Validation of a simplex PCR assay enabling reliable identification of clinically relevant Candida species

Genomic DNA (gDNA) samples of 81 clinically relevant fungal strains; Candida (43), aspergilli (32), Fusarium (4), Lichtemia (1), Rhyzopus (1), Scedosporium (1) and 16 bacteria; Gram-positive (10) and Gram-negative (6) were examined. The reference strains and clinical isolates (Table 1) were maintained at the Department of Microbiology, University of Szeged and at the Department of Medical Microbiology, University of Debrecen. To preserve the viability and purity of fungal and bacterial strains they were maintained in 50% glycerol stock solution at − 80 °C and were periodically subcultured.

All genomic DNA (gDNA) extraction steps were performed in a class II laminar-flow cabinet to avoid environmental contamination.

Annotated sequences of the Candida beta-tubulin genes were extracted from the EMBL/GeneBank databases to make multiple alignments using Clustal Omega. Available Candida beta-tubulin sequences were aligned surveying potential sequence deviations within species. When designing primers, we screened for melting domains covering enough mismatches to enable proper discrimination among the tested strains but comprising 30–40 nucleotide long conserved nucleotides on flanking regions for the very specific hybridization of primers and avoiding cross-reactions with gDNA of other BSI causing fungal, bacterial strains and with the human genomic DNA.

The CanTub forward primer is 37 bp long(5’-CTAAAATCAGAGAAGAATTCCCCTGATAGAATGATGGC-3′), GC content 37.8%, Tm 73.2 °C and the CanTub reverse primer is 43 bp long (5’-CAATTGACCTGGGATAACGTAAAGAAGTAGTAACACCAGACAT-3′), GC content 38%, Tm 74.2 °C.

Temperature gradient assay was performed from 55 to 72 °C for assessing the performance of the primer pair during amplification with a temperature gradient program using the LightCycler® 96 thermal cycler Instrument (Roche Applied Science, Penzberg, Germany). The MgCl₂ optimization was performed by adding different amounts of MgCl₂ within the concentration range 1 to 3.5 mM. The primer optimization assay was performed using 0.2, 0.4, 0.6 μM of the forward and reverse primers.

In order to monitor the accumulation of aspecific amplicons in the CanTub-simplex PCR, primers were tested with 20 ng gDNA of five Candida reference strains (C. albicans ATCC 10231, C. glabrata ATCC 90030, C. parapsilosis ATCC 22019, C. tropicalis ATCC 750, C. krusei ATCC 6258), and two clinical isolates (C. guilliermondii SZMC 1536 and C. dubliniensis SZMC 1470 depicted by ID33 and ID36 in Table 1). The thermocycling reactions were conducted in a Roche LightCycler®Nano instrument (P2). 200 ng of the yielded CanTub PCR amplicons were electrophoresed on 2% TBE-agarose gel stained with ethidium-bromide to investigate aspecific PCR increments.

Real-time PCR assays were conducted on three different PCR platforms (P1, P2, P3). (P1) LightCycler® 96 thermal cycler, (P2) LightCycler® Nano and (P3) LightCycler® 2.0 Instruments (Roche Diagnostics, Risch-Rotkreuz, Switzerland) were used with the LightCycler 480 High-Resolution Melting Master (Roche Applied Science, Penzberg, Germany) (P1) 10 μl reaction volumes consisted of 5 μl 2× LightCycler 480 High Resolution Melting Master, 0.5 μl of each primer (0.4 μM), 1 μl MgCl₂ (2 mM) and 3 μl template DNA. The thermocycling reactions were conducted in a LightCycler 480 Multiwell Plate, (white). (P2), (P3) 20 μl reaction volumes consisted of 10 μl 2× LightCycler 480 High Resolution Melting Master, 0.5 μl of each primer (0.4 μM), 5 μl PCR grade water, 1 μl MgCl₂ (2 mM) and 3 μl template DNA. The thermocycling reactions were conducted in LightCycler® 8-Tube Strips (P2) and in 20 μl glass capillaries (P3). The real-time PCR runs always included at least two controls of reaction mix without DNA (non-template control - NTC). Temperature parameters were set as follows: an initial denaturing step of 95 °C for 10 min followed by 50 cycles of denaturation at 95 °C for 10 s, annealing at 60 °C for 10 s and extension at 72 °C for 10 s. Fluorescent data were collected in the ResolightDye channel (470/514 nm).

