Development of a PCR Assay Followed by Nonradioactive Hybridization Using Oligonucleotides Covalently Bound to CovaLink NH Microwells for Detection of Four Plasmodium Species in Blood Samples from Humans

We developed and evaluated a PCR-based assay to detect four Plasmodium species in 79 blood samples from 56 travelers returning from areas where malaria is endemic. DNA amplification targeting a small region of the 18S rRNA gene was performed with Plasmodium genus-specific primers. The biotinylated P...

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Bibliographic Details
Published in:Journal of Clinical Microbiology 2006-09, Vol.44 (9), p.3279-3284
Main Authors: Machouart, M, Bigois-Delemotte, L, Ajana, F, Brizion, M, Biava, M.F, Collomb, J, Fortier, B
Format: Article
Language:English
Subjects:
Animals
Base Sequence
Biological and medical sciences
Blood - parasitology
Fundamental and applied biological sciences. Psychology
Humans
Infectious diseases
Malaria - diagnosis
Malaria - parasitology
Medical sciences
Microbiology
Molecular Sequence Data
Nucleic Acid Hybridization - methods
Oligonucleotide Probes
Parasitemia - diagnosis
Parasitemia - parasitology
Parasitology
Plasmodium
Plasmodium - classification
Plasmodium - genetics
Plasmodium - isolation & purification
Polymerase Chain Reaction - methods
RNA, Ribosomal, 18S - genetics
Sensitivity and Specificity
Sequence Analysis, DNA
Species Specificity
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Summary:We developed and evaluated a PCR-based assay to detect four Plasmodium species in 79 blood samples from 56 travelers returning from areas where malaria is endemic. DNA amplification targeting a small region of the 18S rRNA gene was performed with Plasmodium genus-specific primers. The biotinylated PCR products were then identified by PCR-colorimetric Covalink NH microwell plate hybridization (CMPH) using species-specific phosphorylated probes covalently bound to a pretreated polystyrene surface. The results from PCR-CMPH showed high specificity, and for 47 of the 56 patients (84%), microscopy and PCR-CMPH results were in agreement. Discordant results were reevaluated with microscopy examination, other molecular methods, and DNA sequencing. Except for one patient, discrepancies were resolved in favor of PCR-CMPH: three mixed infections were detected, four species identification errors were corrected, and two negative results were shown to be positive. Our results indicate that PCR-CMPH is a simple, rapid, and specific method for malaria diagnosis. It employs stable reagents and inexpensive equipment, making it suitable for routine epidemiological use.
ISSN:0095-1137
1098-660X
1098-5530
DOI:10.1128/JCM.00014-06