Accentuated Molecular Detection Technique to Segregate and Identify Helminths of Fish through High Resolution Melting (HRM) Analysis

Abstract

Real-time PCR coupled with high-resolution molecular (HRM) analysis was conducted to target the second internal transcribed spacer (ITS-2) of nuclear ribosomal DNA. The latter acted as a genetic marker to identify and distinguish two anisakid and one cucullanid species parasitizing marine and freshwater fish. Unique and distinct characteristics of HRM patterns were produced for each of the three roundworms investigated. The melt profiles and threshold of the cycles (Ct values), at which amplification commenced, for Anisakis simplex (Rudolphi), Contrcaecum osculatum (Rudolphi) Dujardin, Contrcaecum sp. and Dacnitoides cotylophora (Ward and Magath) were diagnostic for species. The molecular analyses by sequencing and comparing the internal transcribed spacer (ITS) of the ribosomal DNA of Dacnitoides, Anisakis and Contrcaecum assays established their distinct identity. The present investigation propagates molecular-phylogenetic and morpho-molecular analysis to characterize diagnostic constituents of cucullanid and anisakid roundworms.

Monitoring the eradication of the highly invasive topmouth gudgeon (Pseudorasbora parva) using a novel eDNA assay

“…False negatives can also occur during the analysis of eDNA in the laboratory, due to poor primer design, low abundance of target DNA, and/or inadequate amplification (Ficetola et al 2015). There are a number of benefits of using qPCR over traditional end-point PCR for the detection of target species using eDNA, such as greater sensitivity and quicker results (Nathan et al 2014; Wood et al 2013), and our results show that high resolution melt curve analysis (HRM) based on species-specific melt curve profiles (Héritier et al 2017; Jaiswal et al 2017) offers even greater sensitivity.…”

Monitoring the eradication of the highly invasive topmouth gudgeon (Pseudorasbora parva) using a novel eDNA assay

“…This serves to highlight the difficulties of inferring species absences from traditional survey methods (Ficetola et al, 2015;Jerde et al, 2011) sensitivity and the diagnostic nature of the melt peak analysis (Winchell, Wolff, Tiller, Bowen, & Hoffmaster, 2010). Thus, for species-specific assays such as this, the presence of the target species will produce an amplification peak that will melt at a temperature specific of the DNA fragment amplified (Héritier et al, 2017;Jaiswal et al, 2017;Robinson et al, 2018). HRM-qPCR assays can be then validated through sequencing, as we did here.…”

“…Our results show that high resolution melt curve analysis (HRM) based on species-specific melt curve profiles(Héritier, Verneau, Breuil, & Meistertzheim, 2017;Jaiswal, Tripathi, & Malhotra, 2017) offers greater sensitivity and quicker results(Martinou, Mancuso, & Rossi, 2010;Nathan, Simmons, Wegleitner, Jerde, & Mahon, 2014;Wood et al, 2013) than traditional end-point PCR for the detection of P. parva using eDNA. The assay was validated in the field at a location where the species had been confirmed by trapping earlier this year, and all qPCR-HRM assays were validated through sequencing, including positive field samples.…”

“…The qPCR assay described here successfully amplified the target species in very small volumes of sample water (15 ml), albeit with lower sensitivity, which should greatly facilitate the collection of multiple replicated field samples, particularly in remote/inaccessible areas. Our results show that high resolution melt curve analysis (HRM) based on species-specific melt curve profiles(Héritier, Verneau, Breuil, & Meistertzheim, 2017;Jaiswal, Tripathi, & Malhotra, 2017) offers greater sensitivity and quicker results(Martinou, Mancuso, & Rossi, 2010;Nathan, Simmons, Wegleitner, Jerde, & Mahon, 2014;Wood et al, 2013) than traditional end-point PCR for the detection of P. parva using eDNA. The advantages of using an qPCR-HRM approach over end-point PCR include increased…”

Immune response of teleost fish to helminth parasite infection

“…Infections by nematodes are currently diagnosed by visual inspection of larvae parasite and histopathology analysis of fish muscle (GAMBOA et al, 2012). As an alternative, molecular techniques as real-time polymerase chain reaction (PCR) with high-resolution molecular analysis was employed to detect and quantify the presence of Anisakis simplex in fish, enabling even taxa differentiation of other parasites (JAISWAL et al, 2017). For fish products, usually it is used UV illumination, artificial digestion by the utilization of chloridric acid and pepsin, and recently it was developed a new method called TrichinEasy  digestion system, a confirmatory test of the presence of the larvae (CAMMILLERI et al, 2016).…”