TITLE: TaqMan qPCR assay for detecting Batrachochytrium dendrobatidis (Bd)
AUTHORS: Omneya Ahmed Osman, Mats Töpel, Tomas Larsson, Alexander Eiler

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">The fungus Batrachochytrium dendrobatidis (Bd) was first detected in Norway in 2017, and thus indicate the arrival of an invasive black-listed species in the country.  Here we report the details of real time PCR assay which was used to screen for B. dendrobatidis from  water samples collected from different locations in Norway.</div></div>

[STEPS]
?. [DNA extraction]
DNA extraction was performed using Qiagen DNeasy power water sterivex kit. The quality of the extracted DNA was estimated using Nanodrop.   Qiagen DNeasy power water sterivex kit: https://www.qiagen.com/se/resources/resourcedetail?id=c5fe7d5f-070a-4ebe-ac04-4bbf05a13e91&lang=en
?. [Real time PCR]
A primer set of ITS1-3 Chytr (5’- CCTTGATATAATACAGTGTGCCATATGTC-3 ́), 5.8S Chytr (5’- AGCCAAGAGATCCGTTGTCAAA-3’) and probe Chytr MGB2 (FAM-5’ TTCGGGACGACCC-3’-NFQ- MGB) (Boyel et al. 2004). Internal control: http://www.primerdesign.co.uk/assets/files/internal_control_handbook_dna.pdf?timestamp=1469446474SsoAdvanced Universal Probes  Supermix: https://www.bio-rad.com/webroot/web/pdf/lsr/literature/bulletin-10023650.pdf
?. [Real time PCR]
PCR master mix without internal control (IC)  ABCD1ReagentWorking solFinal concVolume (µL)2SsoAdvanced Universal Probes  Supermix 2x1x103ITS1-3 Chytr10 uM0,9 µM1,845.8S Chytr10 uM0,9 µM1,85TaqMan probe10 uM0,25  µM0,56DEPC-water  0,97template  58∑  20
ABCD1ReagentWorking solFinal concVolume (µL)2SsoAdvanced Universal Probes  Supermix 2x1x103ITS1-3 Chytr10 uM0,9 µM1,845.8S Chytr10 uM0,9 µM1,85TaqMan probe10 uM0,25  µM0,56DEPC-water  0,97template  58∑  20
?. A serial dilution of 100, 50, 25, 10, 1, 0.1 and 0.01 genomic equivalents (GE) of the DNA extract of Bd standard was tested and 5 dilutions (100, 25, 10, 1, 0.1 GE) were used for the qPCR assay. Pipette 5 μl of DNA template to reach 20 μl final volume in each well.For negative controls use 5 μl of RNase/DNase free water.
?. [PCR program]
ABCD1 Step Time Temp (C )2Enzyme activation 2 min 953Cycling x50 (step3 & 4)Denaturation 10 sec954Extension and Data  collection *1min60
ABCD1 Step Time Temp (C )2Enzyme activation 2 min 953Cycling x50 (step3 & 4)Denaturation 10 sec954Extension and Data  collection *1min60
?. [qPCR instrument]
QPCR was performed on BioRad qPCR machine CFX96.Analysis of the results was done by CFX maestro softwarehttps://www.bio-rad.com/en-se/product/cfx-maestro-software-for-cfx-real-time-pcr-instruments?ID=OKZP7E15
?. [qPCR instrument]
Quantification cycle (Cq)Positive control of 100 genome equivalent is expected to give a signal at Cq value 27. In case of internal control, Cq values of 27±3 are within the normal range. A control sample with IC and water should be evaluated at each qPCR run.
?. [Real time PCR]
PCR master mix with IC  ABCD1 Working solutionFinal concentrationVolume (µL)2SsoAdvanced Universal Probes  Supermix2x1x12,53ITS1-3 Chytr10 uM0,9 µM2,25045.8S Chytr10 uM0,9 µM2,2505TaqMan probe10 uM0,25  µM0,6256IC primer/probe  17IC-DNA**  0,58DEPC-water  0,8759template  510∑  25
ABCD1 Working solutionFinal concentrationVolume (µL)2SsoAdvanced Universal Probes  Supermix2x1x12,53ITS1-3 Chytr10 uM0,9 µM2,25045.8S Chytr10 uM0,9 µM2,2505TaqMan probe10 uM0,25  µM0,6256IC primer/probe  17IC-DNA**  0,58DEPC-water  0,8759template  510∑  25