Received Date: April 04, 2017 Accepted Date: May 15, 2017 Published Date: May 18, 2017
Citation: Jaiswal N, Tripathi R, Malhotra SK (2017) Accentuated Molecular Detection Technique to Segregate and Identify Helminths of Fish through High Resolution Melting (HRM) Analysis. Single Cell Biol 6: 163. doi:10.4172/2168- 9431.1000163
Copyright: © 2017 Jaiswal N, et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Visit for more related articles at Single Cell Biology
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.
Molecular detection; Single stranded DNA; Helminths; Fish; RT-PCR; Cucullanid; Anisakid
The molecular diagnostic procedures based on RT-PCR have proved to be reliable biotechnological tool from the standpoint of segregation of species and other taxa. Their potential to detect multiple pathogens simultaneously in one setting has put these on a higher pedestal of clinical significance as well. The asymptomatic infections of invasive parasitic protozoans were detected in earlier investigations [1,2]. The inquisitiveness about such infections could thus be PCRcoupled technology has been scarcely used for detection and diagnosis of helminth infections, particularly of zoonotic significance.
The analysis conducted by real-time PCR with SYBR Green1 melting curve analysis in this study proved to be an effective and sensitive method for specific detection and identification of pathogenic parasites of zoonotic significance.
The conservation of specific products at the required specific temperature to generate fluorescence data, and simultaneous denaturation of non-specific products hides the disadvantageous binding characteristic of the dye with all the double stranded DNA comprising primer dimers and non-specific products. Therefore, this fluorescence characteristic and the associated melting point variants have been attributed immense taxonomic significance for taxa differentiation.
The nucleic acid sequence elongation and its conformation is sensitive to the melting point of amplicon. Thus, the melting point curve analysis has thus provided an opportunity to correlate it with sequence length variations to determine species-specific sequence variation. Ct values have been recognized to be the sensitive and specific tools that banished the requirement of electrophoresis of amplicons, and instead assisted the process to detect the adequate number of cycles required to cross the threshold at the time of initiation of the amplification cycle, so that ultimately relative or absolute quantification of nucleic acids could be attained.
The assistance of SEM applications, as well as RT-PCR technology the three genera viz., Dacnitoides, Anisakis and Contrcaecum has been facilitated. The innovative molecular applications have resulted into fast track diagnostics to resolve issues of biodiversity and Systematic Parasitology .
It was recently highlighted  that the nucleotide sequence length and barcode structure are a function of the melting point of an amplicon. Therefore, it is obvious that the diversity of sequence variation within and among samples can be determined by the melting point analysis, thereby confirming the use of melting curve analysis as a tool to establish distinct identity of individual amplicons comprising contrasting sequences .
Since marine fish are the main reservoirs of the nematodes of Anisakidae in the coastal ecosystem at Goa, whose larvae were quite often difficult to discern with from adult worms morphologically, after real-time PCR amplification, HRMA allowed to discriminate clearly the three species according to the different melting temperatures of the amplicons, although they have an identical number of bases . Melting profiles showed minimal variability within samples from the same group. The specificity of the amplification was confirmed by sequence analysis, thus proving that the clustering pattern presented in the curves was representing two different species, as effectively as DNA sequencing. The cost effectiveness of real-time PCR–HRMA is comparable to, or cheaper than, conventional PCR and sequencing, with a very rapid postamplification detection step .
Although the recent investigations to develop advanced methods for detection and assessment of abundance of a variety of trematodes and other parasites, including flatworms, without collection of hosts [8-11], the application of PCR-based tests , standard field protocols need be established as yet, particularly to clearly understand the environmental persistence of DNA . However, there are disadvantages too in the employment of eDNA methods, as they are also prone to false negatives if DNA degradation occurred due to the errors in sampling on error in calculation of timings of infection outbreak or due to spatial or temporal implications.
more rapid, close-tube, melt peak assays were generated from a variety of anisakid nematodes from marine and freshwater fish using RT-PCR technology. The adult roundworms of Anisakis simplex (Rudolphi) and Contrcaecum osculatum (Rudolphi) Dujardin were collected from Sillago sihama, and a sciaenid, Johnius dussumieri, respectively, during February through November, 2014. The larval C. osculatum were recovered from S. sihama, and of Contrcaecum from J. dussumieri. Simultaneously, freshwater catfish, Rita rita was examined during this period from Gangetic riverine ecosystem at Fatehpur, Uttar Pradesh to collect the specimens of Dacnitoides cotylophora (Ward and Magath). The rationale for selecting two anisakids and one cucullanid species for the study was the extension of range of infectivity by the roundworms of Anisakidae and related groups of worms, from marine habitat in the fish hosts of areas of Central West coast of India at Goa to the freshwater fish hosts of Gangetic riverine ecosystems .
