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Diagnosis of Marek's disease by real time PCR from samples ...

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Materials and Methods• Samples were collected <strong>from</strong> three differentchallenge studies at different <strong>time</strong>s after infection• Collection <strong>of</strong> <strong>samples</strong>: Original and in FTA® cardsBlood Feather Pulp Tumors56 36 14


Material and Methods• Real <strong>time</strong> <strong>PCR</strong>:SYBR® green (GAPDH)Taq Man Probe (gB, pol2, HVT)• Primers:MDV serotype 1 (gB)MDV serotype 2 (pol2)MDV serotype 3 HVT gene (spanning internal repeat regions)Chicken genome (GAPDH)• Statistical analysis: Correlation test <strong>of</strong> Pearson


Material and Methods


Real <strong>time</strong> <strong>PCR</strong>Ratio=Spt1Ratio=Stp2Ratio=Stp3Ct(GAPDH)Ct(gB)Ct(GAPDH)Ct(pol2)Ct(GAPDH)Ct(HVT)ThresholdCt


Use <strong>of</strong> FTA cards to measure viral DNA load(serotype 1) in blood and feather pulpFTA®GAPDH/gB FTA = .01209 + .99970 * GAPDH/gB orgnl.Correlation: r = .99987 p


Use <strong>of</strong> FTA cards to measure viral DNA load(serotype 3) in blood and feather pulpFTA®1.21.00.80.60.40.20.0GPDH/HVT FTA = .29615 + .09697 * GAPDH/HVT org.Correlation: r = .0852856 56 <strong>samples</strong> <strong>samples</strong>1.41.21.00.80.60.40.20.0GAPDH/HVT FTA = -.1043 + .94784 * GAPDH/HVT orgCorrelation: r = .49598 p


Use <strong>of</strong> FTA cards to measure viral DNA load(serotype 1) in tumors2.2GPDH/gB FTA = .10263 + .80534 * GPDH/gB orgCorrelation: r = .80333 p


Conclusion• Correlation between the load <strong>of</strong> MDV DNA inoriginal <strong>samples</strong> and <strong>samples</strong> taken in FTA®cards is statistically significant for all threeserotypes. Exception HVT in blood


Practical applications• Imprints <strong>of</strong> tumors taken in FTA® cards can beused for the diagnosis <strong>of</strong> MD• Samples <strong>of</strong> blood taken in FTA® cards can be usedfor early diagnosis <strong>of</strong> MD (wild type virus) and formonitoring serotype 2 MD vaccines• Samples <strong>of</strong> feather pulp taken in FTA® cards canbe used for monitoring serotypes 1, 2, and 3


AcknowledgmentsRicardo CortesTom CiminoThis study was funded <strong>by</strong> Merial Select Inc.


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