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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.