RESUMEN
The whole-genome sequencing of coxsackievirus (CV)-A10 does not follow a conventional experimental protocol. To fully understand the genetic variation and evolution of CV-A10, complete genome amplification is necessary. Most previous studies have concentrated on partial sequences of the CV-A10 genome, such as the VP1 gene. The few studies that have investigated CV-A10 at the genomic level have reported only two complete genome sequences to GenBank. The basic fault may be attributed to the regional nature of the genetics and evolution of CV-A10 and to the lack of laboratory procedures for obtaining the genomes. In this study, we present a robust "three-step" protocol performed with A105UF/A820, EVP4/A6141, and A4879/A1005R for the full-length genome amplification of CV-A10. The results revealed that the method is able to accurately and reproducibly amplify three fragments with overlaps of the full-length genome of eight CV-A10 strains. Compared with other methods, this assay is both quick and specific. In addition, the three-step protocol could be capable of amplifying the full-length genomes of CV-A10 strains isolated from different countries and regions. The specific three-step protocol may be particularly useful for investigating samples co-infected with CV-A10 and other viruses.
Asunto(s)
Enterovirus Humano A/genética , Genoma Viral , ARN Viral/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/métodos , Mapeo Cromosómico , Cartilla de ADN/síntesis química , Cartilla de ADN/genética , GenotipoRESUMEN
Enterovirus 71 (EV71) and coxsackievirus A16 (CA16) have been the primary causative agents of hand, foot, and mouth disease (HFMD) outbreaks in mainland China in the past. Hence, the surveillance of HFMD has mostly focused on these viruses. However, in recent years, coxsackievirus A10 (CA10) has also been associated with the increasing sporadic HFMD cases and outbreaks. Therefore, a sensitive assay for rapid detection of the CA10 RNA is necessary for disease control. Here, we have developed a specific TaqMan real-time RT-PCR assay by analyzing VP1 gene sequences of CA10 strains from different locations. The assay has been shown to be specific, sensitive, and robust through detection of other related viruses, standard curves, and clinical samples, respectively. This is the first report on development of a VP1 gene-based TaqMan real-time RT-PCR assay for rapid diagnosis of CA10 virus.