RESUMEN
Background: Coxsackie B viruses (genus, Enterovirus; family, Picornaviridae) can cause aseptic meningitis, encephalitis, pleurodynia, and fatal myocarditis, and are implicated in the pathogenesis of dilated cardiomyopathy. The differentiation of the group B Coxsackieviruses into their subtypes has potential clinical and epidemiological implications. Objective: In this study, we developed a one-step, single-tube genogroup-specific reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay for the detection of group B Coxsackie genomes targeting 5′ UTR region. Materials and Methods: The amplification can be obtained in less than 1 hour by incubating all the reagents in a single tube with reverse transcriptase and Bst DNA polymerase at 63°C. Detection of gene amplification could be accomplished by agarose gel electrophoresis and the monitoring of gene amplification can also be visualised with the naked eye by using SYBR green I fluorescent dye. Results: A total of 40 samples comprising 31 positive samples and 9 negative samples were used in this study for comparative evaluation. The results were compared with those from Real-Time Polymerase Chain Reaction (RT-PCR). None of the RT-PCR-positive samples were missed by RT-LAMP, thereby indicating a higher sensitivity of the RT-LAMP assay. Conclusion: Thus, due to easy operation without a requirement of sophisticated equipment and skilled personnel, the RT-LAMP assay reported here is extremely rapid, cost-effective, highly sensitive, and specific and has potential usefulness for rapid detection of non-polio enterovirus (NPEV) not only by well-equipped laboratories but also by peripheral diagnostic laboratories with limited financial resources in developing countries.
Asunto(s)
Técnicas de Laboratorio Clínico/economía , Técnicas de Laboratorio Clínico/métodos , Infecciones por Coxsackievirus/diagnóstico , Electroforesis en Gel de Agar , Enterovirus Humano B/genética , Enterovirus Humano B/aislamiento & purificación , Humanos , Técnicas de Amplificación de Ácido Nucleico/economía , Técnicas de Amplificación de Ácido Nucleico/métodos , Compuestos Orgánicos/metabolismo , ARN Viral/genética , ARN Viral/aislamiento & purificación , Sensibilidad y Especificidad , Coloración y Etiquetado/métodos , Temperatura , Factores de TiempoRESUMEN
The level of interferon (IFN) in serum and homogenized cardiac tissue, and the activity of natural killer cells in Coxsackievirus B3 induced myocarditis in BALB/c mice were determined. NK cell cytolytic activity and IFN titers peaked on day 3~ 7 postinoculation (p.i.) and then declined. Virus titers in heart tissue reached a maximum on day 7 p.i. and then declined. These results suggest that the increase of NK cell activity and IFN titers provides some protection against Cox B, induced myocarditis by limiting virus replication in heart tissue.