Faculty of Science

DETECTION OF THE AMPHIBIAN PATHOGEN BATRACHOCHYTRIUM
DENDROBATIDIS ON SPADEFOOTS IN THE KAMLOOPS REGION
2014 | KATIE BENNETT

B.Sc. Honours thesis

DETECTION OF THE AMPHIBIAN PATHOGEN BATRACHOCHYTRIUM DENDROBATIDIS
ON SPADEFOOTS IN THE KAMLOOPS REGION

by
KATIE BENNETT
A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE
OF BACHELOR OF SCIENCE (HONS.)

in the
DEPARTMENT OF BIOLOGICAL SCIENCES

(Animal Biology)

We accept this thesis as conforming to the required standards:

___________________________________________________
Jonathan Van Hamme (Ph.D.), Thesis Supervisor, Dept. Biological Sciences

___________________________________________________
Karl Larsen (Ph.D.), Co-supervisor, Dept. Natural Resource Sciences

___________________________________________________
Matt Reudink (Ph.D.), Dept. Biological Sciences

___________________________________________________
Nancy Flood (Ph.D.), Honours Co-ordinator, Dept. Biological Sciences

Dated this 24th day of April 2014, in Kamloops, British Columbia, Canada

ABSTRACT
Thesis Supervisor: Dr. Jonathan Van Hamme

Batrachochytrium dendrobatidis is a species of chytrid fungus responsible for the
decline and extinction of amphibian populations worldwide (Longcore 1999 and others).
Recently, B. dendrobatidis has been detected on western toads (Bufo boreas) in southwestern
British Columbia (Deguise & Richardson 2009) and has therefore become a concern for
conservationists across the province. Chytrid DNA can be extracted from tissue samples, skin
swabs, and water bodies and subsequently detected using real time PCR techniques. It is
currently unknown whether the chytrid fungus is affecting amphibian populations in the
Kamloops area. This project aimed to first validate an effective real time PCR assay for
detecting the presence of the amphibian pathogen B. dendrobatidis, and second to employ
this real time PCR assay to detect B. dendrobatidis on experimental samples collected in the
Kamloops region.
Specifically, spadefoots were captured during the summer of 2013 from the New
Afton mining site located south of Kamloops, BC. A total of 78 spadefoots were captured,
swabbed, and subsequently weighed, measured and released. Parameters for optimizing an
effective real time PCR assay were tested to detect B. dendrobatidis in experimental samples;
initially a Taqman assay was tried, but due to a lack of success, a SYBR Green assay was
used. DNA was extracted from all samples and 30 of these samples were tested for B.
dendrobatidis using the SYBR Green assay and gel electrophoresis.
B. dendrobatidis DNA was not detected in any of the 30 experimental samples. Melt
curve analysis indicated the presence of multiple PCR products, products which were
observed by gel electrophoresis at lengths less than 150 base pairs. It is possible that nonspecific primer binding occurred, or that primer dimers formed, at low template DNA
concentrations as these bands were not observed above 42 standard DNA copy numbers.
Based on the results in hand, it is possible that (1) chytrid is not present in the ponds at New
Afton mine, (2) chytrid is present but is not affecting the spadefoots, (3) the sensitivity of the
SYBR Green assay was not sufficient to detect chytrid DNA, (4) PCR inhibitors interfered
with the detection of chytrid DNA on samples, and (5) chytrid DNA did not adhere to the
swabs that were used in this work. Future research should employ swabs with smaller tips

ii

and employ the more sensitive Taqman assay using a probe with a non-flourescent quencher
(NFQ) as opposed to a black-hole quencher (BHQ-1) that was used here.

iii

ACKNOWLEDGEMENTS

This project would not have been possible without the help and contributions of Dr.
Jon Van Hamme, Dr. Karl Larsen, and Joanne Hales. Jon was incredibly helpful with the
molecular techniques used to test the environmental samples for chytrid and taught me many
useful skills. He also spent a lot of time helping me to edit my honours proposal, U-REAP
report, and honours thesis. Karl was always available when I had questions about spadefoots
and played an integral role in getting this project started. His vast knowledge of spadefoots
made summer data collection and capturing of the spadefoots much easier. During the
summer, I was lucky enough to work alongside Joanne Hales, a Masters of Sciences student
who is studying the ecology of spadefoots at New Afton. Joanne took me on as an
undergraduate research assistant to help with her project but also helped me to collect data
for my research.
In addition, this project was made more feasible by funding from New Afton, the
Natural Sciences and Environmental Research Council of Canada, and the Undergraduate
Research Experience Program (U-REAP) at Thompson Rivers University.

iv

TABLE OF CONTENTS
1 INTRODUCTION ..................................................................................................... 1
2 MATERIALS AND METHODS .............................................................................. 3
2.1

Field Sampling ............................................................................................................ 3

2.2

Real Time PCR............................................................................................................. 3

2.3

TaqMan assays ........................................................................................................... 5

2.4

SYBR Green assays .................................................................................................... 6

2.5

DNA extractions ......................................................................................................... 7

2.6

Real Time PCR assays............................................................................................... 7

2.7

Gel electrophoresis for experimental samples............................................... 9

3 RESULTS ................................................................................................................... 9
3.1

TaqMan assay ............................................................................................................. 9

3.2

SYBR Green assays ................................................................................................. 14

3.3

Real Time PCR analysis of environmental samples ................................... 19

3.4

DNA Inhibitors ......................................................................................................... 22

4 Discussion ............................................................................................................ 23
5 Literature cited................................................................................................... 26
6 APPENDICES......................................................................................................... 29

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LIST OF FIGURES

Figure 2.1 Batrachochytriumdendrobatidis primer sequences and minor groove binder probe
sequence. ..................................................................................................................... 4
Figure 3.1 TaqMan assay amplification plot for chytrid standard DNA showing only
background noise. Change in fluorescence is plotted against the cycle number. ..... 10
Figure 3.2 Agarose gel of real time PCR products from the first TaqMan assay. Lanes 1
through 8 contain DNA concentrations of 420 000, 42 000, 4 200, 420, 42, 4.2, 0.42 and
0 molecules/reaction respectively. ............................................................................ 11
Figure 3.3 Second TaqMan assay amplification plot for chytrid standard DNA. Change in
fluorescence is plotted against the cycle number. ..................................................... 11
Figure 3.4 Agarose gel of real time PCR products from the second TaqMan assay. Lane 1
contains a molecular weight marker and lanes 3 through 10 contain DNA concentrations
of 420 000, 42 000, 4 200, 420, 42, 4.2, .42, and 0 molecules/reaction respectively.
Arrow indicates expected location of target DNA. ................................................... 12
Figure 3.5 Third TaqMan assay amplification plot for chytrid standard DNA. Change in
fluorescence is plotted against the cycle number. ..................................................... 13
Figure 3.6 Fourth TaqMan assay amplification plot for chytrid standard DNA. Change in
fluorescence is plotted against the cycle number. ..................................................... 13
Figure 3.7 A typical TaqMan assay amplification plot for chytrid standard DNA. Change in
fluorescence is plotted against the cycle number. ..................................................... 14
Figure 3.8 A SYBR Green assay amplification plot for chytrid standard DNA. Change in
fluorescence is plotted against the cycle number. ..................................................... 15
Figure 3.9 Agarose gel of real time PCR products from the first SYBR Green assay. Lanes 1
through 8 contain DNA concentrations of 420 000, 42 000, 4 200, 420, 42, 4.2, .42, and
0 molecules/reaction, respectively. Arrow indicates location of target DNA (146 bp). 16
Figure 3.10 Standard curve generated from a series of dilutions of template DNA using the
SYBR green buffer and a cycling temperature of 66°C. Copy number is on the x-axis
and CQ value is on the y-axis. ................................................................................... 17

