RT-PCR detection of yellow head virus (YHV) infection in Penaeus monodon using dried haemolymph spots.
Sample collection and RNA isolation from shrimp haemolymph for RT-PCR diagnosis of yellow head virus (YHV) infections is crucial for disease control programs for cultivated shrimp in Thailand. Problems with RNA degradation arise when field samples must be collected far from the laboratory by relatively inexperienced personnel who do not have ready access to sophisticated reagents. In an attempt to solve this problem, haemolymph samples from shrimp were collected either by mixing with 10% (w/v) sodium citrate or by spotting on ISOCODE filter paper. RNA was extracted subsequently either by a rapid boiling method or by using TRI reagent and the extracts were used in a semi-quantitative, non-stop, semi-nested RT-PCR assay for YHV. Dried haemolymph spots on ISOCODE filter paper extracted with TRI reagent gave the most reliable and reproducible results. It also allowed longer periods of storage at room temperature.
Kiatpathomchai
Wansika
W
National Center for Genetic Engineering and Biotechnology (BIOTEC), National Science and Technology Development Agency, Pathumthani, Thailand.
Jitrapakdee
Sarawut
S
Panyim
Sakol
S
Boonsaeng
Vichai
V
D000818:Animals; D001483:Base Sequence; D004279:DNA, Viral / Q000235:genetics; D006458:Hemolymph / Q000821:virology; D028381:Nidovirales / Q000235:genetics* / Q000302:isolation & purification*; D030341:Nidovirales Infections / Q000175:diagnosis / Q000662:veterinary* / Q000821:virology; D033561:Penaeidae / Q000821:virology*; D020871:RNA Stability; D012367:RNA, Viral / Q000235:genetics / Q000302:isolation & purification; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014773:Virology / Q000379:methods
D004279:DNA, Viral; D012367:RNA, Viral