A novel 5-Plex qPCR-HRM assay detecting human diarrheal parasites

Intestinal parasitic diseases occur worldwide, and their diagnosis poses considerable challenges. spp., (and, arguably, and spp.) are among the most important and common parasitic protozoans causing diarrhea. Several multiplex real-time PCR assays have been developed for the synchronous detection of...

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Published inGut pathogens Vol. 12; no. 1; p. 27
Main Authors Lamien-Meda, Aline, Schneider, Renate, Walochnik, Julia, Auer, Herbert, Wiedermann, Ursula, Leitsch, David
Format Journal Article
LanguageEnglish
Published England BioMed Central Ltd 29.05.2020
BioMed Central
BMC
Subjects
Analysis
Blastocystis
Cryptosporidium
Deoxyribonucleic acid
Design
Diagnosis
Diarrhea
Dientamoeba
DNA
Entamoeba
Epidemiology
Hydrolysis
Infections
Intestine
Methods
Microscopy
Mortality
Parasites
Parasitic diseases
Plasmids
Protozoa
qPCR-HRM
Temperature
Israel
Cryptosporidium
qPCR-HRM
Diarrhea
Giardia
Dientamoeba
Blastocystis
Entamoeba
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Summary:Intestinal parasitic diseases occur worldwide, and their diagnosis poses considerable challenges. spp., (and, arguably, and spp.) are among the most important and common parasitic protozoans causing diarrhea. Several multiplex real-time PCR assays have been developed for the synchronous detection of these parasites. However, most assays include the use of hydrolysis probes, increasing the cost of stool examination. In this study, we designed and evaluated a real-time PCR protocol, based on high-resolution melting (HRM) curve analysis, to simultaneously detect and differentiate five gastrointestinal parasites. Using a blinded panel of 143 clinical samples with laboratory diagnostic data to evaluate the method, we obtained a 95.8% concordance with conventional methods. Moreover, 4.2% of the samples were positive for and 2.8% additional infections were found with our multiplex assay. Our method is sensitive and specific for the selected parasites with the additional possibility of being run in single-plex as a backup control for mixed infections. The assay is a convenient and cost-effective method that could contribute to a quicker and accurate diagnosis as well as to more targeted therapies of parasite-derived diarrhea. Finally, this new multiplex PCR assay could also be instrumental in epidemiology studies on these parasites.
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ISSN:1757-4749
1757-4749
DOI:10.1186/s13099-020-00365-6