Duplex Real-Time PCR for Rapid Simultaneous Detection of Batrachochytrium dendrobatidis and Batrachochytrium salamandrivorans in Amphibian Samples

Chytrid strains and culture conditions.B. dendrobatidis and B. salamandrivorans were grown in TGhL broth (16 g tryptone, 4 g gelatin hydrolysate, 2 g lactose per liter of distilled water) in 25-cm³ cell culture flasks and incubated at 20°C (B. dendrobatidis) or 15°C (B. salamandrivorans). Homolaphlyctis polyrhiza, Gaertneriomyces semiglobifer, Geranomyces variabilis, Rhizophlyctis rosea, Rhizoclosmatium globosum, Polychytrium aggregatum, Monoblepharis polymorpha, and Podochytrium dentatum (Table 1) were grown in PmTG broth (0.5 g peptonized milk, 0.5 g tryptone, 2.5 g glucose per liter of distilled water) in 25-cm³ cell culture flasks, with incubation at 23°C. To obtain zoospores of B. dendrobatidis and B. salamandrivorans, 2 ml of a 5-day-old culture was transferred to TGhL broth with 1% agar plates and incubated for 5 to 7 days at 20°C (for B. dendrobatidis) or 15°C (for B. salamandrivorans). Zoospores were subsequently collected by flooding the agar plates with 2 ml of filtered (0.2-μm filter) pond water and collecting the fluid. The number of zoospores present in the suspension was determined using a hemocytometer.

Quantitation standards and DNA extracts.Suspensions containing standardized numbers of B. dendrobatidis and B. salamandrivorans zoospores were prepared as described by Boyle et al. (8). Tenfold serial dilution series ranging from 1,000 to 0.01 genomic equivalents (GEs) of zoospores per real-time PCR mixture were prepared for B. dendrobatidis and B. salamandrivorans. DNA of the other described Chytridiomycota species (Table 1) was prepared from growing cultures with DNA extraction in 100 μl of Prepman Ultra reagent (Applied Biosystems, Foster City, CA), following the DNA extraction method described by Hyatt et al. (9).

Primer and probe design.The previously described forward primer STerF (5′-TGCTCCATCTCCCCCTCTTCA-3′) and reverse primer STerR (5′-TGAACGCACATTGCACTCTAC-3′) were used to detect the 5.8S rRNA gene of B. salamandrivorans (6) (GenBank accession number KC762295). The B. salamandrivorans-specific Cy5-labeled probe STerC (5′-ACAAGAAAATACTATTGATTCTCAAACAGGCA-3′), based on the 5.8S rRNA gene of B. salamandrivorans, was developed using Kodon (Applied Maths, Kortrijk, Belgium) (Fig. 1). The primer set ITS1-3 Chytr (5′-CCTTGATATAATACAGTGTGCCATATGTC-3′) and 5.8S Chytr (5′-TCGGTTCTCTAGGCAACAGTTT-3′) and the TaqMan probe Chytr MGB2 (5′-CGAGTCGAAC-3′) described by Boyle et al. (8) were used to detect the ITS1 rRNA gene of B. dendrobatidis. All primers and probes were checked with BLASTN analysis, to ensure that amplification of genes from other organisms or species was unlikely. For B. salamandrivorans, the specificity of the primer set was tested in a SYBR green real-time PCR with DNA extracts of pure B. salamandrivorans culture and negative controls, with melting curve analysis and gel electrophoresis of the real-time PCR products (see below).

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Fig 1

Specific primers and probe for Batrachochytrium salamandrivorans. (A) rRNA gene sequences of the ITS1, 5.8S, and ITS2 regions used for the design of the B. salamandrivorans primers and probe. (B) B. salamandrivorans primer and probe sequences. The sequence used is from GenBank accession number KC762295.

B. salamandrivorans SYBR green real-time PCR.The B. salamandrivorans SYBR green assay was performed on a CFX96 real-time system (Bio-Rad Laboratories, Hercules, CA). A reaction mixture composed of 12.5 μl SYBR green PCR mix (1× SensiMix SYBR No-ROX; Bioline Reagents Ltd., London, United Kingdom), B. salamandrivorans forward primer STerF at a concentration of 0.3 μM, B. salamandrivorans reverse primer STerR at a concentration of 0.3 μM, 5 μl template, and a volume of RNase- and DNase-free water to a total of 25 μl was used in each reaction. Amplification conditions consisted of 95°C for 10 min followed by 40 cycles of 95°C for 15 s and 62°C for 15 s. A temperature gradient from 60°C to 95°C, with plate reads at every temperature increment of 0.5°C, was used to generate melting curve data.

