High throughput detection of by real-time PCR with internal control system and automated DNA preparation-1

the y-axis. "+" in "cell isolation" means isolation success as confirmed by detection of inclusion bodies upon microscopy. B, box plot analysis of threshold cycle values in real-time PCR positive/conventional negative (n = 32) and real-time PCR positive/conventional PCR positive (n = 38) samples. Difference in threshold cycle values are significant (p < 0.05).<p><b>Copyright information:</b></p><p>Taken from "High throughput detection of by real-time PCR with internal control system and automated DNA preparation"</p><p>http://www.biomedcentral.com/1471-2180/8/77</p><p>BMC Microbiology 2008;8():77-77.</p><p>Published online 19 May 2008</p><p>PMCID:PMC2397412.</p><p></p

High throughput detection of by real-time PCR with internal control system and automated DNA preparation-2

Lood (x-axis). A, Qiagen DNA mini kit; B, Qiagen M48 DNA mini kit, used on a Qiagen M48 automated DNA extraction instrument. Each datum point represents the rate of positive results in six replicate tests per concentration. Limits of detection are comparable with both methods of DNA extraction. C, Threshold cycles (y-axis) as a measure of efficiency of PCR amplification for and internal control. Each reaction contained 15 copies of plasmid-derived target gene and variable numbers of internal control plasmid pCoxmimic, as depicted on the x-axis. Results of eight replicate real-time PCR reactions per setting are shown as a result of box-plot analysis, showing the range of results by whiskers, whereby the two central quartiles of data are represented as a box. Solid line with grey boxes, target gene, broken line with white boxes, internal control. No reduced efficiency in amplification is observed for the target gene in presence of up to 100 copies of internal control. D, Correlation of DNA copies per ml as determined by real-time PCR after automated (x-axis) and manual extraction procedure (y-axis).<p><b>Copyright information:</b></p><p>Taken from "High throughput detection of by real-time PCR with internal control system and automated DNA preparation"</p><p>http://www.biomedcentral.com/1471-2180/8/77</p><p>BMC Microbiology 2008;8():77-77.</p><p>Published online 19 May 2008</p><p>PMCID:PMC2397412.</p><p></p

Phylogenetic relationship of studied strains.

<p>Maximum parsimony tree using 1,713 bases in the dataset computed with PAUP* and rooted using Dugway (bold) as outgroup. Plasmid type, genomic group (GG), <i>adaA</i> types and the subgroups of the Q154-deletion type are shown next to the tree. Branch lengths correspond to the number of mutations. Animal strains are written in italic. IP: integrated plasmid; WT: wildtype; D_2.1: deletion 2.1; D₁: deletion 1. The figure was drawn using APE <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0053440#pone.0053440-Paradis1" target="_blank">[38]</a>.</p

Polymorphisms of the <i>adaA</i> genetic region demonstrated using seven <i>C. burnetii</i> reference genomes.

<p>Three deletion events resulting in two main deletion types (Q154 and Q212) and three <i>adaA</i> gene variants (<i>adaA</i>, <i>adaA</i>_SNP and <i>adaA</i>_rep) resulting from independent mutation processes were identified <i>in silico</i>. The organization of the <i>adaA</i> flanking region of the Dugway genome (displayed in the center) is compared to the other six reference genomes. Just for the purpose of visual compactness, the light orange region was pruned between the ORFs CBUD_1100 and CBUD_1122. Gene annotations are obtained from GenBank. Coding genes are drawn in blue, pseudogenes in white and the <i>adaA</i> gene in red. Arrow direction represents the location of the ORFs at the forward and backward strand. Locus tags are shortened by its common prefix and written above the ORFs. If gene names are known, they are written next to their locus tag and in parentheses. Long collinear blocks (LCBs) are shown as colored rectangles at the backbone of the Dugway genome. The pairwise syntenic regions between the genomes are drawn in lighter color, accordingly. The phylogenetic relationship is shown at the left side. Just for visualization purposes, the Dugway genome was rotated to the middle. The length of the branches encodes the number of mutation events. Three different deletions were identified and its affected LCBs are drawn to the phylogenetic branch where the deletion events may have occurred. Dugway and RSA331 were chosen as representative strains to show the intact <i>adaA</i> gene and <i>adaA</i>_SNP gene, respectively. A fragment of strain F10 is exposed in the figure to show the inserted repeat variant (<i>adaA</i>_rep). The figure was drawn using genoPlotR <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0053440#pone.0053440-Guy1" target="_blank">[37]</a>. *Q321 replaces RSA334, which was accidently assigned to the published whole genome data.</p

