Development of a New Internally Controlled One-Step Real-Time RT-PCR for the Molecular Detection of Enterovirus A71 in Africa and Madagascar

Figures

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  FIGURE 1. Sensitivity of the real-time RT-PCR for detecting EV-A71 C4-SEP06 RNAs. The cloned 1DVP1 gene of the C4-SEP06 strain was transcribed in vitro and then subjected to serial 10-fold dilutions from 106 to 0.1 copies/μl in nuclease-free water. The linear regression line is shown with its associated correlation coefficient (R2 value) and its amplification efficiency (shown as a percentage), as given by ABI 7500 software. The negative control (Nuclease-free water) is shown in blue.

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  FIGURE 2. Mutual interference between EV-A71 and IC. (A) RNA genomes were extracted from 10-fold serially diluted C4-SEP06 viral suspensions (from 106 to 0.1 TCID50/ml in DMEM medium) with (green curves) or without (red curves) a fixed amount of IC RNA (105 RNA copies). They were then subjected to amplification in the same duplex real-time RT-PCR. The linear regression line is shown with its associated correlation coefficient (R2 value) and the amplification efficiency (shown as a percentage), as given by the ABI 7500 software. The negative control (DMEM medium) is shown in blue. (B) The IC RNA curves corresponding to the green EV-A71 RNA curves in panel (A).

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  FIGURE 3. Specificity and sensitivity of the real-time RT-PCR assay for different EV-A71 genogroups. (A–E) RNA genomes were extracted from 10-fold serial dilutions of EV-A71 suspensions (from 103 to 1 TCID50/ml in DMEM medium) in the presence of a fixed amount of IC (105 RNA copies), and tested. EV-A71 template (red), negative control (DMEM medium, blue), only EV-A71 amplification is represented. (F) RNA was extracted from undiluted suspensions of reference strains of other enteroviruses in the presence of a fixed amount of IC (105 RNA copies) and tested. Coxsackieviruses A2, A3, A4, A6, A7, A8, A10, A12, A14, A16, coxsackieviruses B1–B6, echoviruses 4, 6, 11, 13, 25, and 30, poliovirus-1 (Sabin strain), EV-D68 and EV-A71 were tested. Enterovirus RNA curves are shown in red, IC curves are shown in green, and the negative control is shown in blue. For clarity, the EV-A71 and IC amplifications are represented on the same graph.

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  FIGURE 4. Application of the real-time RT-PCR for the detection of EV-A71 in stool specimens. Suspensions of stool specimens (n = 10) were spiked with C4-SEP06 EV-A71 (final concentration 101 TCID50/ml) and a fixed amount of IC (105 RNA copies) was added. RNA was then extracted and amplified by real-time RT-PCR. The original C4-SEP06 EV-A71 suspensions at 104 and 101 TCID50/ml in PBS were used as positive control. The stool specimens and PBS without spiking were used as negative controls. Amplification curves for EV-A71 RNA templates and the IC control from the same experiments are shown in panel (A) and (B), respectively.

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  FIGURE 5. Application of the real-time RT-PCR for the detection of EV-A71 in plasma-EDTA samples. Plasma samples (n = 10) were spiked with fixed amounts of C4-SEP06 EV-A71 (final concentration 103 TCID50/ml) and IC (105 RNA copies) was added. PBS alone and C4-SEP06 (103 TCID50/ml) in PBS were used as negative and positive controls, respectively. RNA was extracted and amplified by real-time RT-PCR. EV-A71 RNA curves are shown in (A) and the corresponding IC curves are shown in panel (B).

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  FIGURE 6. Phylogenetic relationships of some identified EV-A71 isolates. Phylogenic tree based on the complete VP1 sequence of EV-A71 retrieved from genbank database (n = 203 sequences), EV-A71 (sub)genogroups are denoted by colored lines (A). Details of the subgenogroups C1 (B), C2 (C), and C4 (D) are shown. The recently identified EV-A71 isolates from Algeria, Tunisia, and Madagascar are indicated by a red dot. EV-A71 from the subgenogroup C1 previously reported in Cameroon and EV-A71 from the subgenogroup C2 previously reported in Senegal, Mauretania and Niger are denoted with orange branches and squares. The evolutionary distances were computed by Neighbor Joining analysis using the Kimura-2-parameter method with 1000-Bootstrap replicates to estimate node consistencies. Only percent of bootstrap >75 are indicated. The length of the branch is proportional to the number of nucleotide divergence.