COMPAS-PCR optimization.

<p>Effect of length variations in the reverse excess primer (P^X) with a 23 bp complementary forward limiting primer (P^L) using 5 ng genomic <i>S</i>. <i>salar</i> DNA and SsoFast Evagreen mastermix. P^L = 5NTS-23F at a concentration of 50 (A & C) or 200 nM (B & D) and P^X at 600 nM (A, B, C & D). In each experiment P^X 3’ end is unchanged, either with no overhang (A & B) or with a 3 nucleotide overhang (C & D) after the forward P^L 5’ complementary end. P^X length is incremented in 5’ from 18 nucleotides until it reaches the 3’ end of P^L at 23 nucleotides resulting in a 100% complementarity between P^X and P^L (A & B), or 26 nucleotides (C & D). A gradient PCR was run varying the annealing temperature (T_a) from 62 to 72°C. The P^X nucleotide length increase is shown on the lower horizontal axis. The difference between melt temperature of the limiting primer (T_m^L) and melt temperature of the excess primer (T_m^X) is reported on the left-hand vertical axis as T_m^L—T_m^X and plotted as triangles. For each P^X, the corresponding optimal T_a is reported on the upper horizontal axis. The resulting Cycle Threshold C_T is reported on the right-hand vertical axis and plotted as circles.</p

COMPAS-PCR principles.

<p>Asymmetric concentration effect for 5S rDNA qPCR highly complementary primers tested on <i>S</i>. <i>salar</i> L235 and <i>S</i>. <i>trutta</i> T107. Concentration of the forward primer 5SNTS-23F in A and B ranges from 0.6 to 0.05 μM while a constant concentration of 0.6 μM was used for the reverse primer 5SNTS-22R+2 in A and 5SNTS-23R+3 in B.</p

Sample origin and sequence information.

<p>Sample origin and sequence information.</p

Salmonid 5S rDNA primers for COMPAS-PCR.

<p>Salmonid 5S rDNA primers for COMPAS-PCR.</p

COMPAS-PCR using highly complementary primers applied to 5S rDNA direct repeat genes.

<p>Non Transcribed Sequence lengths are indicated according to sequence access numbers S73107 and LN835408-LN835422, varying from 137 and 138 bp for <i>S</i>. <i>salar</i> to 160 bp for <i>S</i>. <i>trutta</i>.</p

Multiplex PCR for the simultaneous detection of the Enterobacterial gene wecA, the Shiga Toxin genes (stx1 and stx2) and the Intimin gene (eae)

Objectives The aetiology of several human diarrhoeas has been increasingly associated with the presence of virulence factors rather than with the bacterial species hosting the virulence genes, exemplified by the sporadic emergence of new bacterial hosts. Two important virulence factors are the Shiga toxin (Stx) and the E. coli outer membrane protein (Eae) or intimin, encoded by the stx and eae genes, respectively. Although several polymerase chain reaction (PCR) protocols target these virulence genes, few aim at detecting all variants or have an internal amplification control (IAC) included in a multiplex assay. The objective of this work was to develop a simple multiplex PCR assay in order to detect all stx and eae variants, as well as to detect bacteria belonging to the Enterobacteriaceae, also used as an IAC. Results The wecA gene coding for the production of the Enterobacterial Common Antigen was used to develop an Enterobacteriaceae specific qPCR. Universal primers for the detection of stx and eae were developed and linked to a wecA primer pair in a robust triplex PCR. In addition, subtyping of the stx genes was achieved by subjecting the PCR products to restriction digestion and semi-nested duplex PCR, providing a simple screening assay for human diarrhoea diagnostic

Simulation of larval dispersal.

<p>The spatial distribution of the 369 landed Pacific oyster (<i>Crassostrea gigas</i>) larvae in Swedish and Norwegian coastal waters in total for the simulated years (1990, 1998, 2002, 2006, 2007, 2010), summed per coastal grid cell (50x50 km). Number of landed larvae (super-individuals) per grid cell is shown (see legend). The location and names of the sampled DNA stations in this study are indicated (black circles, cf. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0177481#pone.0177481.t001" target="_blank">Table 1</a>). For simulation details see [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0177481#pone.0177481.ref050" target="_blank">50</a>]. Reprinted from Rinde et al. 2016 under a CC BY license, with permission from NIVA, original copyright 2016. The map is produced using ESRIs GIS software ArcMap v 10.4.1 (<a href="http://www.esri.com" target="_blank">www.esri.com</a>), and the country dataset GISCO NUTS 2010.</p

Rapid expansion of the invasive oyster <i>Crassostrea gigas</i> at its northern distribution limit in Europe: Naturally dispersed or introduced?

<div><p>The Pacific oyster, <i>Crassostrea gigas</i>, was introduced to Europe for aquaculture purposes, and has had a rapid and unforeseen northward expansion in northern Europe. The recent dramatic increase in number of <i>C</i>. <i>gigas</i> populations along the species’ northern distribution limit has questioned the efficiency of Skagerrak as a dispersal barrier for transport and survival of larvae. We investigated the genetic connectivity and possible spreading patterns between Pacific oyster populations on the southern Norwegian coast (4 localities) and Swedish and Danish populations by means of DNA microsatellite analysis of adult oysters, and by simulating larvae drift. In the simulations we used a 3D oceanographic model to explore the influence of recent climate change (1990–2010) on development, survival, and successful spreading of Danish and Swedish Pacific oyster larvae to Norwegian coastal waters. The simulations indicated adequate temperature conditions for development, survival, and settlement of larvae across the Skagerrak in warm years since 2000. However, microsatellite genotyping revealed genetic differences between the Norwegian populations, and between the Norwegian populations and the Swedish and Danish populations, the latter two populations being more similar. This patchwork pattern of genetic dissimilarity among the Norwegian populations points towards multiple local introduction routes rather than the commonly assumed unidirectional entry of larvae drifted from Denmark and Sweden. Alternative origins of introduction and implications for management, such as forecasting and possible mitigation actions, are discussed.</p></div

Genotyping of 262 <i>Crassostrea gigas</i> individuals using six microsatellite loci in two multiplex PCR.

<p>Genotyping of 262 <i>Crassostrea gigas</i> individuals using six microsatellite loci in two multiplex PCR.</p

Discriminant Analysis of Principal Components (DAPC).

<p>Scatter plot with (a) and without (b) location N_B in the analysis. Sampling locations are internally connected with lines to the center of each ellipses. The Danish and Swedish samples are indicated by blue colors (D_A, dark blue and S_S, light blue), the Norwegian outlier location (N_B, green) is differentiated from the remaining Norwegian samples (N_I, N_O, and N_G) represented by red color.</p