Stable Yellowhead Virus (YHV) RNA Detection by qRT-PCR During Six-Day Storage
Coastal Sciences, Gulf Coast Research Laboratory
Storage conditions of haemolymph samples which contain yellowhead virus (YHV) may result in a decline of YHV RNA concentration or false-negative results in the detection of YHV. We evaluated the stability of YHV RNA in haemolymph stored at different temperatures for 6 d with conventional RT-PCR and TaqMan qRT-PCR. Specific pathogen free individuals of Litopenaeus vannamei were challenged with YHV92TH isolate, and haemolymph samples of 3 groups of 10 pooled moribund shrimp were aliquoted and stored at 4 and 25 degrees C for 0, 2,6,12,24,48,72,96,120, and 144 h. All samples were evaluated by conventional RT-PCR and qRT-PCR. After the optimization of experimental conditions, TaqMan qRT-PCR showed a very strong linear relationship between the log scale of the standard DNA copy number and the C-T values (R-2 = 0.999) over a 7-log range from 10(2) to 10(8) copy number per reaction. Even though the haemolymph was stored at either 4 or 25 degrees C for a 6-day period, the viral load number at 4 degrees C was not significantly different from that stored at 25 degrees C. The only difference was between the samples stored for 144 h at either 4 or 25 degrees C and those stored at -80 degrees C. We conclude that shrimp haemolymph can be drawn from shrimp at farms or in the wild and stored at either 4 or 25 degrees C for 3-5 d without a significant reduction in measured YHV RNA levels and without having to immediately freeze the samples. (C) 2008 Elsevier B.V. All rights reserved.
Overstreet, R. M.,
Jovonovich, J. A.
(2008). Stable Yellowhead Virus (YHV) RNA Detection by qRT-PCR During Six-Day Storage. Aquaculture, 278(1-4), 10-13.
Available at: http://aquila.usm.edu/fac_pubs/1479