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Figure 3.11 Amplification plot produced from a rtPCR SYBR green assay using standard
DNA and a cycling temperature of 66°C. ................................................................. 18
Figure 3.12 Melt curve analysis of a real time PCR trial using the SYBR green buffer run at a
cycling temperature of 66˚C. Target DNA is located at approximately 73˚C. ......... 18
Figure 3.13 Gel electrophoresis produced using real time PCR products yielded from a
cycling temperature of 66OC. Lanes 1 through 8 contain DNA concentrations of 420
000, 42 000, 4 200, 420, 42, 4.2, .42, and 0 molecules/reaction respectively. Arrow
indicates location of target DNA (146 bp). ............................................................... 19
Figure 3.14 Amplification plot produced from a real time PCR SYBR green assay using both
standard DNA and experimental DNA. .................................................................... 20
Figure 3.15 Melt curve analysis of a real time PCR trial using a SYBR Green buffer with
both experimental samples and chytrid standard DNA. ........................................... 21
Figure 3.16 Standard curve plot produced from a real time PCR SYBR Green assay using
experimental DNA and chytrid standard DNA at concentrations of 420 000, 42 000, 4
200, 420, 42, and 0.42 molecules/reaction. Open squares indicate standard DNA and
solid circles indicate environmental samples. ........................................................... 21
Figure 3.17 Gel electrophoresis produced using real time PCR products. Lanes 1 and 30
contain a molecular weight marker, lanes 2-4 and 13-29 contain environmental samples,
and lanes 5 through 12 contain DNA concentrations of 420 000, 42 000, 4 200, 420, 42,
4.2, .42, and 0 molecules/reaction respectively. Arrow indicates location of target DNA
(146 bp). .................................................................................................................... 22

vii

LIST OF TABLES

Table 2.1 Volumes and concentrations of components used in the TaqMan real time PCR
assays. ......................................................................................................................... 5
Table 2.2 Volumes and concentration of components used in the SYBR Green real time PCR
assays. ......................................................................................................................... 6
Table 2.3 Experimental setup for a real time PCR assay using environmental samples spiked
with B. dendrobatidis plasmids................................................................................... 8
Table 3.1 Amplification Cycle for environmental samples spiked with B. dendrobatidis
plasmids using a real time PCR SYBR Green assay. ............................................... 22

viii

1

INTRODUCTION

Batrachochytrium dendrobatidis is a species of chytrid fungus responsible for the
decline and extinction of amphibian populations worldwide (Longcore 1999 and others). The
fungus may be present in water bodies where it infects the keratinized cells of amphibians
causing chytridomycosis; a disease that interrupts the ability of amphibians to osmoregulate
(maintain proper water movement across skin) (Daszek et al. 1999 and others). Chytrid acts
as a pathogen by disrupting electrolyte transport by inhibiting epithelial Na+ channels; in
severe cases, systemic depletion of Na+, K+, and Cl- causes cardiac arrest (Campbell et al.
2011). B. dendrobatidis infects the skin of adult amphibians and the mouthparts of anuran
larvae (Garner et al. 2005).
Currently, the chytrid fungus is associated with the rapid decline or extinction of
approximately 200 amphibian species around the globe in an array of climates (Skerratt et al.
2007). The prevalence of B. dendrobatidis in an African population of Xenopus since the
1930’s has been well documented. It is thought that international trade of infected Xenopus
laevis by humans has acted as a vector for the transmission of chytrid to unaffected
amphibian populations worldwide (Vredenburg et al. 2013). Presently, B. dendrobatidis is
affecting amphibians on all continents; 52 of 82 countries sampled for the presence of the
chytrid fungus have tested positive while the pathogen has been detected in 42 percent of
amphibian species (Olson et al. 2013).
Recently, B. dendrobatidis has been detected on northern leopard frogs (Rana
pipiens) in southeast British Columbia (Voordouw et al. 2010) and on western toads (Bufo
boreas) in southwestern British Columbia (Deguise & Richardson 2009) and has therefore
become a concern for conservationists across the province. This species of chytrid fungus can
be detected in water bodies, sediments, and on amphibians through the use of real time PCR
techniques (Boyle et al. 2004, Kirshtein et al. 2007). Two commonly used assays are the
TaqMan assay and the SYBR Green assay. The TaqMan assay is frequently used (Boyle et
al. 2004, Garner et al. 2006, and others) because it has a high specificity for the target DNA
as it utilizes both a set of PCR primers as well as a nucleotide probe targeting a specific DNA
sequence between the forward and reverse primer sites. The SYBR green assay has been
1

proven to be as equally sensitive as the TaqMan assay (Kirshteinet al. 2007), but is less
common because it can have reduced specificity for the target DNA as it does not include the
TaqMan probe, only the forward and reverse PCR primers. Chytrid may also be identified
using histological techniques but this has proved less effective (Kriger et al. 2006).
Real time PCR is a highly efficient method for detecting B. dendrobatidis because it
not only detects the presence or absence of the fungus in samples, but also provides
information on the amount of fungus present in the original sample. It is currently unknown
whether the chytrid fungus is affecting amphibian populations in the Kamloops area, and so
this project aimed to firstly validate an effective real time PCR program that can be used to
detect for the presence of the amphibian pathogen B. dendrobatidis, and secondly to employ
this modified real time PCR program to determine whether this species of chytrid fungus is
affecting Great Basin Spadefoots found on the New Afton mining site operated by New Gold
Inc. located south of Kamloops, BC. New Afton’s tailing ponds, as well as other ephemeral
and permanent water bodies in the area, are commonly used as breeding ponds by spadefoots.
The Great Basin Spadefoot is an endangered amphibian found throughout the dry,
arid region of western North America (Cannings 1999). There is very little known about the
ecology of this species as it spends the majority of its life cycle in underground burrows. The
ability to live in arid environments is unique to the spadefoots; their ability to absorb water
from the soil (Ruibal et al. 1969) renders them capable of living underground for long
periods of time. The spadefoots emerge from their burrows during breeding season; thunder
and spring rains trigger the spadefoots to travel to breeding ponds (Ruibal et al. 1969).
Breeding ponds may be ephemeral or permanent aquatic bodies including both large ponds
present year-round and small puddles. Even minimal amounts of water in tire tracks left by
vehicles can be sufficient for spadefoot breeding (Personal Observation). Tadpole
development is rapid in order to increase their chances of survival, this is particularly
important in ephemeral water bodies.
While some research studying B. dendrobatidis and amphibians has been undertaken
in western North America (Deguise & Richardson 2009), no observations on the effects of B.
dendrobatidis on the Great Basin Spadefoot have been made. Early detection of this parasitic
fungus is crucial in helping to treat and protect endangered populations. In particular, the

2

knowledge of the relationship between B. dendrobatidis and the Great Basin Spadefoot can
aid in development of a management plan to conserve this spadefoot species.