Total genomic DNA was extracted from these worms, using DNeasy Tissue Kit (Qiagen) in a final buffer volume of 50 μm. A 950 base pair (bp) fragment of the Inter Transcribed Spacer – 2 gene from the extracted DNA was amplified using primers SS2, 5’-TTGCAGACACATTGAGCACT-3’; and NC2, 5’-TTAGTTTCTTTTCCTCCGCT-3’  using a Thermal Cycler IQ5 Real Time PCR Detection System Biorad Laboratories Inc., Hercules, CA, USA. The total PCR volume was 20 um and contained 1 microlitre template DNA, 10 micorlitre SYBR Green ER Supermix and 0.4 microlitre of each primer (0.2 micromolar). Amplification was performed using the cycling profile 94°C initial denaturation (5min), 35 cycles of 94°C denaturation (30 sec), 53°C annealing (30 sec) and 72°C extension (30 sec)(extension), followed by a final extension at 72°C for 5 min. Fluorescence was measured during the annealing step of each cycle.
The fluorescence of intercalating dyes utilized in conducting qPCR, is attained on their being bound to double-stranded DNA (dsDNA), while the presence of single stranded DNA (ssDNA) does not induce these to fluoresce. Once the completion of amplification cycles is achieved, the melt curve is derived following the programmed instructions. The amount of fluorescence is then measured by thermal cycler at a preset temperature (usually above the primer Tm) at the termination of the qPCR run. The denaturation of dsDNA commenced to transform into single-stranded DNA resulting into simultaneous dissociation of the dye, after incremental increase in temperature, while simultaneous measure of fluorescence continued. The commencement of melting of the dsDNA ensures melting of unstable (i.e. G/C poor) prior to G/C rich regions until the temperature is sufficiently high to cause it to melt.
Threshold cycles (Ct values) for A. simplex, C. osculatum and D. cotylophora assays were consistent over time, and gave significantly comparable values that could enable to establish taxa differentiation. The findings of experimentation to denote peaks of melting point curves of ITS2 gene to distinctly establish the separate identity of adult specimens of A. simplex (Figure 1a, 79.59°C), adult (Figure 1b, 85.5°C) and larvae (Figure 1c, 87°C) of Contracaecum sp. larvae from J. dussumieri (Figure 1d, 86°C and 75°C), and adult worms of D. cotylophora (Figure 1e, 86.5°C) recovered from freshwater (R. rita) fish are shown in Figure 1e. As many as three replicates of each species were done for HRM analysis. But only the representative depiction of derivative melt curves all of these three replicates was included (Figure 1c and Figure 1d) to avoid repetition of illustrations, particularly because, these figures were for the larvae of the only genus Contracaecum that were collected from two different marine fish species, namely, larval C. osculatum from S. sihama (Figure 1c), as well as larval C. osculatum from Johnius dussumieri (Figure 1d). Simultaneously, the three replicates of aligned melt curves of larval C. osculatum from S. sihama, as well as larval C. osculatum from Johnius dussumieri have been presented in Figure 2c and Figure 2d, respectively.
Figure 1: Profiles of the melt peak curves (derivative melt curves) showing denaturation profile of ITS2 amplicons a: Adult Anisakis simplex from Sillago sihama, b: Adult Contracaecum osculatum from Sillago sihama, c: Larval C. osculatum from Sillago sihama, d: Larval C. osculatum from Johnius dussumieri e: Adult worms of Dacnitoides cotylophora recovered from freshwater Rita rita
Figure 2: Profiles of the melt peak curves (aligned melt curves) showing denaturation profile of ITS2 amplicons. a: Adult Anisakis simplex from Sillago sihama, b: Adult Contracaecum osculatum from Sillago sihama, c: Larval C. osculatum from Sillago sihama, d: Larval C. osculatum from Johnius dussumieri, e: Adult worms of Dacnitoides cotylophora recovered from freshwater Rita rita.
Ct values analysed for D. cotylophora were 15.3-16.85°C, whereas these were 23.13-23.44°C for adult worms of C. osculatum, and 20.71- 20.9°C for their larvae, and 11.72-14.02°C for the larvae of Contracaecum sp. These values were 18.9-19.19°C for adult specimens of A. simplex.
