RESUMEN
A PCR assay for the detection of hepatitis A virus (HAV) in shellfish is described. The procedure involves the concentration of viral particles with the use of polyethylene glycol (PEG), followed by viral RNA extraction and purification with oligo(dT) cellulose. Reverse transcriptase-PCR detection was accomplished in a single step with the use of primers specific for the VP3-VP1 region of the genome. The procedure detected one 50% tissue culture infective dose (0.6 PFU) per 25 g of shellfish homogenate. Heminested PCR was then carried out to verify the specificity of the PCR products. The method was used to detect HAV in shellfish samples from EU categories B and C and to evaluate the quality of shellfish in routine monitoring for HAV in view of the relevant public health implications of this foodborne disease.
Asunto(s)
Bivalvos/microbiología , Virus de la Hepatitis A/aislamiento & purificación , ARN Viral/aislamiento & purificación , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/métodos , Mariscos/microbiología , Animales , Seguridad de Productos para el Consumidor , Virus de la Hepatitis A/genética , Sensibilidad y Especificidad , Factores de TiempoRESUMEN
The hepatitis A virus (HAV) is the most common cause of viral infection linked to shellfish consumption. The lack of correlation between the fecal coliform indicators and the presence of enteric viruses in shellfish and their harvesting waters points to the need for molecular methods to detect viruses. We compared two RT-PCR based techniques currently available for the detection of the hepatitis A virus (HAV) in shellfish. Both approaches involve extraction of viral particles by glycine buffer and concentration of virus particles by one or two PEG precipitation steps. One procedure involves as RNA extraction method the use of oligo (dT) cellulose to select poly (A) RNA, and the other uses a system in which total RNA is bound on silica membrane. Comparison of the two RT-PCR based methods highlighted the efficiency of the first approach which is less time-consuming and technically demanding than the second.
Asunto(s)
Bivalvos/virología , Virus de la Hepatitis A/genética , Hepatitis A/prevención & control , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/métodos , Mariscos/virología , Animales , Hepatitis A/transmisión , Virus de la Hepatitis A/aislamiento & purificación , Membranas Artificiales , ARN Mensajero/análisis , ARN Mensajero/aislamiento & purificación , ARN Viral/análisis , ARN Viral/aislamiento & purificación , Sensibilidad y Especificidad , Dióxido de SilicioRESUMEN
New promising and versatile materials for the development of in situ sustained release systems consisting of thin films of either poly(2-hydroxyethyl methacrylate) or a copolymer based on poly(ethylene-glycol diacrylate) and acrylic acid were investigated. These polymers were electrosynthesized directly on titanium substrates and loaded with ciprofloxacin (CIP) either during or after the synthesis step. X-ray photoelectron spectroscopy was used to check the CIP entrapment efficiency as well as its surface availability in the hydrogel films, while high-performance liquid chromatography was employed to assess the release property of the films and to quantify the amount of CIP released by the coatings. These systems were then tested to evaluate the in vitro inhibition of methicillin-resistant Staphylococcus aureus (MRSA) growth. Moreover, a model equation is proposed which can easily correlate the diameter of the inhibition haloes with the amount of antibiotic released. Finally, MG63 human osteoblast-like cells were employed to assess the biocompatibility of CIP-modified hydrogel coatings.
Asunto(s)
Ciprofloxacina/farmacología , Materiales Biocompatibles Revestidos/síntesis química , Técnicas Electroquímicas/métodos , Hidrogel de Polietilenoglicol-Dimetacrilato/síntesis química , Infecciones Relacionadas con Prótesis/prevención & control , Titanio/efectos adversos , Antibacterianos/farmacología , Adhesión Celular/efectos de los fármacos , Movimiento Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Cromatografía Líquida de Alta Presión , Humanos , Cinética , Staphylococcus aureus Resistente a Meticilina/efectos de los fármacos , Pruebas de Sensibilidad Microbiana , Microscopía Fluorescente , Osteoblastos/citología , Osteoblastos/efectos de los fármacos , Osteoblastos/ultraestructura , Espectroscopía de Fotoelectrones , Infecciones Relacionadas con Prótesis/microbiologíaRESUMEN
This review describes the ecological, clinical and epidemiological features of emerging vibrios and discusses what laboratory methods are being used for the detection of pathogenic vibrios in clinical, environmental and food samples. After selecting articles illustrative of the current scientific research on pathogenic vibrios, the review focuses on the need for better insight into the risk factors of emerging infections to establish adequate prevention procedures.