2

MATERIALS AND METHODS

2.1

Field Sampling
Collection of water and skin samples took place during the summer of 2013 on the

New Afton mining site operated by New Gold Inc. Materials for collecting skin swabs
included: sterile fine tip cotton swabs (Puritan Medical Products, Maine, part no. 25-806
1WC FDNA), alcohol, microcentrifuge tubes (Sarstedt, part no. 72.693.005), and sterile
disposable, powder free vinyl gloves (Siedman Associates, New York, part no. GVP9-MD1). Vinyl gloves were chosen as they have proven to have minimal toxicity for tadpoles
relative to latex and nitrile gloves (Cashins et al. 2008). Each spadefoot was caught and
handled using clean gloves. A cotton swab was repeatedly run over the specimen’s abdomen,
sides, drink patch, thighs, and feet webbing as outlined in Hyatt et al. (2007). The tip of the
cotton swab was then clipped using scissors into a clean microcentrifuge tube and placed in a
cooler on ice until it could be stored in a -20OC freezer in the microbiology lab at Thompson
Rivers University. Each spadefoot was weighed and its snout-vent length was measured to
the nearest 0.5 mm using a measuring tape. The location and time of capture was also
recorded.
2.2

Real Time PCR
This research took place in the microbiology lab at Thompson Rivers University

during the fall of 2013 under the supervision of Dr. Jon Van Hamme. An Illumina Real Time
PCR (rtPCR) instrument with Eco software was used for all rtPCR experiments. Materials
ordered for this work included: B. dendrobatidis (Bd) rDNA control plasmids (Pisces
Molecular, Colorado), Taqman Universal Master Mix II (Applied Biosystems, Ontario),
SYBR Green buffer (Applied Biosystems, Ontario, part no. 4472908), a probe (Chytr
MGB2), and forward and reverse PCR primers (ITS1-3 Chytr and 5.8S Chytr) (Alpha DNA,
Quebec) (Figure 2.1). The primer and probe sequences chosen were taken from Boyle et al.

3

(2004) with the exception of a black hole quencher (BHQ-1) being attached to the probe
instead of a non-fluorescent quencher (NFQ).

Primer Sequences
ITS1-3 Chytr

29 bases

5’- CCTTGATATAATACAGTGTGCCATATGTC -3’
5.8S Chytr

22 bases

5’- AGCCAAGAGATCCGTTGTCAAA -3’

Minor groove binder probe sequence
Chytr MGB2

15 bases

5’- 6FAM GCAGTCGAACAAAAT BHQ1 -3’
Figure 2.1 Batrachochytrium dendrobatidis primer sequences and minor groove binder
probe sequence.
The 7 Bd control plasmid solutions ordered from Pisces Molecular ranged in
concentration from 2.1 x 100 to 2.1 x 106 molecules/µL. Prior to rtPCR experiments, the
control plasmids were each aliquoted into 10 microcentrifuge tubes and stored in a non-frost
free freezer at -20°C in order to reduce the number of freeze-thaw cycles each standard was
subjected to. Lyophilized PCR primers were briefly centrifuged and reconstituted in sterile,
nuclease free water to a stock concentration of 100µM. Each stock was diluted to 900 nM
and aliquoted into microcentrifuge tubes that were then stored at -20°C. The Chytr MGB2
probe was diluted with water to a final concentration of 250 nM and stored as well. Prior to
each experiment, the primers, probe, and Bd plasmids were thawed on ice, vortexed and
centrifuged. All equipment used for preparing PCR plates (e.g. pipettors, pipette tips) were
placed under a UV light for approximately 2 minutes, and all reactions were prepared in a
sterile biological safety cabinet using filter tips (Neptune Scientific, Ontario).
Following each rtPCR assay, PCR products were further analyzed using gel
electrophoresis. The agarose gels used for B. dendrobatidis control plasmids were each
prepared using 50 mL of Tris-borate EDTA (TBE) buffer and 0.75 g of agarose. Once the
agarose was added to the TBE, the mixture was heated in a microwave until the agarose

4

dissolved (approximately 1 min 15 s). Next, 2.5 µL of GelRed (10 000 × Biotium, Lot
10G1026) was added to the mixture that was then allowed to cool prior to pouring into a gel
tray (90 cm3) containing an 8 or 15 well comb. Once the gel was set, the comb was removed
and the gel was placed in an electrophoresis unit containing TBE. 2 µL loading dye (10× Fast
digest buffer, Fermentas, Lithuania, Lot 00050741) was added to each PCR product, and 10
µL from each was pipetted into the agarose gel. Additionally, 5 µL of molecular weight
marker (20mg/µL, Invitrogen, Ontario, part no. 15628-050) was added to a single lane.
Electrophoresis was carried out for 60 min at 95 V.
2.3

TaqMan assays
For the first TaqMan experiment, an rtPCR assay was conducted following the

methods outlined in Boyle et al.(2004) with the exception of using 15 µL reaction volumes
as opposed to 25 µL reaction volumes. The 15 µL reactions contained 7.5 µL TaqMan buffer,
900 nM (0.54 µL) of each primer, 250 nM (0.75 µL) of probe, 5.47 µL of water, and 0.2 µL
of a DNA control plasmid or water. The PCR amplification conditions were 2 min at 50OC
then10 min at 95OC; this was followed by 20 s at 95OC and 1 min at 60OC for 50 cycles. In
subsequent rtPCR assays using the TaqMan buffer, the annealing temperature was altered
(Table 2.1) but all other amplification conditions remained the same. In addition to
experimenting with an array of annealing temperatures, the probe and DNA volumes used
were modified (Table 2.1).
Table 2.1 Volumes and concentrations of components used in the TaqMan real time PCR
assays.
Primer
volume
(µL)
0.54
0.54
0.54
0.54
0.54
0.54
0.54

Primer
concentration
(nM)
900
900
900
900
900
900
900

Probe
concentration
(nM)
250
250
250
250
250
250
250

DNA
volume
(µL)
0.2
0.2
0.2
0.2
0.2 – 2.2
0.2
0.2

DNA concentration
(molecules/rxn)

900

Probe
volume
(µL)
0.75
0.75
0.75
0.75
0.75
0.75
0.75 –
2.5
0.75

0.54

250

900

2.5

250

0.2, 0.6,
0.8
0.2

4.2 × 105, 1.26 × 106,
1.68 × 106
4.2 × 10-1 – 4.2 × 105

0.54

4.2 × 10-1 – 4.2 × 105
4.2 × 10-1 – 4.2 × 105
4.2 × 10-1 – 4.2 × 105
4.2 × 10-1 – 4.2 × 105
4.2 × 105 – 4.62 × 106
4.2 × 10-1 – 4.2 × 105
4.2 × 105

Annealing
Temperature
(°C)
60
58
62
64
66
60
60
59
59

5

0.54

900

2.5

250

0.54

900

2.5

250

2.4

0.2, 0.6,
0.8
0.2, 0.6,
0.8

4.2 × 105

59

4.2 × 105

57

SYBR Green assays
The first SYBR Green rtPCR experiment conducted used 15 µL reaction volumes.

