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% compare 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 is is an easy and simple method, the LAMP is potentially applicable for point-of-care diagnosis.

Development of Mitochondrial Loop-Mediated Isothermal Amplification for Detection of the Small Liver Fluke Opisthorchis viverrini (Opisthorchiidae; Trematoda; Platyhelminthes)

“…More effective methods to detect every life stage of this dangerous worm are required. Until now, a variety of microscopic, immunological, and PCR (and real-time PCR) approaches have been used to detect metacercariae in fish and to detect eggs and adult worms as well as antibodies in late-stage opisthorchiasis patients (24), including a LAMP assay using the internal transcribed spacer 1 (ITS-1) in the nuclear ribosomal gene cluster as a target (2). However, it would be more reasonable to use a mitochondrial target for a LAMP assay, due to its stability and the likely higher copy number for provision of DNA template.…”

“…LAMP has been successfully developed for rapid detection of a range of zoonotic platyhelminths, such as Fasciola hepatica and Fasciola gigantica (1), Clonorchis sinensis (4), Schistosoma japonicum (29), Taenia spp. (16), Paragonimus westermani (6), and, recently, O. viverrini (2). For these LAMP reactions, nuclear genes were mainly chosen as the target regions.…”

Molecular Testing for Clinical Diagnosis and Epidemiological Investigations of Intestinal Parasitic Infections

“…A similar approach with primers and a hydrolysis probe designed from the C. sinensis ITS1 sequence showed specific (with the exception of O. viverrini) and sensitive detection of C. sinensis in a small number of fecal samples and fish tissue samples (370) Primer sets for the detection of O. viverrini DNA in stool samples using LAMP were designed from the mitochondrial nad1 sequence and from the ribosomal ITS1 sequence of O. viverrini (373,374). The ITS1-based LAMP assay showed a 100% sensitivity compared to microscopy when tested on stool samples (n ϭ 50) from schoolchildren from an area of endemicity in Thailand, compared to a remarkably low sensitivity of only 24% using conventional PCR on the same target (373).…”

Visual Detection of Isothermal Nucleic Acid Amplification Using pH-Sensitive Dyes

“…Furthermore, calcein requires inclusion of the ionic form of manganese (14,18), which can inhibit polymerase reactions. Malachite green features an additional color change in the negative reactions, with a change from dark blue to clear (16,17).…”