Rapid detection of Opisthorchis viverrini copro-DNA using loop-mediated isothermal amplification (LAMP)

• Published in: Parasitology international Vol. 61; no. 1; pp. 178 - 182
• Main Authors: Arimatsu, Yuji, Kaewkes, Sasithorn, Laha, Thewarach, Hong, Sung-Jong, Sripa, Banchob
• Format: Journal Article
• Language: English
• Published: Netherlands Elsevier Ireland Ltd 01.03.2012
• Subjects: Animals; Anopheles sinensis; Clonorchis sinensis; Coinfection - diagnosis; detection limit; Diagnosis, Differential; DNA Primers; DNA, Helminth - analysis; DNA, Ribosomal Spacer; eggs; feces; Feces - parasitology; flukes; Gastroenterology and Hepatology; Humans; Infectious Disease; Internal transcribed spacer 1; Internal transcribed spacer 2; internal transcribed spacers; LAMP; Loop-mediated isothermal amplification; Naphthalenesulfonates - chemistry; Nephelometry and Turbidimetry; Nucleic Acid Amplification Techniques - methods; Opisthorchiasis - diagnosis; Opisthorchiasis - parasitology; Opisthorchis - genetics; Opisthorchis - isolation & purification; Opisthorchis viverrini; parasitology; Parasitology - methods; people; ribosomal DNA; Sensitivity and Specificity; Stool examination; Trematoda; trematode infections; Trematode Infections - diagnosis; turbidity; Cambodia; Thailand; Vietnam; Loop-mediated isothermal amplification; Internal transcribed spacer 2; Internal transcribed spacer 1; LAMP; Opisthorchis viverrini; Stool examination
• ISSN: 1383-5769; 1873-0329
• DOI: 10.1016/j.parint.2011.08.009

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.