The 15 µL reaction volumes contained 7.5 µL SYBR Green buffer, 900 nM (0.54 µL) of
each primer (ITS1-3 and 5.8S), 6.42 µL water, and 0.2 µL DNA control plasmid or water.
The PCR amplification conditions were 2 min at 50°C then 2 min at 95°C; this was followed
by 15 s at 95°C and 1 min at 59°C for 50 cycles. In addition, all but the first SYBR Green
assay used a melt curve analysis of 15 s at 95°C, 15 s at 55°C, and 15 s at 95°C. Subsequent
SYBR Green rtPCR experiments followed the same amplification conditions with the
exception of the annealing temperature (Table 2.2). Finally, the last SYBR Green rtPCR
experiment used four different primer concentrations and only the highest plasmid DNA
concentration (4.2 × 105 molecules/reaction) (Table 2.2). This was done to explore an
association between the primer concentrations used and the amount of primer dimer
produced.
Table 2.2 Volumes and concentration of components used in the SYBR Green real time PCR
assays.
Primer volume

Primer

DNA concentration

DNA volume

Annealing

(µL)

concentrations

(molecules/rxn)

(µL)

temperature (°C)

(nM)
0.54

900

4.2 × 10-1 – 4.2 × 105

0.2

60

0.54

900

4.2 × 10-1 – 4.2 × 105

0.2

60

0.54

900

4.2 × 10-1 – 4.2 × 105

0.2

60

0.54

900

4.2 × 10-1 – 4.2 × 105

0.2

60

0.54

900

4.2 × 10-1 – 4.2 × 105

0.2

62

0.54

900

4.2 × 10-1 – 4.2 × 105

0.2

66

0.54

900

4.2 × 10-1 – 4.2 × 105

0.2

68

0.3, 0.5, 0.7, 0.9

900

4.2 × 105

0.2

66

6

2.5

DNA extractions
DNA extractions were completed in the microbiology lab at Thompson Rivers

University under the supervision of Dr. Jon Van Hamme during the winter of 2014. DNA
extractions followed the methods outlined in Boyle et al. (2004) with the exception of 50 µL
of PrepMan Ultra, rather than 40 µL, being used. In addition, Boyle et al. (2004) used whole
toes to extract DNA from and we used cotton swabs as described by B.C. Ministry of
Environment Wildlife Health Program (2008). Materials used for the extractions include:
PrepMan Ultra and Zirconium/silica beads (0.5 mm in diameter). The cotton swabs used for
sampling in the summer were quite large and so prior to nucleic acid extraction with
PrepMan Ultra, fragments (approximately 0.001 g dry weight) were removed from the tip of
each swab using a razor blade. This was carried out in a sterile biological safety cabinet using
clean gloves. Between each swab, the razor blade and tweezers were placed in alcohol then
flamed using a Bunsen burner. This helped to avoid cross-contamination of each sample.
Each cotton sample was placed in a clean, labeled microcentrifuge tube containing 30 to 40
mg zirconium/silica beads. The samples were then stored in a non-frost free freezer at -20°C.
Fifty µL PrepMan Ultra was added to each prepared sample, the sample was then
homogenized for 45 s in a mini beadbeater. This was followed by a brief centrifugation (30 s
at 13 × 103 × g) to recover all the material to the bottom of the tube. The homogenization and
centrifugation steps were repeated once more prior to immersing the samples in a boiling
water bath (approximately 102OC) for ten minutes. After being cooled for 2 min in an ice
water bath, the samples were centrifuged at 13 000 × g for 3 min. Twenty µL of supernatent
was recovered to a clean tube in a sterile biological safety cabinet and stored at -80°C.

2.6

Real Time PCR assays
Thirty environmental DNA samples were analyzed for B. dendrobatidis using a real

time PCR SYBR Green assay. 15 µL reactions were prepared with each containing 7.5 µL
SYBR Green buffer, 900 nM (0.54 µL) of each primer (ITS1-3 and 5.8S), 6.42 µL water, and
0.2 µL environmental DNA or B. dendrobatidis control plasmids. For each trial,
environmental samples were run in triplicate and 2 standard curves were generated. The PCR
7

amplification conditions were 2 min at 50°C then 2 min at 95°C; this was followed by 15 s at
95°C and 1 min at 66°C for 50 cycles. In addition, a melt curve analysis consisting of a
single cycle of 15 s at 95°C, 15 s at 55°C, and 15 s at 95°C was used.
To test for the presence of DNA inhibitors in environmental samples, a single rtPCR
trial was run containing B. dendrobatidis plasmids alone as well as environmental samples
spiked with plasmid DNA (Table 2.3). This experiment utilized both a SYBR Green buffer
(as was used previously) and a SYBR Green Fastmix buffer (Quanta Biosciences,
Gaithersburg, MD, part no. 95072-125) to compare the results of each buffer. The PCR
amplifications used were 2 min at 50°C then 2 min at 95°C followed by 15 s at 95°C and 1
min at 66°C for 50 cycles.
Table 2.3 Experimental setup for a real time PCR assay using environmental samples spiked
with B. dendrobatidis plasmids.
Lane

A

Column

Plasmid DNA

Plasmid DNA concentration

Environmental

(µL)

(molecules/rxn)

DNA (µL)

1

0.2

4.2 × 105

0

SYBR Green Fastmix

2

0.2

4.2 × 104

0

SYBR Green Fastmix

3

0.2

4.2 × 10

3

0

SYBR Green Fastmix

4

0.2

4.2 × 102

0

SYBR Green Fastmix

5

0.2

4.2 × 101

0

SYBR Green Fastmix

0.2

4.2 × 10

0

0

SYBR Green Fastmix

-1

0

SYBR Green Fastmix

6

B

7

0.2

4.2 × 10

8

0

0

1

0

SYBR Green Fastmix

4.2 × 10

6

2.0

SYBR Green Fastmix

6

1.0

SYBR Green Fastmix

2

0.2

4.2 × 10

3

0.2

4.2 × 106

0.5

SYBR Green Fastmix

0.2

4.2 × 10

6

0.25

SYBR Green Fastmix

6

0

SYBR Green Fastmix

4

C

0.2

Buffer

5

0.2

4.2 × 10

1

0.2

4.2 × 106

2.0

SYBR Green

0.2

4.2 × 10

6

1.0

SYBR Green

6

0.5

SYBR Green

2
3

0.2

4.2 × 10

4

0.2

4.2 × 106

0.25

SYBR Green

5

0.2

4.2 × 106

0

SYBR Green

8

2.7

Gel electrophoresis for experimental samples
Each of the 30 environmental DNA real time PCR products was analyzed using gel

electrophoresis to confirm results of the rtPCR assay. Agarose gels were each prepared using
200 mL of tris-borate EDTA (TBE) and 3.0 g of agarose. Once the agarose was added to the
TBE, the mixture was heated in a microwave until the agarose dissolved (approximately 1
min 50 s). Next, 10 µL of GelRed (Biotium, 10 000×) was added to the mixture that was then
allowed to cool prior to pouring into a gel tray containing two 30 well combs. Once the gel
was set, the comb was removed and the gel was placed in an electrophoresis unit containing
TBE. Loading dye (2 µL of 10× Fast digest buffer, Fermentas) was added to each PCR
product, and 5 µL from each was pipetted into the agarose gel. Additionally, 5 µL of
molecular weight marker (20 mg/µL, Invitrogen, Ontario) was added to 4 lanes.
Electrophoresis was carried out for 60 min at 95 V.

3

RESULTS

3.1

TaqMan assay
The TaqMan assay did not detect the Bd plasmid DNA and so a standard curve that

could be used to detect fungal DNA in environmental samples was not produced. The first
experiment followed the PCR program outlined in Boyle et al. (2004) and used DNA
concentrations of 420 000, 42 000, 4 200, 420, 42, 4.2, and 0.42 molecules/reaction.
Amplification plots (Figure 3.1) showed that no PCR products were produced and only
background noise was detected. Following the real time PCR, agarose gel electrophoresis
was used to visualize the quality of the PCR products. The agarose gel revealed unbound
probe and primer dimer (Figure 3.2). The targeted 146 base pair region (Boyle et al. 2004)
was not apparent. A possible reason for large amounts of unbound probe is the 60ºC
annealing temperature was too high.

