1. Identifying Amphibian Pathogens Using Quantitative PCR
Alexis Gramera*, Jessica Krempp, and Gregory Ruthig, Ph. D.
Department of Biology, North Central College, Naperville, IL
Abstract
Water molds are a major source of mortality
for amphibians. It is essential to understand
the diseases that contribute to these
amphibian declines and to develop
methodology for the identification of
amphibian pathogens. We designed
techniques to identify the presence and
abundance of pathogens in the
environment. Pathogens were isolated from
frog egg samples (either Lithobates
catesbeianus or L. clamitans). The abundance
of a pathogen was determined using real-
time quantitative PCR analysis. The
formation of this technique provides future
researchers a less costly and more time
efficient method for water mold
identification.
Introduction
• Amphibian populations have declined
drastically since 1989 (Wilkinson 2003).
• Water molds are amphibian pathogens
that can live on many different host
species (Ruthig and Provost-Javier 2012).
• It is difficult to identify pathogenic water
mold species using morphology.
• A critical component of the study of
water molds includes the detection of the
pathogen on infected amphibians, as well
as in the environment (Ruthig and
DeRiddler 2012).
Methods
• We collected frog egg masses from bodies of water in
Naperville, IL (A-B).
• We isolated water molds from infected eggs onto
cornmeal agar (C-D).
• We extracted and amplified the ITS regions of the
genomes of collected pathogens.
• PCR products were purified and sequenced at the
University of Chicago.
• Using MEGA6.06, we aligned our sequences with
known sequences from the database, Genbank to
determine the taxonomic identity of our strains.
• We designed a qPCR probe that is species specific, that
does not bind to other strains.
• We are now testing the ability of our qPCR probe to
determine its efficacy. We want to determine if:
1. The probe works on the strain for which it was
designed.
2. The probe works on other water mold strains.
A B
DC
Results
E
Acknowledgements
• Thank you to Dr. Gregory Ruthig for his assistance and guidance throughout this project.
• Thank you to North Central for making this project possible.
• Thank you to Chris Boffa and Jacquelyn Pfaff for their laboratory assistance.
• Thank you to Andrew DuBois, Joel DiBernardo, and Katy Reese for their contribution to the Ruthig Lab.
• Thank you to Dr. Jonathan Visick, Dr. Stephen Johnston, and Dr. Jennifer Sallee for their guidance.
Leptolegnia sp. EM32A
Leptolegnia sp. NT
Leptolegnia sp. SP
Achlya papillosa
Achlya oligacantha
Achlya racemosa
Achlya colorata
Leptolegnia sp. SAP248
Achlya aquatica
Achlya primoachlya
Achlya americana
Achlya intricata
Achlya ambisexualis
Achlya heterosexualis
2014 Gramera RvWk Mating B ITS 1 (Leptolegnia sp.)
Saprolegnia semihypogyna
Achlya sp. O3EG1
Achlya sp. DG
Leptolegnia sp. CBS
Saprolegnia diclina
2014 Gramera RvWk Mating A ITS 1 (Saprolegnia sp.)
Saprolegnia salmonis
Saprolegnia hypogyna
Saprolegnia parasitica
2014 Gramera Field Saprolegnia ferax ITS 1
Saprolegnia oliviae
Saprolegnia ferax
Saprolegnia sp. AESB
Saprolegnia bulbosa
Saprolegnia anomalies
Saprolegnia longicaulis
Aphanomyces sp. NVA
Aspergillus tubingensis
Pythium sp. OAK
Pythium erinaceum
Pythium parvum
Pythium takayamanum
2014 Gramera RvWk Mating C ITS 1 (Pythium sp.)
2014 Gramera Field Phytophthora sp. ITS 1
Phytophthora brassicae
Phytophthora botryosa
Phytophthora tropicalis
Phytophthora riparia
96
58
52
24
65
97
19
38
100
87
98
52
99
100
27
40
19
18
9
18
12
78
55
7893
36
50
0.1
1. We collected three egg masses.
2. We obtained DNA sequences from the ITS
region of fourteen water mold isolates (E).
3. We classified these isolates using Maximum
Likelihood Phylogenetic analysis (F).
4. We designed a locked nucleic acid (LNA)
primer/probe set for qPCR using Primer
Express 3.0 for strain 2014 Gramera RvWk
Mating A, a likely member of the genus
Saprolegnia.
5. We are currently testing the LNA probe’s
efficacy, with the goal that the probe will
only amplify our desired strain (G).
F
Forward Primer
[TTGCTTGTGCTTCGGTACGA]
Reverse Primer
[ATTTCGGCGAGGCTGTTG]
LNA Probe
[TGGACATATATTGCTTTTTG]
(Bold letters indicate LNAs)
G
B
Arrow indicates
samples collected
Positive
Amplifications
• Quantitative PCR
(qPCR) is a molecular
technique that allows
researchers to identify
the presence and
abundance of a
particular pathogen
strain.