Opisthorchis viverrini: Detection by polymerase chain reaction (PCR) in human stool samples

Umesha, Kanasinakatte R.; Kumar, Sanath; Parvathi, Ammini; Duenngai, Kunyarat; Sithithaworn, Paiboon; Karunasagar, Indrani

doi:10.1016/j.exppara.2008.09.004

Cited by 24 publications

(19 citation statements)

References 25 publications

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“…This problem is even more evident when the eggs are degraded (Briski et al, 2011). Thus, researchers choose to lyse the eggs for release of larvae prior to DNA extraction using heat shock (Traub et al, 2009), saline (Buathong et al, 2010), autoclave techniques (Stensvold et al, 2006;Duenngai et al, 2008;Umesha et al, 2008) or crushing (Sato et al, 2009). Using this new method based on Chelex ® 100 resin described in this study, we could neutralize inhibitors, since regardless of the number of eggs, all samples were satisfactorily amplified.…”

Section: Resultsmentioning

confidence: 99%

Novel DNA extraction assay for molecular identification of Aedes spp eggs

Freitas¹,

Gomes-Júnior²,

Batista³

et al. 2014

Genet. Mol. Res.

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ABSTRACT.Aedes aegypti and A. albopictus represent the two most important species of mosquitoes in relation to dengue virus transmission both in the Americas and Asia. However, the study of theses species generally requires the establishment of a colony for the larvae to hatch, or waiting for the adult development to perform its taxonomic classification, which is time consuming. Thus, the establishment of new methods aimed at obtaining DNA directly from the mosquito eggs is relevant. Accordingly, we compared a new approach based on Chelex ® 100 resin with the standard STE method to extract DNA from the eggs of Aedes spp to molecularly identify these vectors. The Chelex ® 100 resin approach was very efficient, as satisfactory amounts of DNA were obtained, making it possible to amplify and sequence a mitochondrial DNA barcode region widely used to identify species. The STE protocol yielded substantial amounts of DNA, but the 260/280 optical density ratio indicated a low quality, precluding amplification. This new method proved quite effective in obtaining DNA from even a single mosquito egg, and it can thus be applied in population genetic studies of various vector insects to enhance monitoring programs.

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Section: Resultsmentioning

confidence: 99%

Novel DNA extraction assay for molecular identification of Aedes spp eggs

Freitas¹,

Gomes-Júnior²,

Batista³

et al. 2014

Genet. Mol. Res.

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“…Detection of O. viverrini -specific sequences by cPCR for the diagnosis of human opisthorchiasis in fecal samples has yielded a varying sensitivity, [13][14][15][16] which shows the need for a more rapid, sensitive, and specific method. We used OV-F and OV-R primers for diagnosis of O. viverrini infection in human fecal specimens by cPCR.…”

Section: Discussionmentioning

confidence: 99%

“…Regarding the specificity of the procedure, no fluorescence was visible and primers did not amplify the 162-basepair band when DNA extracted from other parasitepositive stools and negative control samples was tested, which indicated a specificity of 100%. The real-time FRET PCR protocol offers an alternative to immunologic 26 or molecular methods [13][14][15][16] for detection of O. viverrini in fecal specimens. By virtue of this rapid method, the entire protocol of the real-time FRET PCR can be completed in less than one hour (after extraction of genomic DNA).…”

Section: Discussionmentioning

confidence: 99%

“…Furthermore, this method is time-consuming. To overcome the constraints of the microscopic method, various sensitive and specific molecular methods such as classic polymerase chain reaction (cPCR)-based techniques capable of DNA amplification from helminthic eggs in stool samples have been reported, [12][13][14][15][16] but these methods still lack the desired reliability.…”

Section: Introductionmentioning

confidence: 99%

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Rapid Molecular Detection of Opisthorchis viverrini in Human Fecal Samples by Real-Time Polymerase Chain Reaction

Intapan

Thanchomnang²,

Lulitanond³

et al. 2009

The American Journal of Tropical Medicine and Hygiene

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Abstract. Real-time fluorescence resonance energy transfer (FRET) polymerase chain reaction (PCR) supplemented with melting curve analysis is a highly sensitive and fast method offering a high throughput. We report the development of a real-time FRET PCR for molecular detection of Opisthorchis viverrini in human fecal samples. The diagnostic sensitivity, specificity, accuracy, and positive and negative predictive values of this method were 97.5%, 100%, 98.9%, 100%, and 98.2%, respectively. The sensitivity was not significantly different from that of the quantified formalin-ethyl acetate concentration technique, the gold standard ( P > 0.05). The procedure has potential for diagnosis of human opisthorchiasis in disease-endemic areas, for large epidemiologic investigations involving at risk populations, and monitoring eradication programs of the liver fluke, which causes hepatobiliary diseases and induces cholangiocarcinoma.

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“…Most recently, PCR tests were developed to discriminate between O. viverrini, H. taichui, and another food-borne trematode, Clonorchis sinensis (22,31,32). Some studies using PCR tests on human stool samples were validated with light microscopy (27,32,33). A more accurate method such as the identification of adult flukes would be valuable to validate PCR tests.…”

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confidence: 99%