9

Figure 3.1 TaqMan assay amplification plot for chytrid standard DNA showing only
background noise. Change in fluorescence is plotted against the cycle number.

10

Figure 3.2 Agarose gel of real time PCR products from the first TaqMan assay. Lanes 1
through 8 contain DNA concentrations of 420 000, 42 000, 4 200, 420, 42, 4.2, 0.42
and 0 molecules/reaction respectively.
In the second TaqMan trial, a lower annealing temperature (58°C) was used to see if
the probe would bind the targeted 146 base pair region more efficiently. All other parameters
of the PCR program as well as the concentrations of the primers and probe plus DNA
volumes remained the same as in the first experiment. The amplification plot (Figure 3.3)
showed that no PCR products were produced and only background noise was detected. Gel
electrophoresis was used to visualize the quality of the PCR products. As in the first
experiment, a large amount of unbound probe was seen (Figure 3.4) and amplified Bd DNA
was not apparent.

Figure 3.3 Second TaqMan assay amplification plot for chytrid standard DNA. Change in
fluorescence is plotted against the cycle number.

11

1 2 3 4 5 6 7 8 9 10

Figure 3.4 Agarose gel of real time PCR products from the second TaqMan assay. Lane 1
contains a molecular weight marker and lanes 3 through 10 contain DNA
concentrations of 420 000, 42 000, 4 200, 420, 42, 4.2, .42, and 0 molecules/reaction
respectively. Arrow indicates expected location of target DNA.
In the third TaqMan assay, a higher annealing temperature (62°C) was used to see if
this enabled the probe and primers to bind the targeted region of DNA. All other parameters
of the PCR program as well as the concentrations of the primers and probe plus DNA
concentrations remained the same. The amplification plot (Figure 3.5) showed that minor
PCR product was produced in one reaction but in all other reactions only background noise
was detected, as in the previous two assays. This experiment was then repeated using an
annealing temperature of 66°C, however, this assay yielded similar results (Figure 3.6).

12

Figure 3.5 Third TaqMan assay amplification plot for chytrid standard DNA. Change in
fluorescence is plotted against the cycle number.

Figure 3.6 Fourth TaqMan assay amplification plot for chytrid standard DNA. Change in
fluorescence is plotted against the cycle number.

13

Overall, annealing temperatures between 58°C and 62°C were tested but did not
improve the results. Greater concentrations of DNA were also tested (4.2 × 105, 8.4 × 105,
1.26 × 106, 1.68 × 106, 2.1 × 106, 2.52 × 106, 2.94 × 106, 3.36 × 106, 3.78 × 106, 4.2 × 106,
and 4.62 × 106 molecules/reaction) such as in the fifth TaqMan rtPCR assay. But, again, the
target PCR product was not generated as shown in both amplification plots (Figure 3.7) and
gel electrophoresis imaging.

Figure 3.7 A typical TaqMan assay amplification plot for chytrid standard DNA. Change in
fluorescence is plotted against the cycle number.
Given the lack of success, and the cost of the MGB-labelled probe, a SYBR green
assay was tried. Other amplification plots and gels produced from the experimental TaqMan
assays can be found in appendix A.

3.2

SYBR Green assays
Since it appeared that some PCR product was being generated in the latter TaqMan

assays, but no fluorescent signal was being detected, a SYBR Green assay was tested using
the PCR program described in Boyle et al. 2004 with DNA concentrations of 0.42, 4.2, 42,
14

420, 4 200, 42 000, and 420 000 molecules/reaction. PCR product was detected by rtPCR as
shown in the amplification plot (Figure 3.8). Gel electrophoresis was used to visualize the
quality of the PCR products; a large amount of product was seen (Figure 3.9) but there was
also non-specific binding and primer dimer. In the following three experiments, this PCR
program was repeated and the same concentrations of DNA, primer, and buffer were used to
see if the results from the first trial would persist. In addition, a melt curve analysis was
added to help determine the quality of the PCR products. All experiments yielded similar
results.

Figure 3.8 A SYBR Green assay amplification plot for chytrid standard DNA. Change in
fluorescence is plotted against the cycle number.

15

1 2 345 6 78

Figure 3.9 Agarose gel of real time PCR products from the first SYBR Green assay. Lanes 1
through 8 contain DNA concentrations of 420 000, 42 000, 4 200, 420, 42, 4.2, .42,
and 0 molecules/reaction, respectively. Arrow indicates location of target DNA (146
bp).
In subsequent experiments, annealing temperatures of 62°C, 66°C, and 68°C were
used to find an ideal temperature that would limit the amount of nonspecific binding and
primer dimer formation. The optimal real time PCR conditions were found to be 50°C for 2
min then 95°C for 2 min followed by 50 cycles of 95°C for 2 min and 66°C for 1 min,
followed by a melt curve analysis. The 15 µL reactions yielded the best results when they
contained 7.5 µL SYBR Green buffer, 900 nM (0.54 µL) of each primer (ITS1-3 Chytr and
5.8S Chytr), 6.22 µL of water, and 0.2 µL of standard DNA.
The detection limit of this real time PCR program is 42 copies (Figure 3.10). Positive
amplification above the threshold (CT) after 35 cycles was not considered positive as nonspecific PCR product was repeatedly detected in control wells (Figure 3.11). DNA
concentrations at and above 42 molecules/reaction resulted in strong fluorescent signals
before 35 cycles, excellent melt curves, and little or no non-specific PCR product (Figure

16

3.10, Figure 3.11, Figure 3.12, Figure 3.13). Points on a standard curve graph produced by
fluorescent signals after 35 cycles resulted in poor R2 values indicative of an inefficient PCR
program. The melt curve analysis (Figure 3.12) revealed that the 5 reactions containing the
greatest concentrations of DNA (420 000, 42 000, 4 200, 420, and 42 molecules/reaction)
had PCR product melting at the same temperature (approximately 73OC). This coincides with
the gel images produced from the PCR products. In lanes 1 through 4 there is a large amount
of amplified DNA with a length close to 146 base pairs, indicating this is the target DNA. In
lanes 5 through 7 there is some PCR product but also a large amount of nonspecific product
and in lane 8 there is no PCR product but lots of nonspecific product.

Figure 3.10 Standard curve generated from a series of dilutions of template DNA using the
SYBR green buffer and a cycling temperature of 66°C. Copy number is on the x-axis
and CQ value is on the y-axis.

17

Figure 3.11 Amplification plot produced from a rtPCR SYBR green assay using standard
DNA and a cycling temperature of 66°C.

Target peak

Nonspecific PCR product

Figure 3.12 Melt curve analysis of a real time PCR trial using the SYBR green buffer run at
a cycling temperature of 66˚C. Target DNA is located at approximately 73˚C.

18

Gel images produced from the real time PCR products helped to confirm the
detection limit of the PCR program. The use of gel electrophoresis also revealed the
formation of primer dimer and nonspecific binding (Figure 3.13).