Possible cross reactions of the CanTub-HRM assay were tested with approximately 20–25 ng purified (OD_260/2801.82–1.97) gDNA of human placenta (Sigma Aldrich, Missouri, USA) and the gDNA samples of 32 aspergilli; 6 reference strains (Aspergillus terreus FGSC A1156, A. fumigatus Af293, A. lentulus CBS117885, A. flavus NRRL11611, A. niger CBS 113.46, A. tubingensis CBS 134.48), further 26 gDNA of Aspergillus (ID39–64), Fusarium (ID65–68), Lichtemia corymbifera (ID69), Rhyzopus oryzae (ID70), Scedosporium aurantiacum (ID71) clinical isolates, finally with the gDNA of 10 Gram-positive bacteria; 5 reference strains (Staphylococcus aureus ATCC 25923, 29,213, 43,300, Enterococcus faecalis ATCC 29212, Enterococcus hirae ATCC 8043), 5 Gram-positive clinical isolates (ID72–76) and with 6 Gram-negative bacteria; Enterobacter aerogenes ATCC13048 reference strain and five Gram-negative clinical isolates (ID77–81) in Table 1.

Amplification reactions were carried out on three different PCR platforms; P1, P2, P3. Duplicate PCRs were performed at every dilution of the Candida EDTA-WB reference panel extracts. Quantification cycle (Cq) values were subtracted and melting curves were analyzed to estimate the lowest template DNA concentration by which the appropriate, species descriptive melting peaks were interpretable (LoRD). To study the correlation between the Cq-s and the genomic load standard curves were obtained by plotting Cq values against the log of cell number; 10⁶–10 CFU/1 ml WB which is equivalent to 2 × 10⁵–0.2 GE/PCR. Mean Cq data were calculated and standard curves were built where these plots determined the linear dynamic ranges. Efficiency was calculated per the following formula, E = (10–1/slope) than was converted to percentage efficiency by using the formula, E% = (E-1) × 100 [44]. Amplification efficiency, (correlation coefficient; R²) was also calculated.

PCRs were conducted on P1 and T_m data were subtracted.

Based on the sequence alignment data we presumed that the amplicons generated by CanTub-simplex primers will display enough sequence divergence between closely related Candida species but at the same time may be conserved enough barcoding the different clinical isolates within species. Primer matches with annotated beta-tubulin genes from C. albicans, C. tropicalis, C. dubliniensis and C. glabrata are indicated in [see Additional file 3] with the sizes of the amplicons.

Optimal reaction conditions were determined as described in the methods section. 0.4 μM primers-, 2 mM MgCl₂-concentrations and annealing at 60 °C proved to be optimal. The size of the amplified CanTub-simplex PCR bands were verified on five Candida reference strains and on two clinical isolates. All the amplified CanTub-simplex PCR fragments are smaller than 300 bp in size and we did not experience any aspecific PCR increments (data not shown).

No cross-amplification was detected with the human genomic DNA and with the Gram-negative and Gram-positive bacteria. Furthermore, no cross-amplification was observed with A. terreus, A. lentulus, A. tubingensis, A. viridinutans and A. udagawae strains. Mild cross reaction was detected with the A. fumigatus, A. flavus and A. niger providing T_m peaks at 86.35 ± 0.16 °C and displaying inconclusive HRM curves and peaks. Moderate false positivity with plain, inconsistent peaks were detected in the case of Rhyzopus oryzae (77.38 °C), Scedosporium aurantiacum (75.73 °C) generating cycle threshold (Cq) values greater than Cq > 42 (data not shown).