<i>adaA</i> genotypes of human <i>Coxiella burnetii</i> isolates from acute and chronic Q fever.

<p>Chronic isolates are listed from entry F1 onwards.</p>a<p>DNA was isolated from paraffin-embedded material.</p>b<p>no plasmid detected, but plasmid-related sequences present in genomic DNA.</p>c<p><i>adaA</i>: intact <i>adaA</i> gene (reference RSA493); <i>adaA</i>_SNP: <i>adaA</i> gene with A/T SNP at position 431; <i>adaA</i>_rep: <i>adaA</i> with 226 bp tandem repeat; Q154-del: Q154-type deletion of <i>adaA</i>; Q212-del: Q212-deletion type of <i>adaA.</i></p

<i>adaA</i> genotypes in 86 animal-derived <i>Coxiella burnetii</i> strains.

a<p>the plasmid type had been described elsewhere <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0053440#pone.0053440-Stein1" target="_blank">[6]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0053440#pone.0053440-Mallavia1" target="_blank">[9]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0053440#pone.0053440-Willems2" target="_blank">[40]</a>.</p>b<p><i>adaA</i>: intact <i>adaA</i> gene; <i>adaA</i>_SNP: <i>adaA</i> gene with A/T SNP at position 431; <i>adaA</i>_rep: <i>adaA</i> with 226 bp tandem repeat; Q154-del: Q154-type deletion of <i>adaA</i>; Q212-del: Q212-deletion type of <i>adaA</i> (see also <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0053440#pone-0053440-g001" target="_blank">Figure 1</a>). The GenBank accessions for the Q154-type deletion are JQ713146 (Z 3574), JQ713148 (Namibia), <a href="mailto:AAUP02000002@complement" target="_blank">AAUP02000002@complement</a> (10798.12509) (Priscilla Q177), and JQ713151 (Z 5a). The accession for the Q212-type deletion is JQ713157 (Z 11).</p

Sequence-based discrimination of the Q154 <i>adaA</i> gene deletion type.

<p>This special deletion type is defined by deletion 1 (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0053440#pone-0053440-g001" target="_blank">Figure 1</a>). Differential bases (SNPs and insertion) of analyzed strains are shown in reference to the published genome of Q154, position 834,296 to 836,007. Vertical numbers display the respective position relative to position in the genome of strain Q154. The large deletion 1 is indicated with an asterix (*). Three conserved distinctions defining the subgroup B are marked in dark grey. The insertion of a ‘T’ is located between position 834,978 and 834,979 of the Q154 genome. Subgroup B can be further divided into two groups B₁ and B₂ (light grey lines) by 5 conserved SNPs (marked light grey).</p

Context of seropositivity and investigated factors of the study populations.

<p>Context of seropositivity and investigated factors of the study populations.</p

Positions of the sampled villages all over Egypt.

<p>The map of Egypt showing the position of each randomly selected sampling site (green dots) in each governorate (grey) where animals were sampled. The sampling site ‘Halayeb’, highlighted by a brown dot, is located in the territory disputed between Egypt and Sudan.</p

Numbers of animals sampled per governorate.

<p>Numbers of animals sampled per governorate.</p