1 2 3 4 5 6 7 8

Figure 3.13 Gel electrophoresis produced using real time PCR products yielded from a
cycling temperature of 66OC. Lanes 1 through 8 contain DNA concentrations of 420
000, 42 000, 4 200, 420, 42, 4.2, .42, and 0 molecules/reaction respectively. Arrow
indicates location of target DNA (146 bp).
3.3

Real Time PCR analysis of environmental samples
The real time PCR SYBR Green assay detected DNA in all experimental samples as

well as wells containing standard DNA at concentrations of 420 000, 42 000, 4 200, 420, 42,
4.2, and 0.42 molecules/reaction (Figure 3.14). Though it appears as though there is chytrid
in environmental samples, melt curve analyses (Figure 3.15) revealed the amplification of
non-chytrid DNA in both control and experimental wells. The melt curve analysis for each
rtPCR trial revealed that the 5 reactions containing the greatest concentrations of chytrid
standard DNA (420 000, 42 000, 4 200, 420, and 42 molecules/reaction) had PCR product
melting at the same temperature (approximately 73°C), however, all other products melted at

19

a greater temperature (approximately 78.5°C). This was consistent with the standard curve
generated (Figure 3.16).

Figure 3.14 Amplification plot produced from a real time PCR SYBR green assay using both
standard DNA and experimental DNA.
Target peak

Nonspecific DNA

20

Figure 3.15 Melt curve analysis of a real time PCR trial using a SYBR Green buffer with
both experimental samples and chytrid standard DNA.

Detection Limit

Figure 3.16 Standard curve plot produced from a real time PCR SYBR Green assay using
experimental DNA and chytrid standard DNA at concentrations of 420 000, 42 000, 4
200, 420, 42, and 0.42 molecules/reaction. Open squares indicate standard DNA and
solid circles indicate environmental samples.
Gel electrophoresis confirmed results of the melt curve analysis and rtPCR standard
curve. Product was present in the wells containing standard DNA at concentrations of 420
000, 42 000, 4 200, 420, and sometimes 42 molecules/reaction (Figure 3.17) Thick bands
below the targeted DNA sequence are likely non-specific PCR product. Other examples of
gel images are in Appendix 1.

21

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30

Figure 3.17 Gel electrophoresis produced using real time PCR products. Lanes 1 and 30
contain a molecular weight marker, lanes 2-4 and 13-29 contain environmental
samples, and lanes 5 through 12 contain DNA concentrations of 420 000, 42 000, 4
200, 420, 42, 4.2, .42, and 0 molecules/reaction respectively. Arrow indicates location
of target DNA (146 bp).
3.4

PCR Inhibitors
Spiking different volumes of environmental DNA samples with B. dendrobatidis

standards revealed the presence of PCR inhibitors in the DNA extracted from swabs. For
both the SYBR Green FastMix buffer and the regular SYBR Green buffer, samples
containing smaller volumes of environmental DNA had earlier amplification cycles while
those with greater volumes of environmental DNA either had later amplification cycles or
did not have any amplification (Table 3.1). As the volume of environmental DNA in the
samples decreased, the amplification cycles occurred earlier indicating the presence of more
DNA in those wells.
Table 3.1 Amplification Cycle for environmental samples spiked with B. dendrobatidis
plasmids using a real time PCR SYBR Green assay.
Lane

A

Column

1
2

Plasmid DNA

Plasmid DNA concentration

Environmental

(µL)

(molecules/rxn)

DNA (µL)

0.2

4.2 × 105

0

17

0.2

4

0

22

4.2 × 10

Amplification Cycle

22

3
4

B

4.2 × 103

0

25

0.2

4.2 × 10

2

0

26

1

0

26

5

0.2

4.2 × 10

6

0.2

4.2 × 100

0

26

7

0.2

4.2 × 10

-1

0

26

8

0

0

0

27

1

0.2

4.2 × 106

2.0

No amplification

0.2

4.2 × 10

6

1.0

No amplification

6

0.5

No amplification

2
3

0.2

4.2 × 10

4

0.2

4.2 × 106

0.25

17

0.2

4.2 × 10

6

0

17

6

2.0

43

5
C

0.2

1

0.2

4.2 × 10

2

0.2

4.2 × 106

1.0

27

0.2

4.2 × 10

6

0.5

20

6

0.25

18

0

17

3
4

0.2

4.2 × 10

5

0.2

4.2 × 106

In addition, the regular SYBR Green buffer proved more effective at detecting
standard DNA than the SYBR Green FastMix buffer. The regular SYBR Green buffer
detected product in all wells while the SYBR Green FastMix buffer only detected DNA in 2
wells.

4

DISCUSSION

Batrachochytrium dendrobatidis has negative implications for amphibian species
around the globe as it infects keratinized skin cells causing chytridiomycosis; a condition that
ultimately leads to mortality (Daszek et al.1999 and others). Due to the detection of chytrid
on all continents (Olsen et al. 2013), many amphibian species are at risk of declining
population sizes and even extinction. B. dendrobatidis can be detected using both histological
and molecular techniques, however, molecular techniques have proven to be more effective
(Kriger et al. 2006).
Real time PCR techniques using either a TaqMan or SYBR green assay are most
commonly used to detect B. dendrobatidis. The rtPCR assay employed by Boyle et al. 2004

23

is often referenced. In their work, they compared the sensitivity and specificity of a TaqMan
assay to histological detection methods, concluding that the TaqMan assay is more accurate
than histology for detecting chytrid. Boyle et al. consistently generated standard curves with
an R2 value close to one (indicative of an efficient assay) and a detection limit of 0.1 genome
equivalents. This is similar to the 10 copy detection limit for the SYBR Green and TaqMan
assays employed by Kirshtein et al. 2007, but is lower than our detection limit of 42 copies.
Kirshtein et al. experimented with a SYBR Green assay following the PCR program outlined
in Boyle et al. 2004 and used melt curve analysis and gel electrophoresis to ensure PCR
amplification did not result in the formation of primer dimer. This is the only study I found
that mentioned the use of gel electrophoresis or melt curve analysis to ensure efficacy of the
PCR program. No studies were found that included gel images or melt curve analysis
descriptions in their results.
Real time PCR can be used to detect B. dendrobatidis in water bodies (Hyman &
Collins 2012, Kirshtein et al. 2007), on amphibian skin swabs (Forzan et al. 2010, Hyman &
Collins 2012, Kriger et al. 2006), and on amphibian toe clips (Deguise & Richardson 2006,
Garner et al. 2006). In the present study, we chose to use skin swabs as opposed to toe clips
because collecting skin swabs is considerably less invasive than toe clipping. In addition, toe
clips may not provide sufficient sample for detecting the fungus (Boyle et al. 2004), while
swab-PCR techniques have proven to be highly sensitive (Forzan et al. 2010, Hyman &
Collins 2012, Kriger et al. 2006). In the literature, researchers have used wooden toothpicks
(Hyman & Collins 2012) and rayon swabs (Kriger et al. 2006) to collect swab samples from
animals. However, due to a limited budget, we opted to use a relatively large cotton swab.
This posed difficulties as the cotton soaked up very large volumes of PrepMan Ultra, thus
diluting the concentration of experimental DNA. To avoid the use of such large volumes of
PrepMan Ultra, we shaved small amounts of cotton off the swab and extracted DNA from
these pieces. In the future, it would be beneficial to use a rayon swab (Medical Wire &
Equipment, MW 100-100).
Given previous experiments that detected chytrid using rtPCR, and through extensive
trial and error, we successfully validated an effective real time PCR program that can be used
to detect for the presence of the amphibian pathogen B. dendrobatidis. While the TaqMan
assay yielded no results, the SYBR green assay was effective as it detected the control B.

