Simplified procedure for detection of enteric pathogenic viruses in shellfish by RT-PCR.
Epidemiological evidence linking the transmission of enteric viral disease to shellfish has been known for a long time. A variety of methods have been described for the detection of viral contaminants in shellfish using RT-PCR. However, these methods generally include numerous, often fastidious and time consuming steps for virus release from shellfish tissues and viral RNA isolation. A simplified procedure based on the enzymatic liquefaction of shellfish digestive tissues without any mechanical homogenisation step, followed by a simple clarification of the lysate using dichloromethane extraction, was developed. Viral RNA is isolated directly from the shellfish extract by a guanidium thiocyanate-silica extraction method, adapted for the use of a vacuum manifold system. Virus-specific RT-PCR assays were set up for detection of genomic sequences of the predominant viral pathogens, HAV, Astrovirus and Norwalk-like viruses (from genogoups I or II). The specificity of the amplicons is confirmed finally by hybridisation with DIG-labelled specific probes. The overall procedure applied to shellfish samples spiked with HAV particles allowed a detection of 20 pfu of HAV per g of hepatopancreas. In addition, up to 20 samples can be tested within 24 h.
Legeay
O
O
Service R&D, Institut Pasteur de Lille, 1 rue du Professeur Calmette, BP 245, 59019 Cedex, Lille, France.
Caudrelier
Y
Y
Cordevant
C
C
Rigottier-Gois
L
L
Lange
M
M
D000818:Animals; D049872:Bivalvia / Q000821:virology; D006507:Hepatovirus / Q000235:genetics / Q000302:isolation & purification; D015151:Immunoblotting; D001257:Mamastrovirus / Q000235:genetics / Q000302:isolation & purification; D009663:Norwalk virus / Q000235:genetics / Q000302:isolation & purification; D010125:Ostreidae / Q000821:virology; D012328:RNA Viruses / Q000235:genetics* / Q000302:isolation & purification*; D012367:RNA, Viral / Q000032:analysis / Q000302:isolation & purification; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012680:Sensitivity and Specificity; D012758:Shellfish / Q000821:virology*
D012367:RNA, Viral