Appendix B. Standard and real-time PCR assays for Batrachochytrium dendrobatidis.
Ethanol preserved tissue was soaked in 1XTE buffer (10mM Tris.Cl pH7.6, 1mM EDTA pH 8.0) prior to extraction of genomic DNA using either a DNeasy Tissue Kit (Qiagen, Valencia, California, USA) or Prepman Ultra (Applied Biosystems, Foster City, California, USA). DNA amplification was conducted using either standard or real-time PCR. Primers were anchored in the ribosomal DNA (rDNA) internal transcribed spacers (ITS). Although B. dendrobatidis displays within-individual sequence variability through ITS, these primers were designed in regions that were conserved across isolates originating from four countries (USA, Australia, Panama and Ecuador; J.A.T. Morgan, unpublished data). For standard PCR a 235 base fragment was amplified between forward primer CHYITS1F3 5’-ACAAAATTTATTTATTTTTTCGAC-3’, located in ITS1 and reverse primer CHYITS2R2 5’-CATGGTTCATATCTGTCCAG-3’, located in ITS2. PCR reactions contained 0.5µM of each primer, combined with 10-100ng of extracted DNA, 1XTaq buffer, 0.8mM dNTP, 2.5mM magnesium and 0.05 units/µL of Taq polymerase (Roche Molecular Systems, Pleasanton, California, USA). This mix was thermocycled for 30 cycles. Cycle 1 was 95º C for 60 s, 50º C for 45 s, and 72º C for 90 s. This was followed by 29 shorter cycles, 95º C for 30 s, 50º C for 30 s, and 72º C for 90 s. The mix was held at 72º C for 7 min to complete extension then held at 4º C. Products were viewed on an ethidium bromide stained 1.5% agarose and TAE gel.
The primers used for real-time PCR assays amplify a 95 base fragment between forward ITS-1Chytr3 5’-CCTTGATATAATACAGTGTGCCATATGTC-3’, located in ITS1, and 5.8Schytr 5’ TCGGTTCTCTAGGCAACAGTTT-3’, located in 5.8S (Boyle et al. 2004) . Assays were performed using the probe ChytrMGB2 5’-CGAGTCGAACAAAAT-3’ following Boyle et al. (2004) with the following modifications. A 20 µL final reaction volume was used and assays were conducted in triplicate on a DNA Engine Opticon 2 System (MJ Research, San Francisco, California, USA). The threshold value was manually set at 0.05 for all assays, the baseline was averaged over the cycle range then subtracted and blanks were not subtracted. Standards gave reproducible results with R2 values above 0.99 for all assays.
Samples were determined to be positive for B. dendrobatidis infection if standard PCR yielded a 235 base band or if the real-time PCR assay detected one or more genomic equivalents in the reaction volume (translating to 80 genomic equivalents in the total extracted tissue). This lower limit for the real-time PCR assay was a conservative measure taken to minimize the chance of calling an uninfected individual positive due to low-level contamination. Negative extraction and PCR controls were run alongside all samples.
LITERATURE CITED
Boyle, D. G., D. B. Boyle, V. Olsen, J. A. T. Morgan, and A. D. Hyatt. 2004. Rapid quantitative detection of chytridiomycosis (Batrachochytrium dendrobatidis) in amphibian samples using real-time Taqman PCR assay. Diseases of Aquatic Organisms 60:141–148.