title: Rapid detection of Opisthorchis viverrini copro-DNA using loop-mediated isothermal amplification (LAMP).
abstract: Opisthorchis viverrini and other foodborne trematode infections are major health problem in Thailand, the Lao People's Democratic Republic, Vietnam and Cambodia. Differential diagnosis of O. viverrini based on the microscopic observation of parasite eggs is difficult in areas where Clonorchis sinensis and minute intestinal flukes coexist. We therefore established a rapid, sensitive and specific method for detecting O. viverrini infection from the stool samples using the loop-mediated isothermal amplification (LAMP) method. A total of five primers from seven regions were designed to target the internal transcribed spacer 1 (ITS1) in ribosomal DNA for specific amplification. Hydroxy naphthol blue (HNB) was more effective to detect the LAMP product compared to the Real-time LAMP and turbidity assay for its simple and distinct detection. The LAMP assay specifically amplified O. viverrini ITS1 but not C. sinensis and minute intestinal flukes with the limit of detection around 10(-3)ng DNA/μL. The sensitivity of the LAMP was 100% compared to egg positive samples. While all microscopically positive samples were positive by LAMP, additionally 5 of 13 (38.5%) microscopically negative samples were also LAMP positive. The technique has great potential for differential diagnosis in endemic areas with mixed O. viverrini and intestinal fluke infections. As it is an easy and simple method, the LAMP is potentially applicable for point-of-care diagnosis.
authors.affiliation: Tropical Disease Research Laboratory, Division of Experimental Pathology, Department of Pathology, Faculty of Medicine, Khon Kaen University, Khon Kaen 40002, Thailand.
authors.forename: Yuji
authors.initials: Y
authors.lastname: Arimatsu
authors.forename: Sasithorn
authors.initials: S
authors.lastname: Kaewkes
authors.forename: Thewarach
authors.initials: T
authors.lastname: Laha
authors.forename: Sung-Jong
authors.initials: SJ
authors.lastname: Hong
authors.forename: Banchob
authors.initials: B
authors.lastname: Sripa
mesh_terms: D000818:Animals; D060085:Coinfection / Q000175:diagnosis; D017931:DNA Primers; D018509:DNA, Helminth / Q000032:analysis*; D021903:DNA, Ribosomal Spacer; D003937:Diagnosis, Differential; D005243:Feces / Q000469:parasitology*; D006801:Humans; D009282:Naphthalenesulfonates / Q000737:chemistry; D009391:Nephelometry and Turbidimetry; D021141:Nucleic Acid Amplification Techniques / Q000379:methods*; D009889:Opisthorchiasis / Q000175:diagnosis* / Q000469:parasitology; D009891:Opisthorchis / Q000235:genetics / Q000302:isolation & purification*; D010274:Parasitology / Q000379:methods*; D012680:Sensitivity and Specificity; D014201:Trematode Infections / Q000175:diagnosis
chemical_list: D017931:DNA Primers; D018509:DNA, Helminth; D021903:DNA, Ribosomal Spacer; D009282:Naphthalenesulfonates; C540250:trisodium 3-hydroxy-4-((2Z)-2-(2-oxo-4-sulfonatonaphthalen-1-ylidene)hydrazinyl)naphthalene-2,7-disulfonate