Detection of hepatitis A virus in shellfish by nested reverse transcription-PCR

• Published in: International journal of food microbiology Vol. 48; no. 1; pp. 67 - 71
• Main Authors: Croci, L, De Medici, D, Morace, G, Fiore, A, Scalfaro, C, Beneduce, F, Toti, L
• Format: Journal Article
• Language: English
• Published: Amsterdam Elsevier B.V 01-04-1999; Elsevier
• Subjects: Animals; Base Sequence; Biological and medical sciences; Bivalvia - virology; COQUILLAGES ET CRUSTACES; DIAGNOSIS; DIAGNOSTIC; DIAGNOSTICO; DNA Primers - chemistry; Electrophoresis, Agar Gel; Fish and seafood industries; Food industries; Food Microbiology; Fundamental and applied biological sciences. Psychology; GENERO HUMANO; GENRE HUMAIN; Guanidines - chemistry; guanidinium isothiocyanate; HAV; HEPATITE; HEPATITIS; Hepatitis A - prevention & control; Hepatitis A virus; Hepatovirus - genetics; Hepatovirus - isolation & purification; Isothiocyanates - chemistry; MANKIND; MARISCOS; METHODE; METHODS; METODOS; Mollusca; Mussels; Nested-RT-PCR; PCR; polymerase chain reaction; Reverse Transcriptase Polymerase Chain Reaction - methods; RNA, Viral - analysis; RNA, Viral - isolation & purification; Sensitivity and Specificity; Sequence Analysis, DNA; SHELLFISH; Shellfish - virology; Water Microbiology; HAV; Nested-RT-PCR; Mussels; Virus; Polymerase chain reaction; Hepatitis A virus; Microbiological testing; Shellfish; Picornaviridae; Pathogenic; Reverse transcription; Hepatovirus
• ISSN: 0168-1605; 1879-3460
• DOI: 10.1016/S0168-1605(99)00028-8

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 TCID
50/10 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 TCID
50/10 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.