The application of RT-PCR technique not only enables detection and quantification in diagnostics, but also the identification of specific DNA sequences [16,17]. The use of real-time PCR for molecular diagnostics is attractive because it is objective, rapid, versatile and cost-effective and can be performed on small tissue samples. The melt peak and Tm values analysis enables identification of species. The finding on twin peaks at 75°C and 86.5°C in the present study were closer to the similar pattern of twin peaks of the specific melting temperature peaks at 80.1°C and 85°C recorded earlier , where a multiplex real time PCR assay was developed for the distinctive detection of E. histolytica, Giardia lamblia and C. parvum in stool samples. Two melting domains were reported recently  in a 550 bp amplicon of the hydroxytryptamine receptor 2A gene. Amplicons with multiple melting domains have been used to differentiate species of Giardia (660 bp amplicon from the gdh gene)  and Naegleria (350-400 bp amplicons from the intergenic spacer).
The intercalating dyes produce fluorescence only when bounded with double stranded DNA and the thermal cycler produces the melt curve after the process of amplification is completed. When the temperature is raised, the DNA denatures and thus the fluorescence decreases. The change in slope of the curve verses temperature obtains the melting curve. All the samples taken under study were identified using RT-PCR. The consistent Tms were obtained for replicates of each species, and each species has a different Tm value leading to its identification. The representative three replicates of melt point curves i.e. derivative curves (Figure 1c, C. osculatum larvae from S. sihama, Figure 1d, larval C. osculatum from J. dussumieri) as well as aligned curves (Figure 2c, C. osculatum larvae from S. sihama, Figure 2d, larval C. osculatum from J. dussumieri) have been shown, so as to avoid repetition of replicates each time, for all the three worms. This is particularly because the larvae of Contracaecum were encountered repeatedly in two different fish species in the marine ecosystem. The melting point is a function of GC content, therefore, the higher the GC content, the more would be the melting point and this forms the basis of differentiation between the different species. The results in this study showed highest melting point values for the adults (23.13-23.44°C) and larvae of C. complanatum (20.71-20.9°C), reflecting higher G:C content in these parasites of marine origin. However, the anisakid, A. simplex (18.9-19.19°C), and the cucullanid species, D. cotylophora (15.3-16.85°C) had lower Tm values, while another Contracaecum sp. larvae had the lowest (11.72-14.02°C). The melting characteristics of ITS2 amplicons from all species were assessed by plotting two different curves (Figures 2a-2e). In the present study, the normalized fluorescence curves i.e. aligned melt curve (Figures 2a-2e) and derivative melt curve (Figure 2a) produced uniquely different plots that were easily distinguishable for each species. It would mean that although the melting profiles of different species (Tm) were very close to each other, they could clearly be discerned by the plotting of normalized melting curves (Figures 2e). The melting of DNA being a multi-state process (dsDNA and ssDNA) has been discussed already , and the possibility of an intermediate state occurring to explain twin peaks as the melting events of the two amplicons, was put forth .
It has been recently asserted  that for species having almost similar melting curves and temperature-shifted fluorescence difference, a sharp decrease in fluorescence was detected in denatured DNA that was consistent for such species, with its respective melting point. The binding of intercalating dyes to any double stranded DNA is its drawback and henceforth melting point curve is a point for analysis, as non specific DNA is denatured at this point and specific DNA products remain intact. This associates the melting point to the composition and sequence of the nucleotides and characterizes sequence variation within the samples. The specific diagnosis made by the melting point curve is of importance in elucidating the genetic diversity, and will assist in study of differences in the biology, ecology and in the transmission of the parasites .
Conclusively, therefore, the findings of this investigation revealed the potential role of real-time PCR in rapid diagnosis of cucullanids and agents of anisakidosis, as a step better than simple PCR technique.It exhibited sensitivity compatible to that of traditional PCR but had the advantage of real-time detection of PCR products and the requirement of restriction digestion or sequence analysis for species differentiation or genotyping was kept in abeyance. Hence, real-time PCR has emerged as an ideal tool for screening for the detection and genotyping of parasites of zoonotic significance, in clinical laboratories.
NJ is thankful to UGC for a Post Doctoral Fellowship under which the current research work was carried out. The authors are grateful to Professor Sandeep K. Malhotra Head, Department of Zoology, University of Allahabad for RT-PCR technology facilities in the Department.