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Susceptibility to Ranavirus Through Frogs and Salamanders Using q-PCR For Detection and Quantification
Thomas Brigman Department of Biology, York College of Pennsylvania
Results
Introduction
Effective MCP
Primers
 Amphibian populations is declining globally and the die-offs seem to be
linked to infectious diseases (Gary et al. 2009, Blaustein and Kiesecker
2002). Ranaviruses is the highest reported reason for mortality in
amphibians (Green et al. 2002).
 It has been reported that 43% of the reported die-offs of amphibians from
2000 to 2005 are from the Ranaviruses (Gary et al. 2009).
 Ranavirus is in the family Iridoviridae, which are large, double-stranded
DNA viruses, with a noticeable icosahedral shape that is mostly noticeable
Figure 1. A 1% agarose gel. lane one is
100 bp ladder. Lane 2 is water and
primers PCR. Lane 3 is plasmid and
primers PCR. (576bp)
in the cytoplasm of infected cells (Chinchar 2002 and Green et al. 2002).
 The major capsid protein (MCP) is highly conserved in the Ranavirus, and
is commonly sequenced at 500bp rejoin at the 5’ to identify Ranavirus
(Gary et al. 2009 and Chinchar 2002).
Standard Curve of MCP Plasmid
 Because of the potentially devastating effect of Ranavirus infections
Table 1. CT Values of DNA Samples
DNA Sample
AMb 400
AM 401
AM 402
AM 403
AM 404
AM 405
RSc 400
RS 401
RS 402
RS 403
RS 404
RS 405
RS 406
CT Mean
33.53
24.52
40.00
32.30
26.00
29.19
36.19
34.46
30.25
34.54
40.00
32.84
30.81
a is
Standard Deviation
b is
Abystoma maculatum (Spotted Salamander)
c
CT Stda
2.12
0.94
0.61
2.65
0.12
1.73
1.52
1.12
0.56
3.26
0.10
0.79
0.26
is Rana sylvatica ( Wood Frog)
among susceptible amphibians species, a lot of effort has been put into
early and rapid detection of the virus (Chinchar 2002).
and salamanders, has been sequenced. FV3 replication is rapid and can be
CT Value
 A strain of Ranavirus, FV3, which is known to effect common frogs, toads
Conclusions
 Ranavirus can be detected using PCR and q-PCR protocol that was
developed and can help in rapid detection.
detected within 2 hours post infection (Chinchar 2002 ).
 It could not be proven that Ranavirus was more susceptible in frogs or
 Since Ranavirus is known to effect frogs and salamanders, determining
which species is more susceptible to infection could play an important role
in early detection of infection within a environment.
Quantity(ug)
Figure 2. CT value of MCP plasmid dilutions(n=5) at known quantity (ug).
Line represents liner regression..
salamanders. (Fisher's exact test p=0.0909). This could be due to a low
sample size.
Objectives
 To prepare a new rapid technique to detect for Ranavirus within frogs and
Amplification Amount of Positive
Frog Sample
salamanders.
 To determine if salamanders or frogs, within a vernal pool located in York,
Collect frogs(n=6)
and salamanders(n=6)
DNA
Extract MCP plasmid
also be beneficial for early detection.
Cycles
Figure 3. Amount of amplification of MCP primers of known infected frog
DNA sample( 3 replicates), on Log scale over a period of cycles
q-PCR with MCP
plasmid and DNA
Test with positive
sample
short period of time.
 Coming up with a proper way of testing the water for Ranavirus could
Amplification Log Scale
Methods
Develop Standard
Curve
 Consider increasing sample size over a long period of time instead of a
 Toads should also be studied with regards to susceptibility to Ranavirus.
Pennsylvania, is more susceptible to Ranavirus.
Run PCR with MCP
plasmid
Future Studies
Test with DNA
samples
Acknowledgements
I would like to give a special thanks to my mentors Dr. Meda Higa and Dr. Bridgette Hagerty for the guidance.
Also I would like to thank Victor Chinchar for the supply of the MCP plasmid and Carrie Reall for the DNA samples.