Clonorchis sinesis and Opisthorchis viverrini: development of a mitochondrial-based multiplex PCR for their identification and discrimination
Le, Thanh, Van De, Nguyen, Blair, David, Sithithaworn, Paiboon, and McManus, Donald (2006) Clonorchis sinesis and Opisthorchis viverrini: development of a mitochondrial-based multiplex PCR for their identification and discrimination. Experimental Parasitology, 112 (2). pp. 109-114.
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We report a single, one-step PCR approach for detection and discrimination of Clonorchis sinensis and Opisthorchis viverrini in different life-stage forms (adults, metacercariae, and eggs) from fish intermediate hosts and from infected patients. Primers designed for species-specific PCR, amplifying portions of the mitochondrial (mt) genome, were also suitable for a multiplex PCR. The latter was a single, one-step reaction under high stringency conditions, using simultaneously 2 pairs of primers (1 pair for C. sinensis—product size 612 bp, and 1 pair for O. viverrini—product size 1357 bp). Assays using serially diluted templates demonstrated that as little as 0.78 ng of genomic DNA of either species could yield amplicons. Genomic DNA extracted from different life-stage forms including adult worms (of both species), eggs (of O. viverrini), eggs possibly of several trematode species (collected from patients infected with C. sinensis in Vietnam) and mixed metacercariae of common trematodes (collected from fishes in the C. sinensis endemic areas), yielded specific bands of the correct size and their identity was confirmed by sequence analysis. The multiplex PCR approach described here proved to be a species-specific, sensitive and fast tool for accurate diagnosis of clonorchiasis and/or opisthorchiasis, permitting the detection of their metacercariae in infected fishes or adult/eggs from patients in endemic areas.
|Item Type:||Article (Refereed Research - C1)|
|Keywords:||Mitochondrial DNA; Clonorchis sinensis; Opisthorchis viverrini; Metacercariae; multiplex PCR; specificity; sensitivity|
|Date Deposited:||26 Nov 2009 05:29|
|FoR Codes:||10 TECHNOLOGY > 1099 Other Technology > 109999 Technology not elsewhere classified @ 100%|
|SEO Codes:||92 HEALTH > 9202 Health and Support Services > 920203 Diagnostic Methods @ 100%|
|Citation Count from Web of Science||