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...

Full description

Bibliographic Details
Main Authors: 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
Format: Article
Language:English
Published: MDPI AG 2015-07-01
Series:International Journal of Molecular Sciences
Subjects:
Online Access:http://www.mdpi.com/1422-0067/16/7/16085
id doaj-05db7725731f4ed59245466a9153c39b
record_format Article
spelling 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
work_keys_str_mv AT hanysady detectionofschistosomamansoniandschistosomahaematobiumbyrealtimepcrwithhighresolutionmeltinganalysis
AT heshammalmekhlafi detectionofschistosomamansoniandschistosomahaematobiumbyrealtimepcrwithhighresolutionmeltinganalysis
AT romanongui detectionofschistosomamansoniandschistosomahaematobiumbyrealtimepcrwithhighresolutionmeltinganalysis
AT wahibmatroosh detectionofschistosomamansoniandschistosomahaematobiumbyrealtimepcrwithhighresolutionmeltinganalysis
AT ahmedkaldelaimy detectionofschistosomamansoniandschistosomahaematobiumbyrealtimepcrwithhighresolutionmeltinganalysis
AT nabilanasr detectionofschistosomamansoniandschistosomahaematobiumbyrealtimepcrwithhighresolutionmeltinganalysis
AT salwadawaki detectionofschistosomamansoniandschistosomahaematobiumbyrealtimepcrwithhighresolutionmeltinganalysis
AT awatifmabdulsalam detectionofschistosomamansoniandschistosomahaematobiumbyrealtimepcrwithhighresolutionmeltinganalysis
AT initithoi detectionofschistosomamansoniandschistosomahaematobiumbyrealtimepcrwithhighresolutionmeltinganalysis
AT yvonneallim detectionofschistosomamansoniandschistosomahaematobiumbyrealtimepcrwithhighresolutionmeltinganalysis
AT kekhengchua detectionofschistosomamansoniandschistosomahaematobiumbyrealtimepcrwithhighresolutionmeltinganalysis
AT joharisurin detectionofschistosomamansoniandschistosomahaematobiumbyrealtimepcrwithhighresolutionmeltinganalysis
_version_ 1725484059344240640