MS2 titer and EMPA or PMPA concentrations after separation of artificial CB mixed sample n°2.

<p>The CB mixed sample n°2 consisted of a fixed amount of MS2 bacteriophage (5×10⁷ genome copies in 20 µL) mixed with 25 µg/ml of EMPA and PMPA and either spiked in a liquid, sand or soil matrix. After filtration through a 30 kD filtration device, MS2 bacteriophage titers and EMPA and PMPA concentrations were measured by qPCR and LC-TOFMS, respectively. RNA extraction was carried out using NucliSens kit. (A) Mean Ct and SD (n = 3) values in the filtrate and in the unfiltered control. (B) and (C) Average concentrations and SD (n = 3) of EMPA and PMPA compounds in the filter retained fraction and in the unfiltered control.</p

Standard curves.

<p>qPCR standard plasmid DNA dilution curves from: (A) recombinant MS2 bacteriophage (pCR4-MS2), (B) AcNPV (pCR4-AcNPV). Mean Ct values (Y axis) are plotted against logarithm (log₁₀) of virus genome copies number. Each data point represents Ct average with standard deviation (SD, n = 3).</p

Quantification methods of biological (MS2 and AcNPV) viral agents and chemical (EMPA and PMPA) compounds: results and Coefficient of Variation (CV).

<p>nd = not determined (technical replicates not used in the experiment).</p

MS2 titers and PEG-400 concentrations after separation of CB mixed sample n°1.

<p>A fixed amount of MS2 bacteriophage (5×10⁸ genome copies in 20 µL) was mixed with increasing amounts of PEG-400. After filtration through a 30 kD filtration device, MS2 bacteriophage titers and PEG-400 concentration were measured by qPCR and LC-TOFMS, respectively. RNA extraction was carried out using NucliSens kit. (A) Mean Ct and SD (n = 3) values in filtrate and retained fractions. (B) Average percentages and SD (n = 3) measured in filtrate and retained fractions. (C) Mean percentage and SD (n = 3) of MS2 RNA recovery from filter and retained fractions.</p

Comparative efficiency of MS2 and AcNPV viral simulants recovery.

<p>Recovery and extraction efficiencies were assessed by qPCR. Tris-Mg-Ca buffer (A and B) is used to recover viruses spiked in different matrices and to extract nucleic acids using different extraction kits, as indicated. One-tailed Wilcoxon test was used to assess if extraction efficiency was significantly lower with NucliSens and Invisorb as compared to EZ1 extraction kit (*p = 0.05). Mean percentage and SD (n = 3) of MS2 RNA (A) and AcNPV DNA extraction efficiency (B) from a liquid, sand or soil matrix after data normalization with the EZ1 extraction kit. (C and D) virus recovery and extraction from a soil matrix using NucliSens extraction kit, and assessing the impact of different types of recovery buffers, as indicated (one-tailed Wilcoxon, *p = 0.05). (C) Mean percentage and SD (n = 3) of MS2 RNA extraction comparing the effect of glycine and Tris-Mg-Ca recovery buffers. (D) Mean percentage and SD (n = 3) of AcNPV DNA extraction: for virus recovery, glycine buffer alone was compared to glycine buffer supplemented with BSA, NaCl or both.</p

Blotting papers showing six 6 spots with dried liquid exudate from pustules collected in one patient.

<p>One spot (arrow) was removed and processed for DNA-based identification of causative agents of the rash illness.</p

Map of West Kasai Province.

<p>Areas affected by rash illness outbreaks are highlighted as follows: ▴: Bena Tshiadi healthcare zone (13 patients in 7 different villages). All cases were confirmed as monkeypox cases. Hourglass Symbol: Yangala healthcare zone (8 patients clustered in Tshikongo village). All were confirmed as varicella cases.♦: Ndesha healthcare zone in the outskirts of Kananga (3 patients clustered in Lubuyi village, several kilometers north of Kananga city). All were confirmed as varicella cases.</p

Multiple alignment of 14 kD DNA targets and design of primers and probes for the qPCR assay.

<p>Primers for qPCR and for the conventional PCR are shown in plain boxes whereas the pan-orthopoxvirus and specific variola virus probes are highlighted in a dashed boxes. The monkepoxvirus specific SNP (C→T) is arrowed. The pyrosequencing probe is indicated by a plain arrow.</p

DNA-based identification of causative agents of rash illness outbreaks (2008 and 2009) in West Kasai province.

<p>Legend: Patients originated from the following healthcare districts: Bena Tshiadi, 1–13; Yangala, 14–22; Ndesha, 23–25.</p

Figure 4

<p><b>A)</b> Gel electrophoresis of the conventional PCR amplification products showing the undigested 384-bp OPV amplicon DNA ladder (lane 1); negative controls (lanes 2–3); human specimens from 13 patients of the Bena Tshiadi healthcare district (lanes 4–13 and 15–18), and from 1 patient of the Yangala healthcare district (lane 14). <b>B)</b><i>BsrGI</i> digestion profile of 384-bp MPXV amplicons: banding patterns with 210- and 174-bp fragments.</p