24

dendrobatidis DNA and a good standard curve could be generated from the data. Note that
the quencher used on the probe in the TaqMan experiments was a black hole quencher
(BHQ), while the probe used in the literature was a non-fluorescent quencher (NFQ) (Boyle
et al. 2004). This may have been a factor in the lack of results produced in the TaqMan
assay, although we have no evidence to support this. The PCR program outlined in Boyle et
al. 2004 was used with the exception of a 66˚C cycling temperature instead of a 60˚C cycling
temperature.
Once the SYBR Green assay and gel electrophoresis methods were validated, they
was used to identify the presence of the chytrid fungus on Great Basin Spadefoots found on
the New Afton mining site located south of Kamloops, BC. Of the 30 environmental samples
analyzed, none appeared to contain chytrid DNA. Possible explanations for the absence of
chytrid are: (1) chytrid is not present in the ponds at New Afton mine, (2) chytrid is present
but is not affecting the spadefoots, (3) the sensitivity of the SYBR Green assay was not
enough to detect chytrid DNA that was potentially on the swabs, (4) PCR inhibitors
prevented chytrid from being detected in the rtPCR assays, and (5) chytrid DNA did not
adhere to the swabs.
Chytrid may not be present in the ponds at New Afton mine due to the ephemeral
nature of many of the ponds. B. dendrobatidis requires a vector to establish itself in water
bodies and so if the ponds are only present for short periods of time, it is possible that chytrid
does not have enough time to grow in the ponds and in turn affect amphibian hosts.
Alternately, chytrid may be present in the ponds at New Gold (particularly the permanent
water bodies) but is not affecting the spadefoots. Spadefoots employ the ponds as breeding
sites for only short periods each year; this may protect them from becoming infected as they
have less contact with the water and pathogens within it (i.e. B. dendrobatidis) than other
amphibians. A third explanation for chytrid not being detected on the environmental swabs is
the sensitivity of the SYBR Green assay was not enough to detect chytrid DNA on the swabs.
The unknown DNA present on environmental swabs was minimal (Appendix D) and the
SYBR Green assay was only able to detect as little as 42 copies. Chytrid may also have not
been detected due to polymerase inhibitors interfering with the copying of DNA. Polymerase
inhibitors are detrimental to PCR results because they produce false negatives. The
mechanism of action of PCR inhibitors varies; while some interfere with the annealing ability

25

of PCR primers, others act by degrading PCR polymerases (Schrader et al. 2012). PCR
inhibitors may have originated in the field when the samples were collected, or in the
laboratory during DNA extraction and processing. Finally, chytrid DNA may not have
adhered to the swabs because we used swabs made of a cotton material as opposed to swabs
made of rayon as used in the literature.
Future research should use a different type of swab (Medical Wire & Equipment,
MW 100-100) and employ the more sensitive Taqman assay using a probe with a nonflourescent quencher (NFQ) as opposed to a black-hole quencher (BHQ-1). In addition,
taking water samples and skin swabs from spadefoot anuran larvae and other amphibian
species throughout the spring, summer, and fall could help to gain further insight into the
association between chytrid and spadefoots in the Kamloops region. The samples collected
could be tested for B. dendrobatidis using real time PCR techniques and gel electrophoresis.
Research conducted by Deguise and Richardson in 2009 revealed the presence of the
chytrid fungus in a breeding population of Western Toads (Anaxyrus boreas) near Squamish,
British Columbia. Given the close proximity of the study site with Kamloops, it is possible
that chytrid may already be affecting amphibian populations near Kamloops or will
potentially affect them in the future. Future research in the Kamloops region would improve
our knowledge of the relationship between B. dendrobatidis and spadefoots as well as other
amphibians in the area. This information could therefore aid in the development of a
management plan to conserve amphibian species affected by the chytrid fungus. Possible
management could involve the creation of man-made ponds containing chytrid-free water for
spadefoot breeding or building toad fences around ponds containing chytrid.

5

LITERATURE CITED

B.C. Ministry of Environment [Internet]. 2008. Victoria, BC: B.C. Wildlife Health Program;
[cited
2014
Apr
16].
Available
from:
http://www.env.gov.bc.ca/wld/documents/wldhealth/Collection%20Protocol.pdf
Boyle, D.G., Boyle, D.B., Olsen, V., Morgan, J.A.T., Hyatt, A.D. 2004. Rapid quantitative
detection of chytridomycosis (Batrachochytriumdendrobatidis) in amphibian samples
using real-time Taqman PCR assay. Diseases of Aquatic Organisms 60: 141-148

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Campbell, C.R., Voyles, J., Cook, D.I., Dinudom, A. 2011. Frog skin epithelium: electrolyte
transport and chytridiomycosis. Int J Biochem Cell Biol. 44(3): 431-434
Cannings SG, Ramsay LR, Fraser DF, Fraker MA. 1999. Rare amphibians, reptiles, and
mammals of British Columbia. Victoria, BC: BC Ministry of Environment, Lands and
Parks. 190 p
Cashins, S.D., Alford, R.A., Skerratt, L.F. 2008. Lethal effect of latex, nitrile, and vinyl
gloves on tadpoles. Herpetological Review 39(3): 298-301
Daszak, P., Berger, L., Cunningham, A.A., Hyatt, A.D., Green, D.E., Speare, R. 1999.
Emerging infectious diseases and amphibian population declines. Emerging
Infectious Diseases 5: 735-748
Deguise, I., Richardson, J.S. 2009. Prevalence of the chytrid fungus
(Batrachochytriumdendrobatidis) in western toads in southwestern British Columbia,
Canada. Northwestern Naturalist 90: 35-38
Garner, T.W.J., Perkins, M.W., Govindarajulu, P., Seglie, D., Walker, S., Cunningham, A.A.,
Fisher, M.C. 2006. The emerging amphibian pathogen Batrachochytrium
dendrobatidis globally infects introduced populations of the North American bullfrog,
Rana catesbeiana. Biology Letters 2:455-459
Hyatt, A.D., Boyle, D.G., Olsen, V., Boyle, D.B., Berger, L., Obendorf, D., Dalton, A.,
Kriger, K., Hero, M., Hines, H., Phillott, R., Campbell, R., Marantelli, G., Gleason,
F., Colling, A. 2007. Diagnostic assays and sampling protocols for the detection of
Batrachochytrium dendrobatidis. Diseases of Aquatic Organisms 73:175-192
Kirshtein, J.D., Anderson, C.W., Wood, J.S., Longcore, J.E., Voytek, M.A. 2007.
Quantitative PCR detection of Batrachochytriumdendrobatidis DNA from sediments
and water. Diseases of Aquatic Organisms 77: 11-15
Kriger, K.M., Hines, H.B., Hyatt, A.D., Boyle, D.G., Hero, J-M. 2006. Techniques for
detecting chytridiomycosis in wild frogs: comparing histology with real-time Taqman
PCR. Diseases of Aquatic Organisms 71:141-148
Longcore, J.E., Pessier, A.P, Nichols, D.K. 1999. Batrachochytriumdendrobatidisgen. et sp.
nov., a chytrid pathogenic to amphibians. Mycologia 91(2): 219-227
Olson, D.H., Aanesen, D.M., Ronnenberg, K.L., Powell, C.I., Walker, S.F., Bielby, J.,
Garner, T.W.J., Weaver, G., The Bd Mapping Group, Fisher, M.C. 2013. Mapping
the global emergence of Batrachochytriumdendrobatidis, the amphibian chytrid
fungus. PLoS ONE 8(2):e56802. doi:10.1371/journal.pone.0056802
Schrader, C., Schielke, A., Ellerbroek, L., Johne, R. 2012. PCR inhibitors- occurrence,
properties and removal. Journal of Applied Microbiology 113: 1014-1026