Duplex real-time TaqMan PCR assay optimization.The B. dendrobatidis and B. salamandrivorans real-time PCR assays were first optimized as simplex assays. Assays were performed on a CFX96 real-time system (Bio-Rad Laboratories, Hercules, CA). Amplification conditions for the simplex and duplex assays consisted of 10 min at 95°C followed by 40 cycles of melting (95°C for 15 s) and annealing/extension (62°C for 1 min). Primer concentrations were optimized in a checkerboard system, with a standard probe concentration of 250 nM. Subsequently, the probe concentrations were optimized with the previously determined optimal primer concentrations. After optimization of the simplex assays, the two PCRs were combined to form the duplex real-time PCR. The precision of the developed duplex real-time PCR assay was evaluated by determining intra- and interassay variability, expressed as the mean coefficient of variation. For the interassay variability, three replicates of the quantitation standard were run in three separate assays; for the intra-assay variability, three replicates were run in one assay. The specificity of the duplex real-time PCR was evaluated by assaying DNA extracts of a wide range of Chytridiomycota species (Table 1). Real-time PCR efficiency, slope, and r² values were calculated with Bio-Rad CFX Manager v1.6 (Bio-Rad Laboratories, Hercules, CA), with the baseline-subtracted curve-fit setting. Slope and r² were calculated with the standard curves determined with the quantitation standards described earlier. Efficiency was calculated as 10^−1/slope − 1. After optimization of the duplex real-time PCR, a protocol that includes adding bovine serum albumin (BSA) to the PCR mixture was validated as an alternative to diluting samples, in order to alleviate PCR inhibition that could arise due to the nature of amphibian samples (9, 10). BSA (Sigma-Aldrich Inc., Bornem, Belgium) was added to the PCR mixture at a concentration of 400 ng μl⁻¹, as this is the optimal concentration to relieve PCR inhibition (11). Four replicates of the described quantitation standards of B. dendrobatidis and B. salamandrivorans with added BSA and four replicates without added BSA were run. Mean quantification cycle (C_q) values generated for the two conditions were compared to evaluate any significant effect of the addition of BSA on basic PCR results.

Amphibian samples.To validate the use of the real-time PCR to detect B. dendrobatidis and B. salamandrivorans in skin samples from amphibians, we applied the optimized protocol to samples from (i) 10 fire salamanders (S. salamandra) experimentally inoculated with B. salamandrivorans, (ii) 41 fire salamanders (S. salamandra) from the declining population in the Netherlands, (iii) 51 fire salamanders (S. salamandra) from a stable Belgian population, and (iv) 27 yellow-bellied toads (Bombina variegata) from a healthy Dutch population with known B. dendrobatidis infection (see Table 4). The B. salamandrivorans infection experiment with fire salamanders was carried out with approval of the ethics committee of the Faculty of Veterinary Medicine, Ghent University (approval no. EC2013/10). Skin swabs were collected by gently rubbing a sterile cotton-tipped swab 10 times across the ventral abdomen, inner thigh, and hind limb digits (9, 12). From dead amphibians, pieces of skin taken from the ventral abdomen (approximate size, 0.25 cm²) were collected for analysis. DNA was extracted from skin swabs in 100 μl Prepman Ultra (Applied Biosystems, Foster City, CA) (9). DNA was extracted from skin tissue using proteinase K digestion, following the protocol of Bandi et al. (13). After DNA extraction, 1:10 dilutions were prepared, to minimize possible PCR inhibition (9), and were stored at −20°C until further use. All tested samples were run in both simplex and duplex real-time PCR assays, for comparison of the variability in C_q values between the simplex and duplex runs. Samples that did not generate a signal were assigned a C_q value of 40, corresponding to the maximum number of cycles run in this real-time PCR setup. For positive samples, the number of GEs per swab or total skin tissue was calculated with the quantitation standards. A sample was considered positive when the number of GEs per swab/skin tissue exceeded 20, which, because of the dilution of the sample in the process of DNA extraction, corresponded to a detection limit of 0.1 GE per real-time PCR.