Application of reverse transcriptase-nested-PCR for detection of poliovirus in mussels.
In order to identify polioviruses in molluscs, we hereby propose a method based on precipitation with PEG 6000 followed by the use of a commercial kit (RNAfast II-Molecular System-San Diego) for the extraction and purification of viral RNA. The RT-PCR phase is followed by a second amplification using nested primers to increase the sensitivity and specificity of the method. Tests were carried out on mollusc samples spiked with Poliovirus 1. Results showed that in samples subjected only to one round of PCR it was possible to detect Poliovirus concentrations as small as 10(3)TCID50/ml. The use of nested-PCR makes the system more sensitive and specific enabling the identification of Poliovirus concentrations as small as 1 TCID50/ml.
De Medici
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D
Laboratorio Alimenti, Istituto Superiore di Sanità, Roma, Italy. demedici@net.iss.it
Beneduce
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F
Fiore
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A
Scalfaro
C
C
Croci
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L
D000818:Animals; D049872:Bivalvia / Q000821:virology*; D017955:Poliovirus / Q000302:isolation & purification*; D016133:Polymerase Chain Reaction / Q000379:methods*; D012367:RNA, Viral / Q000032:analysis
D012367:RNA, Viral