would not amplify Aspergillus DNA and could be used to differentiate eight medically important Candida pathogens in real-time PCR assays based on their melting profiles, with a sensitivity of detection as low as 10 fg of Candida genomic DNA. Moreover, we further evaluated and selected species-specific primer sets covering Candida albicans, Candida glabrata, Candida tropicalis, and Candida dubliniensis and show that they had high sensitivity and specificity. These real-time PCR primer sets could potentially be assembled into a single PCR array for the rapid detection of Candida species in various clinical settings, such as corneal transplantation.

A Real Time PCR strategy for the detection and quantification of Candida albicans in human blood

“…20 copies/reaction, which corresponds approximately to one-fifth of a single CFU, considering approximately 110 copies of the sequence/CFU (diploid genome) 8 . Other authors who used C albicans culture samples in qPCR observed similar results 14,15 . 7 11.89 ± 0.77 10 5 11.02 ± 0.30 10 7 17.54 ± 0.84 * mean of ten assays; ** mean of three assays; *** mean of five assays; ND -non detected…”

Rapid and Accurate Identification of <b><i>Candida albicans</i></b> and <b><i>Candida dubliniensis</i></b> by Real-Time PCR and Melting Curve Analysis

“…The other method is a nested PCR involving a first round of amplification with pan-Candida primers followed by real-time PCR amplification with 8 different species-specific primer pairs for the identification of 8 different Candida species including C. albicans and C. dubliniensis [36]. The additional requirement of the nested step in this procedure [36] is time-consuming, increases cost and the possibility of false-positive results due to amplicon carry-over. Our real-time PCR assay involves only one round of amplification and the method can also be used as a thermal PCR assay with detection of amplicons by agarose gels (Fig.…”

“…0.5 ° C) or negligible which may lead to misidentification of some isolates since ITS region sequences vary among clinical C. albicans and C. dubliniensis isolates [5,7,31]. The other method is a nested PCR involving a first round of amplification with pan-Candida primers followed by real-time PCR amplification with 8 different species-specific primer pairs for the identification of 8 different Candida species including C. albicans and C. dubliniensis [36]. The additional requirement of the nested step in this procedure [36] is time-consuming, increases cost and the possibility of false-positive results due to amplicon carry-over.…”

“…The target chosen in this study was 18S rRNA gene, the DNA was obtained from Candida isolates by using a commercial DNA extraction kit and PCR amplification was performed in LightCycler with pan-Candida primers followed by detection of amplicons by hybridization with 4 separate species-specific probe primers labeled with a fluorescent dye [33]. Three real-time PCR assays using SYBR Green dye have also been described [34][35][36]. Two of these real-time PCR assays used panfungal primers targeting the ITS region of rDNA [34,35].…”

Species-specific detection of Candida tropicalis using evolutionary conserved intein DNA sequences

“…; Susever and Yegenoglu ; Zhang et al . ). Alternatively, the use of functional genes (DNA topoisomerase II and phospholipase B gene) for detection of C. tropicalis are also reported (Kanbe et al .…”