Detection of Schistosoma mansoni and Schistosoma haematobium by Real-Time PCR with High Resolution Melting Analysis
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 th...
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doaj-05db7725731f4ed59245466a9153c39b2020-11-24T23:48:54ZengMDPI AGInternational Journal of Molecular Sciences1422-00672015-07-01167160851610310.3390/ijms160716085ijms160716085Detection of Schistosoma mansoni and Schistosoma haematobium by Real-Time PCR with High Resolution Melting AnalysisHany Sady0Hesham M. Al-Mekhlafi1Romano Ngui2Wahib M. Atroosh3Ahmed K. Al-Delaimy4Nabil A. Nasr5Salwa Dawaki6Awatif M. Abdulsalam7Init Ithoi8Yvonne A. L. Lim9Kek Heng Chua10Johari Surin11Department of Parasitology, Faculty of Medicine, University of Malaya, 50603 Kuala Lumpur, MalaysiaDepartment of Parasitology, Faculty of Medicine, University of Malaya, 50603 Kuala Lumpur, MalaysiaDepartment of Parasitology, Faculty of Medicine, University of Malaya, 50603 Kuala Lumpur, MalaysiaDepartment of Parasitology, Faculty of Medicine, University of Malaya, 50603 Kuala Lumpur, MalaysiaDepartment of Parasitology, Faculty of Medicine, University of Malaya, 50603 Kuala Lumpur, MalaysiaDepartment of Parasitology, Faculty of Medicine, University of Malaya, 50603 Kuala Lumpur, MalaysiaDepartment of Parasitology, Faculty of Medicine, University of Malaya, 50603 Kuala Lumpur, MalaysiaDepartment of Parasitology, Faculty of Medicine, University of Malaya, 50603 Kuala Lumpur, MalaysiaDepartment of Parasitology, Faculty of Medicine, University of Malaya, 50603 Kuala Lumpur, MalaysiaDepartment of Parasitology, Faculty of Medicine, University of Malaya, 50603 Kuala Lumpur, MalaysiaDepartment of Biomedical Science, Faculty of Medicine, University of Malaya, 50603 Kuala Lumpur, MalaysiaDepartment of Parasitology, Faculty of Medicine, University of Malaya, 50603 Kuala Lumpur, MalaysiaThe 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.http://www.mdpi.com/1422-0067/16/7/16085Schistosoma mansoniS. haematobiumreal-time PCRhigh resolution melting analysis |
collection |
DOAJ |
language |
English |
format |
Article |
sources |
DOAJ |
author |
Hany Sady Hesham M. Al-Mekhlafi Romano Ngui Wahib M. Atroosh Ahmed K. Al-Delaimy Nabil A. Nasr Salwa Dawaki Awatif M. Abdulsalam Init Ithoi Yvonne A. L. Lim Kek Heng Chua Johari Surin |
spellingShingle |
Hany Sady Hesham M. Al-Mekhlafi Romano Ngui Wahib M. Atroosh Ahmed K. Al-Delaimy Nabil A. Nasr Salwa Dawaki Awatif M. Abdulsalam Init Ithoi Yvonne A. L. Lim Kek Heng Chua Johari Surin Detection of Schistosoma mansoni and Schistosoma haematobium by Real-Time PCR with High Resolution Melting Analysis International Journal of Molecular Sciences Schistosoma mansoni S. haematobium real-time PCR high resolution melting analysis |
author_facet |
Hany Sady Hesham M. Al-Mekhlafi Romano Ngui Wahib M. Atroosh Ahmed K. Al-Delaimy Nabil A. Nasr Salwa Dawaki Awatif M. Abdulsalam Init Ithoi Yvonne A. L. Lim Kek Heng Chua Johari Surin |
author_sort |
Hany Sady |
title |
Detection of Schistosoma mansoni and Schistosoma haematobium by Real-Time PCR with High Resolution Melting Analysis |
title_short |
Detection of Schistosoma mansoni and Schistosoma haematobium by Real-Time PCR with High Resolution Melting Analysis |
title_full |
Detection of Schistosoma mansoni and Schistosoma haematobium by Real-Time PCR with High Resolution Melting Analysis |
title_fullStr |
Detection of Schistosoma mansoni and Schistosoma haematobium by Real-Time PCR with High Resolution Melting Analysis |
title_full_unstemmed |
Detection of Schistosoma mansoni and Schistosoma haematobium by Real-Time PCR with High Resolution Melting Analysis |
title_sort |
detection of schistosoma mansoni and schistosoma haematobium by real-time pcr with high resolution melting analysis |
publisher |
MDPI AG |
series |
International Journal of Molecular Sciences |
issn |
1422-0067 |
publishDate |
2015-07-01 |
description |
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. |
topic |
Schistosoma mansoni S. haematobium real-time PCR high resolution melting analysis |
url |
http://www.mdpi.com/1422-0067/16/7/16085 |
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