LoRD of the simplex CanTub-PCR assay was estimated on Candida EDTA-WB standard panel extracts containing serially diluted Candida gDNA in a 6-log range. Linear dynamic ranges were assessed on P1, P2, and P3 with PCR efficiency (E) and coefficient of correlation (R²) were also estimated using the standard curve data (Fig. 1). The analytical sensitivity with the lowest concentration of template DNA where reliable identifications with conclusive melting peaks and HRM curves were attainable in the case of at least one technical duplicate proved to be 0.2 GE/PCR (10 cells/1 ml WB) on all panels and on all platforms except the C. parapsilosis reference panel on P1 and P3, where LoRD proved to be 2 GE/PCR (100 cells/1 ml WB) only. On P2 the LoRD was 2 GE/PCR also for C. parapsilosis, C. dubliniensis and C. guilliermondii reference panels.

CanTub-simplex assay targeting the beta-tubulin genes proved to be accurate and reliable for specific identification of seven Candida species. Thirty eight clinical isolates phenotypically identified as Candida species were subsequently tested in a blinded manner and barcoded due to comparing the melting data to those of the reference strains.

Normalized melting curves (Fig. 2/a) with the difference plot (Fig. 2/b) are shown by using saturating concentrations of a fluorescent double stranded DNA intercalating ResoLight® dye. HRM analyses were performed on Candida EDTA-WB clinical panels and on platform P1.

Whisker plots show the distribution of the T_m values when testing the 38 Candida clinical strains on four separate plates. For every dataset, the median, minimum, maximum T_m values along with the 25th and 75th percentile are also shown in Fig. 3. Based on the distribution of the melting temperature data of the Candida strains tested, we created seven melting clusters for the reliable species level identification on P1-P3 C. dubliniensis cluster_1 T_m with 95% CI: 77.58 ± 0.32 °C (P1), 78.39 ± 0.22 °C (P2), 76.45 ± 0.37 °C (P3). C. tropicalis cluster_2 T_m with 95% CI: 77.96 ± 0.2 °C (P1), 78.77 ± 0.21 °C (P2), 77.09 ± 0.41 °C (P3). C. albicans cluster_3 T_m with 95% CI: 78.64 ± 0.18 °C (P1), 79.16 ± 0.14 °C (P2), 77.61 ± 0.15 °C (P3). C. krusei cluster_4 T_m with 95% CI: 79.17 ± 0.26 °C (P1), 78.64 ± 0.18 °C (P2), 78.17 ± 0.24 °C (P3). C. parapsilosis cluster_5 T_m with 95% CI: with 95% CI: 79.92 ± 0.37 °C (P1), 80.08 ± 0.3 °C (P2), 78.65 ± 0.39 °C (P3). C. guilliermondii cluster_6 T_m with 95% CI: 80.98 ± 0.3 °C (P1), 81.19 ± 0.3 °C (P2), 79.76 ± 0.18 °C (P3). C. glabrata cluster_7 T_m with 95% CI: 81.3 ± 0.31 °C (P1), 81.6 ± 0.32 °C (P2), 82.02 ± 0.32 °C (P3).Using these melting clusters for PCR typifying of unknown Candida strains there is no need for using standards as reference controls.

We estimated the intra-, and inter-assay consistency of the CanTub-simplex PCR on Candida EDTA-WB reference- [see Additional file 1] and on Candida EDTA-WB clinical panels [see Additional file 2]. For measuring the precision of the assay average coefficient of variation was calculated for the technical duplicate T_m values measured on P1 where intra-assay % C. V. proved to be 0.14 (ref. plate_1), 0.19 (ref. plate_2) and 0.08 (ref. plate_3) on Candida EDTA-WB reference plates and 0.14 (clin. plate_1), 0.12 (clin. plate_2), 0.10 (clin. plate_3), 0.13 (clin. plate_4) on Candida EDTA-WB clinical plates accounting for a very high accuracy of the CanTub-simplex PCR. Plate-to-plate consistency was also assessed for the assay between the three Candida EDTA-WB reference plates and the four Candida EDTA-WB clinical panels. Reproducibility was measured and sample coefficient of variations (grand % C.V.-s) between the three reference and four clinical plates were taken, where inter-assay proved to be 0.11between standard [see Additional file 1] and 0.12between clinical panels [see Additional file 2] which is highly acceptable.