Detection of hepatitis A virus in shellfish by nested reverse transcription-PCR.
A method for the detection of HAV in shellfish, based on the use of guanidinium isothiocyanate-containing solution for RNA extraction and purification steps, followed by nested PCR, is hereby proposed. Tests were carried out on mollusc samples spiked with HAV strain FG. Results showed that in samples subjected only to one round of PCR it was possible to detect HAV at concentrations of 10(3)-10(4) TCID50/10 g of mollusc. The use of the nested PCR renders the system more sensitive and specific enabling the identification of HAV concentrations as low as 1 TCID50/10 g of mollusc. Furthermore thus method, in addition to allowing the avoidance of confirming tests, such as hybridization, proved to be inexpensive and simple to perform.
Croci
L
L
Laboratorio Alimenti, Istituto Superiore di Sanità, Rome, Italy. l.croci@iss.it
De Medici
D
D
Morace
G
G
Fiore
A
A
Scalfaro
C
C
Beneduce
F
F
Toti
L
L
D000818:Animals; D001483:Base Sequence; D049872:Bivalvia / Q000821:virology*; D017931:DNA Primers / Q000737:chemistry; D004587:Electrophoresis, Agar Gel; D005516:Food Microbiology; D006146:Guanidines / Q000737:chemistry; D006506:Hepatitis A / Q000517:prevention & control; D006507:Hepatovirus / Q000235:genetics / Q000302:isolation & purification*; D017879:Isothiocyanates / Q000737:chemistry; D012367:RNA, Viral / Q000032:analysis / Q000302:isolation & purification; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D017422:Sequence Analysis, DNA; D012758:Shellfish / Q000821:virology*; D014871:Water Microbiology
D017931:DNA Primers; D006146:Guanidines; D017879:Isothiocyanates; D012367:RNA, Viral; C054435:guanidine isothiocyanate