List of probes developed for each simplex, duplex and triplex assay, as identified in Fig 1.

<p>The first 3–4 letters of probe names designate the taxa being targeted (except for FS1, which designates the marker), while “RC” means reverse complement. In probe sequences, bases preceded by a “+” sign are LNA bases.</p

Example of the approach used to select and design qPCR primers and probes.

<p>Sequence alignment of the COI-5P region targeted for primer and probe design in the context of developing a qPCR assay that amplifies only <i>L</i>. <i>albescens</i> and <i>L</i>. <i>postalba</i> DNA. The primer and probe sequences are shown above the alignments. An ARMS base (red letter) was introduced into each primer to increase specificity. Sequences shown here were either gleaned from public databases or were obtained through specific PCR amplification followed by Sanger sequencing, as described in the Materials and methods section (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0160878#pone.0160878.s001" target="_blank">S1 File</a> for details).</p

Diagram illustrating the principle guiding the design of qPCR probes specific to the North American (N) and Asian (A) alleles of the FS1 nuclear marker [20].

<p>See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0160878#sec012" target="_blank">Results</a> section and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0160878#pone.0160878.s001" target="_blank">S1 File</a> for details.</p

Example of a qPCR validation assay.

<p>Amplification curves obtained in validating the <i>L</i>. <i>albescens/L</i>. <i>postalba</i> assay tested on 61 samples (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0160878#pone.0160878.s001" target="_blank">S1 File</a>, “Duplex assay 1A” tab, for details on samples). Only <i>L</i>. <i>albescens</i> (red traces) and <i>L</i>. <i>postalba</i> (green traces) samples generated positive amplifications (results of three technical replicates are shown for each sample).</p

Amplification of undiluted and diluted (normalized) DNA samples.

<p>Comparison of amplification curves generated using COI lymantriine general primers (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0160878#pone.0160878.s001" target="_blank">S1 File</a>, tab 1) on multiple, undiluted samples <b>(A)</b>, and those obtained from the same samples following dilutions calculated to achieve the Ct of 22–23 <b>(B)</b>.</p

List of primers developed for DNA quantification and for each simplex, duplex and triplex assay, as identified in Fig 1.

<p>The first 3–4 letters of primer names designate the taxa being targeted (except for FS1, which designates the marker), while “F” and “R” refer to forward and reverse. In primer sequences, bold/underlined letters designate degenerate sites while italicized/underlined letters designate ARMS bases.</p

Specimens of target species and closely related ones used for assay development and validation.

<p>Specimens of target species and closely related ones used for assay development and validation.</p

Geographical distribution of FS1 genotypes, as determined using <i>Duplex assay 4</i>, for gypsy moths identified as <i>L</i>. <i>dispar dispar</i> using <i>Simplex assay 3</i>.

<p>Blue circles: homozygous for the FS1-N allele; red circles: homozygous for the FS1-A allele; blue/red circles: heterozygous for the N and A alleles. Black letters near each circle identify specimens identified as <i>L</i>. <i>dispar dispar</i> using <i>Simplex assay 3</i>; red letters designate <i>L</i>. <i>dispar asiatica</i> and <i>L</i>. <i>dispar japonica</i> positive controls (refer to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0160878#pone.0160878.t004" target="_blank">Table 4</a> for details). Box: central Asia region rich in specimens that are homozygous for the FS1-A allele while displaying <i>L</i>. <i>dispar dispar</i> COI barcode. Background map is from <a href="https://commons.wikimedia.org/wiki/" target="_blank">https://commons.wikimedia.org/wiki/</a>.</p

A Multi-Species TaqMan PCR Assay for the Identification of Asian Gypsy Moths (<i>Lymantria</i> spp.) and Other Invasive Lymantriines of Biosecurity Concern to North America

<div><p>Preventing the introduction and establishment of forest invasive alien species (FIAS) such as the Asian gypsy moth (AGM) is a high-priority goal for countries with extensive forest resources such as Canada. The name AGM designates a group of closely related <i>Lymantria</i> species (Lepidoptera: Erebidae: Lymantriinae) comprising two <i>L</i>. <i>dispar</i> subspecies (<i>L</i>. <i>dispar asiatica</i>, <i>L</i>. <i>dispar japonica</i>) and three closely related <i>Lymantria</i> species (<i>L</i>. <i>umbrosa</i>, <i>L</i>. <i>albescens</i>, <i>L</i>. <i>postalba</i>), all considered potential FIAS in North America. Ships entering Canadian ports are inspected for the presence of suspicious gypsy moth eggs, but those of AGM are impossible to distinguish from eggs of innocuous <i>Lymantria</i> species. To assist regulatory agencies in their identification of these insects, we designed a suite of TaqMan^® assays that provide significant improvements over existing molecular assays targeting AGM. The assays presented here can identify all three <i>L</i>. <i>dispar</i> subspecies (including the European gypsy moth, <i>L</i>. <i>dispar dispar</i>), the three other <i>Lymantria</i> species comprising the AGM complex, plus five additional <i>Lymantria</i> species that pose a threat to forests in North America. The suite of assays is built as a “molecular key” (analogous to a taxonomic key) and involves several parallel singleplex and multiplex qPCR reactions. Each reaction uses a combination of primers and probes designed to separate taxa through discriminatory annealing. The success of these assays is based on the presence of single nucleotide polymorphisms (SNPs) in the 5’ region of mitochondrial cytochrome c oxidase I (COI) or in its longer, 3’ region, as well as on the presence of an indel in the “FS1” nuclear marker, generating North American and Asian alleles, used here to assess Asian introgression into <i>L</i>. <i>dispar dispar</i>. These assays have the advantage of providing rapid and accurate identification of ten <i>Lymantria s</i>pecies and subspecies considered potential FIAS.</p></div