Development and validation of probe-based multiplex real-time PCR assays for the rapid and accurate detection of freshwater fish species

Abstract

Reliable species identification methods are important for industrial environmental monitoring programs. Probe based real-time quantitative polymerase chain reaction (qPCR) provides an accurate, cost-effective and high-throughput method for species identification. Here we present the development and validation of species-specific primers and probes for the cytochrome c oxidase (COI) gene for the identification of eight ecologically and economically important freshwater fish species: lake whitefish (Coregonus clupeaformis), yellow perch (Perca flavescens), rainbow smelt (Osmerus mordax), brook trout (Salvelinus fontinalis), smallmouth bass (Micropterus dolomieu), round whitefish (Prosopium cylindraceum), spottail shiner (Notropis hudsonius) and deepwater sculpin (Myoxocephalus thompsonii). In order to identify novel primer-probe sets with maximum species-specificity, two separate primer-probe design criteria were employed. Highest ranked primer-probe sets from both methods were assayed to identify sequences that demonstrated highest specificity. Specificity was determined using control species from same genus and non-target species from different genus. Selected primer-probe sets were optimized for annealing temperature and primer-probe concentrations to identify minimum reagent parameters. The selected primer-probe sets were highly sensitive, with DNA concentrations as low as 1 ng adequate for positive species identification. A decoder algorithm was developed based on the cumulative qPCR results that allowed for full automation of species identification. Blinded experiments revealed that the combination of the species-specific primer/probes sets with the automated species decoder resulted in target species identification with 100% accuracy. We also conducted a cost/time comparison analysis between the qPCR assays established in this study with other species identification methods. The qPCR technique was the most cost-effective and least time consuming method of species identification. In summary, probe-based multiplex qPCR assays provide a rapid and accurate method for freshwater fish species identification, and the methodology established in this study can be utilized for various other species identification initiatives.

Detection of an invasive aquatic plant in natural water bodies using environmental DNA

“…Environmental DNA (eDNA), which is shed by all living organisms in the environment, is increasingly used for the detection of elusive or low abundant species and has been shown to be equally or more sensitive than traditional surveying methods [6, 7]. So far, the majority of developed eDNA single species detection methods have primarily focused on aquatic animals including mammalians [8, 9], fish [8, 10–17] Molluscs [18, 19], crustacea [20–22], amphibians [23, 24] reptiles [25–27] and insects [28–30]. Detection of aquatic plant eDNA has been scarce in comparison [31].…”

A Highly Sensitive and Specific Probe-Based Real-Time PCR for the Detection of Avibacterium paragallinarum in Clinical Samples From Poultry

“…Bacteriological diagnosis of A. paragallinarum is often challenging due to its fastidious nature. It is well-known that real-time PCR (rtPCR) assays employing amplicon-specific probes are highly sensitive and precise for the rapid and accurate detection of pathogens from clinical samples (12)(13)(14).…”

Novel Tetraplex Quantitative PCR Assays for Simultaneous Detection and Identification of Xylella fastidiosa Subspecies in Plant Tissues

“…Inclusivity was evaluated on strains of Xf subspecies and exclusivity on a range of strains chosen to be present in the same plant and insect niches as Xf (Rogers, 2016) or to be genetically closely related to it. With the exception of a few studies (Boureau et al, 2013;Hulley et al, 2019), only 1 to 10 non-target strains are selected to test the specificity of novel molecular detection tools (Francis et al, 2006;Harper et al, 2010;Burbank and Ortega, 2018). Here a larger collection including 30 non-target strains and 39 Xf strains was analyzed to ensure the specificity of the primer and probe combinations based on the advice of the PM 7/98 of the EPPO (2014) and the MIQE of Bustin et al (2009).…”

“…In recent years, multiplexed TaqMan qPCR has become a useful tool for the identification and quantification of pathogens in different areas such as food safety (Köppel et al, 2019;Wei et al, 2019), medical environment (Janse et al, 2013;Kamau et al, 2013), agronomics (Wei et al, 2008;Zitnick-Anderson et al, 2018), GMO detection (Choi et al, 2018;Wang et al, 2018), and the environment (Hulley et al, 2019). For plant pathogens, these methods have been tested on samples of naturally infected plants, spiked samples (Li et al, 2009;Willsey et al, 2018), and on mixtures of plant and pathogen DNAs (Abraham et al, 2018).…”