Rapid identification of clinical common invasive fungi via a multi-channel real-time fluorescent polymerase chain reaction melting curve analysis

Abstract

The incidence of invasive fungal infections (IFIs) has recently increased, and early and accurate diagnosis of IFIs is important for the rational selection of antifungal drugs with high efficacy. We developed a method for rapid and accurate clinical diagnosis of IFIs and provide a reference for personalized drug treatment. We designed and screened fungal internal transcribed spacer regions with universal primers and designed 8 TaqMan detection probes to establish a multi-channel real-time fluorescent polymerase chain reaction (PCR) melting curve analysis (MCA) method. The sensitivity, specificity, and reproducibility of this method were investigated using standard fungal strains and clinical isolates. Candidemia was detected using the MCA method. The limit of detection and assay cut-off (melting temperature [Tm]) for Candida albicans were 0.05 pg/μL and 66.50 °C; Candida glabrata were 0.1 pg/μL and 66.25 °C; Candida tropicalis were 0.1 pg/μL and 60.15 °C; Candida krusei were 0.1 pg/μL and 72.15 °C; Candida parapsilosis were 0.2 pg/μL and 63.10 °C; Candida guilliermondii were 0.1 pg/μL and 61.84 °C; Cryptococcus neoformans were 0.1 pg/μL and 65.50 °C; Aspergillus flavus were 0.05 pg/μL and 71.50 °C; Aspergillus terreus, Aspergillus fumigatus, and Aspergillus niger were 0.05 pg/μL and 76.80 °C. Analytical specificity was evaluated using 13 clinical pathogens including Streptococcus pneumoniae, Staphylococcus aureus, and Haemophilus influenzae, etc. No false-positive results were obtained for any of these samples. The MCA method can detect and identify different candidemia simulations. The limit detection concentration of C albicans was 44 cfu/mL, C glabrata was 73 cfu/mL, C tropicalis was 29 cfu/mL, C parapsilosis was 21 cfu/mL, C krusei was 71 cfu/mL, and C guilliermondii was 53 cfu/mL. The multi-channel real-time fluorescence PCR melting curve analysis displayed high sensitivity and specificity in detecting various clinically invasive fungi. Furthermore, it simultaneously detected the genera Candida, Cryptococcus, and Aspergillus and identified Candida at the species level. Our method can facilitate early and accurate clinical diagnosis and personalized medication regimens.

Optimization of a Quantitative PCR Methodology for Detection of Aspergillus spp. and Rhizopus arrhizus

“…Polymerase chain reaction (PCR)-based methodologies, generally, are directly applied to clinical samples from sterile and non-sterile sites, to detect fungal DNA. These techniques detect the genetic material of the pathogen, in singleplex or multiplex reactions using specific primers, and the PCR products are analyzed through several techniques such as sequencing [30][31][32], fluorescence in situ hybridization (FISH) [33], restriction fragment length polymorphism (RFLP) [20,34], capillary electrophoresis [35], melting curve analysis (MCA) [30,36] or with the direct use of probes [31,37,38]. However, in clinical settings the preferred methodology is quantitative real-time PCR (qPCR), since the reaction occurs in an entirely closed system avoiding contamination, and with low hands-on time.…”

Fungal infections diagnosis – Past, present and future

“…Melting curve analysis (MCA) is a methodology that can be automatically performed after qPCR reaction, with high sensitivity values, based on the association of different amplicons to different melting temperatures. Melting temperatures are mainly determined by the guanine and cytosine content, but also by the size of the amplicon [ 136 , 147 ]. MCA usually accompanies the use of intercalation dyes like SYBR Green, since it binds to all dsDNA present in the sample, all amplicons will be analysed through MCA, and for this reason, it requires a more careful analysis [ 136 , 141 , 147 ].…”

“…Melting temperatures are mainly determined by the guanine and cytosine content, but also by the size of the amplicon [ 136 , 147 ]. MCA usually accompanies the use of intercalation dyes like SYBR Green, since it binds to all dsDNA present in the sample, all amplicons will be analysed through MCA, and for this reason, it requires a more careful analysis [ 136 , 141 , 147 ]. This approach is not the most indicated to medical diagnostics, and so currently for a rigorous monitoring of the amplification in real-time, the probe-based qPCR is used instead.…”

“…Probe-based qPCR techniques can be applied in multiplex situations, although they depend on the capacity of the equipment to read fluorescence at different wavelengths [ 136 ]. In this case, each probe would be associated with the detection of a specific target, with a specific fluorescence, and the equipment would have to be able to detect different fluorescence simultaneously [ 136 , 147 ].…”

Laboratory Diagnosis of Candidiasis

“…The ITS region of ribosomal DNA (rDNA) is the most useful genetic marker for rapid and accurate molecular identification of Candida species and phylogenetic studies due to its region sequence variability among different species [15,[45][46][47]. The ITS 1 and ITS 2 are two vital non-coding regions composed of conservative and variable subregions outside and inside respectively [45].…”