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Skerratt, L.F., Berger, L., Speare, R., Cashins, S., McDonald, K.R., Phillott, A.D., Hines,
H.B., Kenyon, N. 2007. Spread of chytridiomycosis has caused the rapid global
decline and extinction of frogs. EcoHealth. doi:10.1007/s10393-007-0093-5
Voordouw, M.J., Adama, D., Houston, B., Govindarajulu, P., Robinson, J. 2010. Prevalence
of the pathogenic chytrid fungus, Batrachochytrium dendrobatidis, in an endangered
population of northern leopard frogs, Rana pipiens. BMC Ecology 10:6
doi:10.1186/1472-6785-10-6

Vredenburg, V.T., Felt, S.A., Morgan, E.C., McNally, S.V.G., Wilson, S., Green, S.L. 2013.
Prevalence of Batrachochytrium dendrobatidis in Xenopus collected in Africa (18712000)
and
in
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doi:10.1371/journal.pone.0063791

28

6

APPENDICES

A. TaqMan Assays

Fifth TaqMan assay amplification plot for chytrid standard DNA showing only background
noise. Change in fluorescence is plotted against the cycle number.

29

TaqMan assay amplification plot for chytrid standard DNA showing only background noise.
Change in fluorescence is plotted against the cycle number.

Typical TaqMan assay amplification plot for chytrid standard DNA showing only
background noise. Change in fluorescence is plotted against the cycle number.

1

2

3

4

5

6 7

8

9 10

30

Agarose gel of real time PCR products from a TaqMan assay with an annealing temperature
of 62OC. Lane 1 contains a molecular weight marker and lanes 3 through 10 contain DNA
concentrations of 420 000, 42 000, 4 200, 420, 42, 4.2, .42, and 0 molecules/reaction
respectively. Arrow indicates expected location of target DNA.

1 2

3

4

5

6

7

8 9 10

Agarose gel of real time PCR products from a TaqMan assay with an annealing temperature
of 58OC. Lane 1 contains a molecular weight marker and lanes 3 through 10 contain DNA
concentrations of 420 000, 42 000, 4 200, 420, 42, 4.2, .42, and 0 molecules/reaction
respectively. Arrow indicates expected location of target DNA.

31

1

2 3 4 5 6 7

8 9 10

Agarose gel of real time PCR products from a TaqMan assay with an annealing temperature
of 60OC. Lane 1 contains a molecular weight marker and lanes 3 through 10 contain DNA
concentrations of 420 000 molecules/reaction and probe volumes between 0.75 and 2.5
µL/reaction.
B. SYBR Green Assays

32

Amplification plot produced from the second real time PCR SYBR green assay using
standard DNA and a cycling temperature of 60OC.

Amplification plot produced from the third real time PCR SYBR green assay using standard
DNA and a cycling temperature of 60OC.

33

Amplification plot produced from the fourth real time PCR SYBR green assay using standard
DNA and a cycling temperature of 60OC.

Amplification plot produced from the fifth real time PCR SYBR green assay using standard
DNA and a cycling temperature of 60OC.

34

Melt curve analysis of the third real time PCR trial using the SYBR green buffer run at a
cycling temperature of 60OC.

Melt curve analysis of the fourth real time PCR trial using the SYBR green buffer run at a
cycling temperature of 60OC.

35

Melt curve analysis of the fifth real time PCR trial using the SYBR green buffer run at a
cycling temperature of 60OC.

36

1

2 3

4

5

6 7

8

9 10

Agarose gel of real time PCR products from the second SYBR Green assay.Lane 1 contains a
molecular weight marker and lanes 3 through 10 contain DNA concentrations of 420 000, 42
000, 4 200, 420, 42, 4.2, .42, and 0 molecules/reaction respectively. Arrow indicates
expected location of target DNA.

37

1

2 3 4

5 6 7 8

9 10

Agarose gel of real time PCR products from the third SYBR Green assay.Lane 1 contains a
molecular weight marker and lanes 3 through 10 contain DNA concentrations of 420 000, 42
000, 4 200, 420, 42, 4.2, .42, and 0 molecules/reaction respectively. Arrow indicates
expected location of target DNA.

38

1

2

3

4

5

6 7

8

9 10

Agarose gel of real time PCR products from the fourth SYBR Green assay.Lane 1 contains a
molecular weight marker and lanes 3 through 10 contain DNA concentrations of 420 000, 42
000, 4 200, 420, 42, 4.2, .42, and 0 molecules/reaction respectively. Arrow indicates
expected location of target DNA.

39

1

2

3

4

5

6 7

8

9 10 11 12 13

Agarose gel of real time PCR products from the fifth SYBR Green assay.Lanes 1 and 13
contain a molecular weight marker and lanes 3 through 10 contain DNA concentrations of
420 000, 42 000, 4 200, 420, 42, 4.2, .42, and 0 molecules/reaction respectively. Arrow
indicates expected location of target DNA.
C. Real Time PCR analysis of environmental samples

40

Amplification plot produced from a real time PCR SYBR green assay using standard DNA
and 10 environmental samples.

Amplification plot produced from a real time PCR SYBR green assay using standard DNA
and 10 environmental samples.

41

Amplification plot produced from a real time PCR SYBR green assay using standard DNA
and 10 environmental samples.

Melt curve analysis of a real time PCR trial using standard DNA and 10 environmental
samples.

42

Melt curve analysis of a real time PCR trial using standard DNA and 10 environmental
samples.

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30

Gel electrophoresis produced using real time PCR products. Lanes 1 and 30 contain a
molecular weight marker, lanes 2-9 contain DNA concentrations of 420 000, 42 000, 4 200,
420, 42, 4.2, .42, and 0 molecules/reaction respectively and lanes 10-29 contain
environmental samples. Arrow indicates location of target DNA (146 bp).

43

1

2

3

4

5

6 7

8

9 10

11 12 13 14

15 16 17 18 19 20

Gel electrophoresis produced using real time PCR products. Lanes 1 and 20 contain a
molecular weight marker, lanes 2-9 contain DNA concentrations of 420 000, 42 000, 4 200,
420, 42, 4.2, 0.42, and 0 molecules/reaction respectively and lanes 10-19 contain
environmental samples. Arrow indicates location of target DNA (146 bp).
D. DNA Concentration on Environmental Swabs
Sample ID
DP 29/5 (1)
S1 3/6 (1)
PH 25/5 (8)
DP 29/5 (3)
PH 29/5 (1)
DP 30/5 (2)
PH 5/6 (1)
DP 30/5 (1)
DP 4/6 (2)
PH 4/6 (1)
LPH 20/6 (1)
DP 29/5 (7)
PLP 4/6 (1)
DP 29/5 (5)
PH 20/6 (1)
PLP 5/6 (1)
M 5/6 (1)
PH 5/6 (2)
C 4/6 (1)
S 11/6 (1)
E2 16/7 (1)
E2 16/7 (2)
PH 20/6 (2)

DNA Concentration (ng/mL)
1.59
<0.5
20.9
7.18
<0.5
18
<0.5
12.4
25
11.9
4.49
21.9
<0.5
<0.5
19.4
7.74
1.56
<0.5
<0.5
34.8
<0.5
6.96
11.8

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PH 11/6 (1)
PH 5/6 (3)

10.4
8.63

45