RESUMO
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.
Assuntos
Ensaio de Imunoadsorção Enzimática/métodos , Vírus da Hepatite E/isolamento & purificação , Hepatite E/virologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Doenças dos Suínos/virologia , Animais , Biotinilação , Primers do DNA/genética , Ensaio de Imunoadsorção Enzimática/normas , Fezes/virologia , Vírus da Hepatite E/genética , Vírus da Hepatite E/imunologia , Humanos , RNA Viral/análise , RNA Viral/genética , RNA Viral/isolamento & purificação , Reação em Cadeia da Polimerase Via Transcriptase Reversa/normas , Sensibilidade e Especificidade , SuínosRESUMO
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.
Assuntos
DNA Viral/análise , Filtração/métodos , Microbiologia de Alimentos , Genoma Viral , Vírus da Hepatite A , Hepatite A/virologia , Verduras/virologia , Surtos de Doenças , Doenças Transmitidas por Alimentos/virologia , Humanos , Magnetismo/métodos , Polietilenoglicóis , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodosRESUMO
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.
Assuntos
Ensaio de Imunoadsorção Enzimática/métodos , Vírus da Hepatite A/isolamento & purificação , Hepatite A/virologia , Vírus da Hepatite E/isolamento & purificação , Hepatite E/virologia , Reação em Cadeia da Polimerase em Tempo Real/métodos , Vírus da Hepatite A/genética , Vírus da Hepatite E/genética , HumanosAssuntos
Técnicas Biossensoriais/métodos , Carbono/química , Imunoensaio/métodos , Óxidos/química , Vírus/isolamento & purificação , Anticorpos Imobilizados/imunologia , Transferência Ressonante de Energia de Fluorescência , Ouro/química , Nanopartículas Metálicas/química , Poliovirus/isolamento & purificação , Rotavirus/isolamento & purificação , Vírus da Varíola/isolamento & purificaçãoRESUMO
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.
