Detection of Paracoccidioides brasiliensis in Tissue Samples by a Nested PCR Assay

Microorganisms.Six isolates of P. brasiliensis(R-2878 to R-2883), originating from A. Restrepo in Colombia, and the ATCC 60885 strain were grown on potato flakes agar at 30°C for 2 weeks. Their identity as P. brasiliensis was confirmed in the Fungus Testing Lab, San Antonio, Texas. Mycelial colonies were scraped off the agar, dissolved in sterile water, frozen, and stored at −20°C. After thawing, 2 × 200 μl of each suspension was used for DNA extraction. One isolate of Aspergillus fumigatus, three of Blastomyces dermatitidis, one of Candida krusei, two of Cryptococcus neoformans, and three isolates of H. capsulatum were grown on blood or Sabouraud agar and identified by standard methods. Fungal suspensions were prepared as described above.

Tissue samples.BALB/c mice were infected by intranasal instillation of 3 × 10⁶ conidia of P. brasiliensis (ATCC 60885) and sacrificed several days and weeks thereafter, as described in detail elsewhere (3). Lungs were removed from mice under aseptic conditions, weighed, and homogenized in 2 ml of saline; the lung tissue homogenate was serially diluted and plated in duplicate on Sabouraud agar. After 30 days of incubation at 18°C, the number of CFU g of tissue⁻¹ was calculated (3). The remaining lung homogenates were stored frozen (−70°C) for up to 5 years before DNA extraction was done. Lung homogenates of 20 ICR mice intravenously infected with 8 × 10³ CFU of H. capsulatum and sacrificed on days 1, 5, and 11 after infection were used as controls. All lung homogenates were positive by quantitative culture in a range of 1 × 10³ to 7 × 10⁶ CFU of H. capsulatum per g of lung (R. Bialek et al., unpublished data).

DNA extraction.To 200 μl of each fungal suspension or thawed lung homogenate, 180 μl of ATL buffer of the QIAamp tissue kit (Qiagen, Hilden, Germany) and proteinase K (Qiagen) to a final concentration of 1 mg/ml were added. After incubation at 55°C for at least 3 h or overnight, the samples were boiled for 5 min, then exposed to three cycles of freezing in liquid nitrogen for 1 min and boiling for 5 min afterwards to disrupt the fungal cells. After cooling to room temperature, proteinase K (Qiagen) was added again to a final concentration of 1 mg/ml. After incubation at 55°C for 1 h, DNA was extracted using the QIAamp tissue kit (Qiagen) following the manufacturer's instructions. This extraction is based on detergents and proteinase K for solubilization of the tissue, the addition of ethanol and chaotrophic salts to allow binding of DNA to a silica membrane in columns, washing steps to remove protein, and elution of DNA from the silica by an alkaline buffer (pH 9.0). The exact composition of the buffers is part of the manufacturer's patent and the information is unavailable.

Primer design.The sequence of gp43 of P. brasiliensis (1) deposited in GenBank (U26160 ) was screened for primers. The outer primers were para I, 5′-AAC TAG AAT ATC TCA CTC CCA GTC C-3′, and para II, 5′-TGT AGA CGT TCT TGT ATG TCT TGG G-3′, being complementary to positions 846 to 870 and 1200 to 1176 of the GenBank sequence, respectively, defining a 355-nucleotide amplicon. The inner primer set consisted of para III, 5′-GAT CGC CAT CCA TAC TCT CGC AAT C-3′, and para IV, 5′-GGG CAG AGA AGC ATC CGA AAT TGC G-3′, which were complementary to the nucleotide positions 953 to 978 and 1148 to 1124 of the deposited sequence, respectively. They delimit a 196-nucleotide sequence.

PCR assay.The reaction mix of the first PCR consisted of 10 μl of DNA extract in a total volume of 50 μl, with final concentrations of 10 mM Tris-HCl (pH 8.3), 50 mM KCl, 2.5 mM MgCl₂ (10× Perkin-Elmer buffer II plus MgCl₂solution [Roche Molecular Systems, Branchburg, N.J.]), a 1-μM concentration of each primer (Roth, Karlsruhe, Germany), 1.5 U of AmpliTaq DNA polymerase (Perkin-Elmer), and a 100-μM concentration of each deoxynucleotide triphosphate (Promega, Madison, Wis.). The reaction mix of the second PCR was identical, except that 1 μl of the first reaction, a 50-μM concentration of each deoxynucleotide triphosphate, and a 1-μM concentration of each second primer were used. Reactions with the outer primer set were thermal cycled once at 94°C for 5 min, 35 times at 94°C for 30 s, 50°C for 30 s, and 72°C for 1 min, and then once at 72°C for 5 min. For the nested PCR product, reactions were thermal cycled once at 94°C for 5 min, 30 times at 94°C for 30 s and 72°C for 1 min, and then once at 72°C for 5 min. The high melting temperatures of the inner primers allowed a two-step nested PCR with high stringency. The PCR was run in a Primus PCR thermocycler Tc 9600 (MWG Biotech, Ebersberg, Germany). The PCR products were analyzed by electrophoresis on 1.5% agarose gels, stained with ethidium bromide, and visualized on an UV transilluminator.

Controls.A sample of 100 fg of plasmid DNA (∼2 × 10⁴ gene copies) containing the target sequence of theP. brasiliensis gp43 gene was used in every PCR assay as a positive control. Sterile water was included in the DNA extraction and was used as a negative control after every fifth sample in the nested PCR assay to monitor for crossover contaminations. Reaction mix without DNA was run in the first and second cycling to try to detect contaminations. In order to screen for inhibitors, all negative samples were examined a second time after adding 2 μl (∼4 × 10³ gene copies) of the positive control.

DNA extraction was controlled by a PCR using the primer set actin 4 (5′-AGC CAT GTA CGT AGC CAT CCA GGC T-3′) and actin 5 (5′-GGA TGT CAA CGT CAC ACT TCA TGA TGG-3′), amplifying a 450-bp sequence of the mouse actin gene and thus indicating the presence of amplifiable murine DNA. The reaction mix was identical to the mix described above, except that 3 mM MgCl₂, a 1-μM concentration of each primer, and only 5 μl of the DNA extract were used. The reaction was thermal cycled according to the first PCR protocol above, except for an annealing temperature of 57°C.

Sequencing.The nested PCR products were purified by using the QIAquick PCR purification kit (Qiagen), based on DNA binding to a silica membrane. Automated sequencing was done with the BigDye terminator cycle sequencing kit and PCR primers in accordance with the recommendations of the manufacturer and was analyzed on the ABI 373 automated DNA sequencer (Applied Biosystems Division, Perkin-Elmer Biosystems, Foster City, Calif.). Sequences were generated from both strands, edited and aligned with the Sequence Navigator software (Applied Biosystems), and used for a BLAST search in GenBank (National Center for Biotechnology Information, Washington, D.C.).

Cloning.The PCR product of the DNA extracted from the ATCC 60885 P. brasiliensis strain after the first round of amplification was purified by Qiagen spin columns (Qiagen). The amplicon was inserted into the PCR II.1 cloning vector using the Original TA cloning kit following the manufacturer's instructions (Invitrogen, Groningen, The Netherlands). After the bacteria were cultured and harvested, the plasmid DNA was purified by a Qiagen plasmid maxi kit (Qiagen) consisting of alkaline lysis of bacteria, separation and binding of plasmid DNA to anion exchange resin, wash steps, and final elution. The DNA concentration was measured by absorption at 260 nm. Serial dilutions were used to determine the detection limit of the nested PCR assay. The amplified product was sequenced to prove homology to the sequence in GenBank.