SYBR Green Real-Time PCR for the Detection of All Enterovirus-A71 Genogroups

<div><p>Enterovirus A71 (EV-A71) has recently become an important public health threat, especially in South-East Asia, where it has caused massive outbreaks of Hand, Foot and Mouth disease every year, resulting in significant mortality. Rapid detection of EV-A71 early in outbreaks would facilitate implementation of prevention and control measures to limit spread. Real-time RT-PCR is the technique of choice for the rapid diagnosis of EV-A71 infection and several systems have been developed to detect circulating strains. Although eight genogroups have been described globally, none of these PCR techniques detect all eight. We describe, for the first time, a SYBR Green real-time RT-PCR system validated to detect all 8 EV-A71 genogroups. This tool could permit the early detection and shift in genogroup circulation and the standardization of HFMD virological diagnosis, facilitating networking of laboratories working on EV-A71 in different regions.</p></div

Neighbour-Joining tree of full EV-A71 VP1 sequences.

<p>Tree produced using Mega 5.05 software with Kimura-2 model with few full EV-A71 representatives of the 12 subgenogroups: A, B0, B1, B2, B3, B4, B5, C1, C2, C3, C4, C5 aligned using ClustalX 2.1. Bootstrap values (in percentage) were generated by using 1000 replicates. For each strain, the GenBank accession number, the country of origin (ISO 3166 code) and the year are indicated.</p

Dissociation curves of the EV-A71 SYBR Green RT-PCR performed on dilutions of plasmid B1.

<p>Dissociation curves of the EV-A71 SYBR Green RT-PCR performed on dilutions of plasmid B1.</p

Results in Ct of the SYBR Green real-time PCR with various primer concentrations.

<p>All plasmids, at 10⁻⁵ dilution each, were tested at Tm 45°C. ‘C’: number of plasmid copies use as template.</p

Results of SYBR Green real-time PCR on serial dilutions of each plasmids.

<p>Real-time PCR were performed with 2 µM of primers at Tm 50°C. Cells filled in bold indicate the biggest dilution for which both duplicate are PCR positive ( = limit of detection). ‘C’: number of plasmid copies use as template. ‘R’: PCR result, ‘+’: positive, ‘−’: negative.</p

Concentration of each subgenogroup plasmid extract.

<p>Concentration of each subgenogroup plasmid extract.</p

Results in Ct of the Taqman real-time PCR performed on plasmids A, B0 C1.

<p>Plasmids A, B0 and C1, at 10⁻⁵ dilution each, were tested with 2 µM of each primer at Tm 50°C and different concentrations of probe.</p

Results of SYBR Green real-time PCR performed on patient and culture samples.

<p>Results of SYBR Green real-time PCR performed on patient and culture samples.</p

Results in Ct of SYBR Green real-time PCR with Tm 44.6°C, 50.2°C and 55°C.

<p>All plasmids, at 10⁻⁵ dilution each, were tested with 2 µM of each primer. Cells filled in bold indicate lowest Ct value for each plasmid. ‘C’: number of plasmid copies used as template.</p

Consensus sequences, including <i>NotI</i> site, for 12 EV-A71 subgenogroups.

<p>Consensus sequences, including <i>NotI</i> site, for 12 EV-A71 subgenogroups.</p