High-Resolution Melting Analysis as an Appropriate Method to Differentiate between Fasciola hepatica and F. gigantica.

Background
Fasciolosis is a shared disease between humans and livestock caused by hepatic trematodes; Fasciola hepatica and F. gigantica. Differentiate between the two species of this genus is essential. High-Resolution Melting (HRM) Analysis represents a new approach to this issue. This method can be performed right after termination of Real-Time PCR. This technique has not been used for identification of adult F. hepatica and F. gigantica genotypes. The aim of this study was to determine Fasciola species by using HRM in isolates taken from Iran, respectively.


Methods
Ninety-three Fasciola spp. samples were collected from infected slaughtered animals in different regions of Iran, including North West (Ardebil Province) and South East (Zahedan Province) during 2016. Genomic DNA from the samples was extracted using a DNA extraction kit and then after Real-Time PCR amplification, HRM was done.


Results
Overall, 59 and 34 isolates were identified as F. hepatica and F. gigantica, respectively. The percentages of each species from animals were as follows: sheep (F. hepatica, 80.39% and F. gigantica, 19.61%), cattle (F. hepatica, 42.85% and F. gigantica, 57.15%).


Conclusion
HRM technique developed in the present study is a powerful, rapid and sensitive technique for epidemiological survey and molecular identification between F. hepatica and F. gigantica.


Introduction
The common liver flukes Fasciola hepatica and F. gigantica are causative agents of fasciolosis: a global zoonotic parasitic disease. Although fasciolosis commonly occurs in liver and biliary ducts, ectopic fasciolosis may occur in the peritoneal cavity, lungs, subcutaneous tissue, lymph nodes, eye, and other locations (1,2). Besides causing disease in humans, it can induce mortality and morbidity in sheep and cattle industry (3). As these flat worms parasitize mollusks and vertebrate hosts, accurate and fast detection of them is of vital importance for facilitating control and prevention strategies of fasciolosis in humans and animals (4). One of the fast, sensitive and specific approach for detection and evolutionary analysis of Fasciola spp. are molecular techniques. Despite existence of many diagnosis tools like microscopy, radiology, ultrasound, CT, MRI, serological and clinical tests for detection of fasciolosis, unsatisfactory amounts of sensitivity and specificity have been obtained in many studies (5). Emerging of molecular methods was a turning point to accurate and precise detection and species identification of parasites. Molecular methods not only offer an appropriate impediment for detection of Fasciola spp. in mammals, but also in detection of infected snails in the epizootiological studies of these flukes (6). Toward this end, many molecular techniques have been used by researchers that vary from conventional PCR methods like PCR-RFLP, PCR-SSCP and multiplex PCR to quantitative and qualitative real-time PCR (qRT-PCR) technology (7-10). Indeed real-time PCR has brought the detection of parasitic diseases to another level, with significant sensitivity, easier performance, and no post-PCR operation compared to conventional PCR methods. HRM analysis is a novel method that allows us rapid screening and detection of closely related species in a laboratory (11). The steps in HRM analysis entangle amplification of the wanted region in the presence of a specialized dsDNA binding dye and step by step denaturation of amplicons by increasing the temperature in small increments in order to produce a characteristic melting profile called melting analysis (12). To date, the HRM has mostly been used in molecular studies of parasitic protozoa and rarely for parasitic worms (11,13,14). The genes targeted for molecular strategies against Fasciola spp. are the ITSI and ITSII of nuclear DNA and COI from mitochondrial DNA. The COI gene fragment introduced as a most variable marker recommended for future analyses (15,3). In this study, we developed a qualitative real-time PCR method appeared with HRM analysis for the detection of the two Fasciola spp. In samples taken from Ardebil and Zahedan provinces, northwestern and southern Iran, respectively. The study was conducted as a complementary case to previous studies on genotype (16) and morphological verifications (17). In this study, we aimed to develop a qualitative real-time PCR method appeared with HRM analysis for the detection of the two Fasciola spp. Samples were taken from Ardebil and Zahedan provinces, northwestern and southern Iran, respectively.

Sample collection
Ninety-three adult trematodes of Fasciola spp. located within the bile ducts of infected cattle (n=42 samples) and sheep (n=51 samples), were collected from slaughterhouses in two different regions of Iran from Nov 2016 to Jan 2017. The study was conducted in Meshkinshahr City, Ardebil Province, North West and Zabol City, Sistan, and Baluchistan Province, South East, Iran (Fig. 1). The flatworms were extensively washed in physiological saline and preserved in >70° (v/v) ethanol and then frozen at -20 °C until DNA extraction. Ethics Committee of Tehran University of Medical Sciences approved the study.

