title: Detection of Schistosoma mansoni and Schistosoma haematobium by Real-Time PCR with High Resolution Melting Analysis.
abstract: The present study describes a real-time PCR approach with high resolution melting-curve (HRM) assay developed for the detection and differentiation of Schistosoma mansoni and S. haematobium in fecal and urine samples collected from rural Yemen. The samples were screened by microscopy and PCR for the Schistosoma species infection. A pair of degenerate primers were designed targeting partial regions in the cytochrome oxidase subunit I (cox1) gene of S. mansoni and S. haematobium using real-time PCR-HRM assay. The overall prevalence of schistosomiasis was 31.8%; 23.8% of the participants were infected with S. haematobium and 9.3% were infected with S. mansoni. With regards to the intensity of infections, 22.1% and 77.9% of S. haematobium infections were of heavy and light intensities, respectively. Likewise, 8.1%, 40.5% and 51.4% of S. mansoni infections were of heavy, moderate and light intensities, respectively. The melting points were distinctive for S. mansoni and S. haematobium, categorized by peaks of 76.49 ± 0.25 °C and 75.43 ± 0.26 °C, respectively. HRM analysis showed high detection capability through the amplification of Schistosoma DNA with as low as 0.0001 ng/µL. Significant negative correlations were reported between the real-time PCR-HRM cycle threshold (Ct) values and microscopic egg counts for both S. mansoni in stool and S. haematobium in urine (p < 0.01). In conclusion, this closed-tube HRM protocol provides a potentially powerful screening molecular tool for the detection of S. mansoni and S. haematobium. It is a simple, rapid, accurate, and cost-effective method. Hence, this method is a good alternative approach to probe-based PCR assays.
authors.affiliation: Department of Parasitology, Faculty of Medicine, University of Malaya, 50603 Kuala Lumpur, Malaysia. hanysady@yahoo.com.|Department of Medical Laboratories, Faculty of Medical Sciences, Hodeidah University, 3114 Hodeidah, Yemen. hanysady@yahoo.com.
authors.forename: Hany
authors.initials: H
authors.lastname: Sady
authors.affiliation: Department of Parasitology, Faculty of Medicine, University of Malaya, 50603 Kuala Lumpur, Malaysia. halmekhlafi@yahoo.com.|Department of Parasitology, Faculty of Medicine and Health Sciences, Sana'a University, 1247 Sana'a, Yemen. halmekhlafi@yahoo.com.
authors.forename: Hesham M
authors.initials: HM
authors.lastname: Al-Mekhlafi
authors.affiliation: Department of Parasitology, Faculty of Medicine, University of Malaya, 50603 Kuala Lumpur, Malaysia. romano@um.edu.my.
authors.forename: Romano
authors.initials: R
authors.lastname: Ngui
authors.affiliation: Department of Parasitology, Faculty of Medicine, University of Malaya, 50603 Kuala Lumpur, Malaysia. wahib_atrosh@yahoo.com.
authors.forename: Wahib M
authors.initials: WM
authors.lastname: Atroosh
authors.affiliation: Department of Parasitology, Faculty of Medicine, University of Malaya, 50603 Kuala Lumpur, Malaysia. fafo_1979@yahoo.com.
authors.forename: Ahmed K
authors.initials: AK
authors.lastname: Al-Delaimy
authors.affiliation: Department of Parasitology, Faculty of Medicine, University of Malaya, 50603 Kuala Lumpur, Malaysia. nabilnesr@yahoo.com.
authors.forename: Nabil A
authors.initials: NA
authors.lastname: Nasr
authors.affiliation: Department of Parasitology, Faculty of Medicine, University of Malaya, 50603 Kuala Lumpur, Malaysia. saldawaki@gmail.com.
authors.forename: Salwa
authors.initials: S
authors.lastname: Dawaki
authors.affiliation: Department of Parasitology, Faculty of Medicine, University of Malaya, 50603 Kuala Lumpur, Malaysia. fafo_1979@yahoo.com.
authors.forename: Awatif M
authors.initials: AM
authors.lastname: Abdulsalam
authors.affiliation: Department of Parasitology, Faculty of Medicine, University of Malaya, 50603 Kuala Lumpur, Malaysia. init@um.edu.my.
authors.forename: Init
authors.initials: I
authors.lastname: Ithoi
authors.affiliation: Department of Parasitology, Faculty of Medicine, University of Malaya, 50603 Kuala Lumpur, Malaysia. limailian@um.edu.my.
authors.forename: Yvonne A L
authors.initials: YA
authors.lastname: Lim
authors.affiliation: Department of Biomedical Science, Faculty of Medicine, University of Malaya, 50603 Kuala Lumpur, Malaysia. khchua@um.edu.my.
authors.forename: Kek Heng
authors.initials: KH
authors.lastname: Chua
authors.affiliation: Department of Parasitology, Faculty of Medicine, University of Malaya, 50603 Kuala Lumpur, Malaysia. joharisurin@um.edu.my.
authors.forename: Johari
authors.initials: J
authors.lastname: Surin
mesh_terms: D000818:Animals; D017931:DNA Primers; D009691:Nucleic Acid Denaturation; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods*; D012548:Schistosoma haematobium / Q000235:genetics / Q000302:isolation & purification*; D012550:Schistosoma mansoni / Q000235:genetics / Q000302:isolation & purification*; D012552:Schistosomiasis / Q000175:diagnosis / Q000469:parasitology; D012680:Sensitivity and Specificity; D013696:Temperature
chemical_list: D017931:DNA Primers
keywords: S. haematobium; Schistosoma mansoni; high resolution melting analysis; real-time PCR