Comparisons of the prevalence of <i>T</i>. <i>whipplei</i> carriage in children feces between Senegal and Laos.

<p>Comparisons of the prevalence of <i>T</i>. <i>whipplei</i> carriage in children feces between Senegal and Laos.</p

Genetic diversity of 42 genotypes of <i>Tropheryma whipplei</i> obtained from 277 samples.

<p>Phylogenetic trees were constructed using the maximum likelihood method based on the Tamura 3-parameter substitution model. The countries in which the genotype has been detected are presented opposite the genotype, and the number of different individuals in which the genotype has been detected is provided in parentheses. The genotypes of each highly variable genomic sequence (HVGS) have intervened in 22, 13, 17 and 7 different combinations, respectively.</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

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

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

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

EV-A71 primers and probe sequences.

<p>In the first three lines is the system developed by Tan <i>et al.</i> (2008^b) and below the modified sequences that we used in this article for the detection of EV-A71.</p

Concentration of each subgenogroup plasmid extract.

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

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