RESUMO
INTRODUCTION: Virus discovery based on cDNA-AFLP (VIDISCA) is a sequence-independent virus discovery method that was recently developed and successfully used to characterize unknown viruses in cell cultures. Its applicability, however, is limited by its low sensitivity. METHODOLOGY: We evaluated whether the introduction of prior amplification of target sequences by random PCR (rPCR) increases the sensitivity of this method to improve its use on clinical specimens. In addition, ultracentrifugation was added to the protocol to allow for pooling of multiple samples, thereby increasing analytical throughput of the VIDSCA. RESULTS: We showed that rPCR enhanced the sensitivity of VIDISCA by 100-fold for two out of four viruses in different clinical samples, and that the ultracentrifugation step allowed for analyzing samples of large volumes (4 ml) and simultaneous processing of multiple (~40) clinical specimens. CONCLUSIONS: We conclude that this modified VIDISCA protocol is a relatively easy method to use for screening of large numbers of clinical samples that are suspected to contain previously unrecognized pathogens, in settings where ultradeep sequencing platforms are not available.
Assuntos
Análise do Polimorfismo de Comprimento de Fragmentos Amplificados/métodos , Reação em Cadeia da Polimerase/métodos , Viroses/diagnóstico , Viroses/virologia , Vírus/isolamento & purificação , Sequência de Bases , DNA Complementar/genética , DNA Viral/genética , Desoxirribonucleases de Sítio Específico do Tipo II/metabolismo , Humanos , Programas de Rastreamento/métodos , Dados de Sequência Molecular , RNA Viral/genética , RNA Viral/isolamento & purificação , Sensibilidade e Especificidade , Ultracentrifugação , Virologia/métodos , Vírus/genéticaRESUMO
Dengue haemorrhagic fever (DHF) is showing an increasing trend in Viet Nam ever since its detection in 1959. Both north and south Viet Nam though are equally endemic, but they show seasonal variations. While in north Viet Nam the peak incidence is from June to November and then there is interruption of transmission due to winter, in south Viet Nam the transmission occurs throughout the year. Realizing the need for making organized efforts for the control of DF/DHF, the Ministry of Health established a National Programme for Dengue Prevention and Control in 1997. The key elements of the programme include: (i) active surveillance; (ii) emergency response; (iii) longterm vector control, and (iv) clinical diagnosis and management. An action plan for the years 1997-1998 covering the whole country has been put into operation.