Stable yellowhead virus (YHV) RNA detection by qRT-PCR during six-day storage

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 °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 CT values (R2 = 0.999) over a 7-log range from 102 to 108 copy number per reaction. Even though the haemolymph was stored at either 4 or 25 °C for a 6-day period, the viral load number at 4 °C was not significantly different from that stored at 25 °C. The only difference was between the samples stored for 144 h at either 4 or 25 °C and those stored at − 80 °C. We conclude that shrimp haemolymph can be drawn from shrimp at farms or in the wild and stored at either 4 or 25 °C for 3–5 d without a significant reduction in measured YHV RNA levels and without having to immediately freeze the samples.