RESUMEN
This study aimed to develop a specific and sensitive reverse transcription polymerase chain reaction enzyme-linked immunosorbent assay (RT-PCR-ELISA) for detecting hepatitis E virus (HEV). Eight sets of primers and biotinylated probes designed in the ORF2-ORF3 overlapping region of HEV were tested for sensitivity. The ability of nested reverse transcription polymerase chain reaction (RT-PCR) and RT-PCR-ELISA to detect HEV was compared. RT-PCR-ELISA was 10-100 times more sensitive than nested RT-PCR and could detect 0.01 ng/µl HEV in swine stool samples. In terms of specificity, RT-PCR-ELISA did not falsely detect HEV when other viruses such as hepatitis A virus, rotavirus, norovirus genotype I, norovirus genotype II, and Feline calicivirus were present. Therefore, RT-PCR-ELISA appears to be a sensitive and specific method for detecting HEV.
Asunto(s)
Ensayo de Inmunoadsorción Enzimática/métodos , Virus de la Hepatitis E/aislamiento & purificación , Hepatitis E/virología , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/métodos , Enfermedades de los Porcinos/virología , Animales , Biotinilación , Cartilla de ADN/genética , Ensayo de Inmunoadsorción Enzimática/normas , Heces/virología , Virus de la Hepatitis E/genética , Virus de la Hepatitis E/inmunología , Humanos , ARN Viral/análisis , ARN Viral/genética , ARN Viral/aislamiento & purificación , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/normas , Sensibilidad y Especificidad , PorcinosRESUMEN
Hepatitis A is a major infectious disease epidemiologically associated with foodborne and waterborne outbreaks. Molecular detection using real-time RT-PCR to detect the hepatitis A virus (HAV) in contaminated vegetables can be hindered by low-virus recoveries during the concentration process and by natural PCR inhibitors in vegetables. This study evaluated three virus concentration methods from vegetables: polyethylene glycol (PEG) precipitation, ultrafiltration (UF), and immunomagnetic separation (IMS). UF was the most efficient concentration method, while PEG and IMS were very low for the recovery rate of HAV. These results demonstrate that UF is the most appropriate method for recovering HAV from contaminated vegetables and that this method combined with the real-time RT-PCR assay may be suitable for routine laboratory use.
Asunto(s)
ADN Viral/análisis , Filtración/métodos , Microbiología de Alimentos , Genoma Viral , Virus de la Hepatitis A , Hepatitis A/virología , Verduras/virología , Brotes de Enfermedades , Enfermedades Transmitidas por los Alimentos/virología , Humanos , Magnetismo/métodos , Polietilenglicoles , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/métodosRESUMEN
This study aimed to develop a specific and sensitive duplex reverse transcription polymerase chain reaction enzyme-linked immunosorbent assay (duplex RT-PCR-ELISA) for hepatitis A virus (HAV) and hepatitis E virus (HEV). Duplex RT-PCR-ELISA could detect and differentiate HAV and HEV with specific probes. When ELISA technique was used to detect probe-bound RT-PCR products, duplex RT-PCR-ELISA could detect as little as 0.1 ng/µL HAV and HEV from clinical samples. Human norovirus, enterovirus, poliovirus, murine norovirus and feline calicivirus were used for the specificity test; all were negative. Therefore duplex RT-PCR-ELISA can be used for the simultaneous detection of HAV and HEV in contaminated fecal samples.
Asunto(s)
Ensayo de Inmunoadsorción Enzimática/métodos , Virus de la Hepatitis A/aislamiento & purificación , Hepatitis A/virología , Virus de la Hepatitis E/aislamiento & purificación , Hepatitis E/virología , Reacción en Cadena en Tiempo Real de la Polimerasa/métodos , Virus de la Hepatitis A/genética , Virus de la Hepatitis E/genética , HumanosAsunto(s)
Técnicas Biosensibles/métodos , Carbono/química , Inmunoensayo/métodos , Óxidos/química , Virus/aislamiento & purificación , Anticuerpos Inmovilizados/inmunología , Transferencia Resonante de Energía de Fluorescencia , Oro/química , Nanopartículas del Metal/química , Poliovirus/aislamiento & purificación , Rotavirus/aislamiento & purificación , Virus de la Viruela/aislamiento & purificaciónRESUMEN
The prevalence of asymptomatic norovirus (NoV) infection was investigated in children registered for kindergarten in Korea during the winter and summer. Children with no gastrointestinal symptoms, including diarrhea and abdominal pain, during the 2 weeks before and following sample collection were included in this investigation. NoV presence and genetic identification were determined with real-time reverse transcriptase-polymerase chain reaction and conventional nested reverse transcriptase-polymerase chain reaction. The prevalence of NoV in asymptomatic children was 5.5% in the winter and 3.5% in the summer, respectively. GII.4 was the most prevalent NoV genotype, but GII.2 and GI.10 were also found during genetic analysis. This study demonstrates that asymptomatic NoV infection may be an important source of transmission in kindergarten children.