Stable Yellowhead Virus (YHV) RNA Detection by qRT-PCR during Six-Day Storage

Authors

• Hongwei Ma, University of Southern Mississippi
• Robin M. Overstreet, University of Southern MississippiFollow
• Jean A. Jovonovich, University of Southern MississippiFollow

Document Type

Article

Date of this Version

6-2008

Citation

Aquaculture 278:1–4 (June 2008), pp. 10–13.

doi: 10.1016/j.aquaculture.2008.03.028

Comments

Copyright © 2008 Elsevier B.V. Creative Commons Attribution Non-Commercial No Derivatives License.

Abstract

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 C_T 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.