DNA extraction
Before DNA extraction, all individual worms rinsed three times with PBS to remove the ethanol. Genomic DNA (Gdna) was extracted from small portion of apical region of adult trematodes to avoid entry female genitalia that likely to outer sperm. Gdna from individual worms was extracted using a QIAamp DNAeasy, Hilden, Germany, according to the manufacturer's reference protocols with some modifications. The concentration of the extracted DNA was specified by NanoDrop (Thermo Scientific, Rockford, IL, USA), and after that, the samples were stored at -20 °C for further analysis. CAGGAAAAGACC-3' design was carried out using the sequences available on GenBank and in continuation. Primers were obtained using the program PRIMER BLAST (http://www.ncbi.nlm.nih.gov/tools/primerblast/) and rechecked with Beacon Designer8.12, PREMIER BIOSOFT software from the consensus sequence obtained by the multiple alignments, with an expected amplicon size of 266 bp for all the sequences. PCR was performed in 20 Ml final reaction volume containing 10 Ml master mix (Type-it HRM PCR Kit; Qiagen, Hilden, Germany), 5.2 Ml distilled water, 0.4 Ml of each primer, and 4 Ml of template DNA. Enzymatic reaction was performed as follows: the reaction mixture was heated for initial denaturation step at 95 °C for 10 min, followed by 40 cycles of amplification performed at 95 °C for 30 sec for denaturation step, 55 °C for 40 sec, for annealing section, 72 °C for 30 sec related to extension portion, and a final extension step at 72 °C for 6 min after 40 cycles. HRM temperature was raised from 70 °C to 95 °C. During this process, the amplicons obtained from PCR were denatured prior to the development of melting curves in the inflexion point where changes in fluorescence with respect to changes in temperature (Df/Dt) were recorded with a ramp of 0.3 °C/sec (18). Fluorescence dye signaling was measured after each cycle. The kit contained the novel double-stranded DNAbinding fluorescent dye, EvaGreen, and an optimized HRM PCR master mix buffer, consisting of HotStarTaq plus DNA polymerase, Q-Solution, and dNTPs. We used a positive standard control for F. hepatica and F. gigantica available in the Department of Medical Parasitology and Mycology, School of Public Health, Tehran University of Medical Sciences. Real-time PCR was carried out in a Mini Opticon real-time PCR detection system (Applied Biosystems Step One Plus Inc., CA, USA). The Real-Time amplification result and Tm analysis were obtained using the Step One Plus TM software ver.

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. Tm analysis was repeated three times in each run to confirm the repeatability of the Tm assay by estimating the Tm variation within a PCR amplification (intra-assay), and between PCR amplifications (inter-assay). The coefficient of variation (CV) was calculated by dividing the standard deviation (SD) by the arithmetic mean of the measured values of Tm (CV ＝ SD[Le, 2012 #11]/mean value). Furthermore, to check the uniformity of temperature in the cycler block, a number of samples were re-amplified at different positions of the cycler block during the same amplification cycle. The intra-assay CVs represent the mean CVs of the results obtained from the replications of Fasciola spp.

Real-Time PCR amplification and HRM analysis
In accordance with Real-Time PCR and HRM analysis procedure, among 93 samples containing cattle (n=42 samples) and sheep (n=51 samples) were amplified using partial sequence of COI gene of Fasciola spp., and then HRM was performed. Tm analysis was repeated three times in each run to confirm the repeatability of the Tm assay (Table 1).

Discussion
Following previous studies and because of the importance of fascioliasis in Iran, more comprehensive studies on this parasite and the diagnosis of its species are necessary to achieve this goal (19,20). In the previous studies, PCR-RFLP technique on ITSI gene was used to determine the species of Fasciola and compare it with the sequencing and morphology methods (16,17).
In the present assay, we successfully developed a real-time PCR and HRM technique on COI gene for rapid, sensitive and precise differentiation of F. hepatica and F. gigantica using only a single pair of primers. The average Tm variation obtained by melting curve analysis was about 1˚C. This finding indicates a sufficient ability and reliability of the assay for distinguishing these two parasites.

Conclusion
HRM technique developed in present study is a powerful, rapid and sensitive technique for epidemiological survey and molecular identification between F. hepatica and F. gigantica.

Ethical considerations
Ethical issues (Including plagiarism, informed consent, misconduct, data fabrication and/or falsification, double publication and/or submission, redundancy, etc.) have been completely observed by the authors.