Abstract
Background Cystic echinococcosis is a zoonotic disease caused by the metacestode stage of Echinococcus granulosus and occurs worldwide, causing considerable economic losses and public health problems. The currently available methods for the diagnosis of animal hydatidosis are time-consuming and require well-equipped laboratories which make them incompatible with testing in resource-poor settings. This study developed and evaluated a rapid, more sensitive, and specific loop-mediated isothermal amplification combined with a lateral flow dipstick assay for the rapid and sensitive detection of cystic echinococcosis. Results In this study, a specific primer set and FITC-labeled probe targeting the conserved region of the NADH-1 gene were designed. The LAMP reaction was performed at 60°C for 40 minutes, and the amplification products were successfully visualized by LFD strips. The analytical sensitivity of LAMP-LFD was determined using 10-fold serial dilutions of E. granulosus DNA. The minimal concentration detected was 10 fg/μl, and no cross-reactivity was observed with DNA extracted from Taenia solium, Taenia saginata, and Fasciola hepatica. The ability of the developed LAMP-LFD assay to detect cystic echinococcosis was further evaluated with 62 cyst samples from slaughtered cattle in Juja Abattoir, Kiambu County, Kenya. The LAMP-LFD was able to detect 59/62 (95.2%, 95% CI 0.87–0.98) as positive samples of E. granulosus compared to 53/62 (85.5%, 95% CI 0.75–0.92) by nested PCR assay. Conclusion Our results indicated that the developed LAMP-LFD technique was more sensitive than the nested PCR assay, rapid, and easy to perform with a simple visual detection of products. Therefore, it could be an important point-of-care diagnostic tool for cystic echinococcosis.
Highlights
Cystic echinococcosis is a zoonotic disease caused by the metacestode stage of Echinococcus granulosus and occurs worldwide, causing considerable economic losses and public health problems. e currently available methods for the diagnosis of animal hydatidosis are time-consuming and require well-equipped laboratories which make them incompatible with testing in resource-poor settings. is study developed and evaluated a rapid, more sensitive, and specific loop-mediated isothermal amplification combined with a lateral flow dipstick assay for the rapid and sensitive detection of cystic echinococcosis
Cystic echinococcosis (CE) has an economic implication because any organ found to be infected with E. granulosus during meat inspection is condemned and judged to be unfit for human consumption, and this may lead to food insecurity due to the loss of meat for human consumption [2, 3]. e life cycle of E. granulosus involves two animal hosts, the dog being the main host for the definitive life cycle stage, whereas the hydatid cyst is found in many warm-blooded animals including cattle, sheep, goats, and camels, as well as in humans [4]
Successful amplification of the target gene (NADH-1 gene) confirmed the specificity of the primers for E. granulosus. e optimum temperature for the Loop-mediated isothermal amplification (LAMP) reaction was 63°C, and the optimal time was 40 minutes. e MgSO4 concentration of 6 mM and 0.4 mM of dNTP mix was optimum for the LAMP assays
Summary
Cystic echinococcosis is a zoonotic disease caused by the metacestode stage of Echinococcus granulosus and occurs worldwide, causing considerable economic losses and public health problems. e currently available methods for the diagnosis of animal hydatidosis are time-consuming and require well-equipped laboratories which make them incompatible with testing in resource-poor settings. is study developed and evaluated a rapid, more sensitive, and specific loop-mediated isothermal amplification combined with a lateral flow dipstick assay for the rapid and sensitive detection of cystic echinococcosis. E ability of the developed LAMP-LFD assay to detect cystic echinococcosis was further evaluated with 62 cyst samples from slaughtered cattle in Juja Abattoir, Kiambu County, Kenya. Our results indicated that the developed LAMP-LFD technique was more sensitive than the nested PCR assay, rapid, and easy to perform with a simple visual detection of products. Cystic echinococcosis (CE) is a neglected parasitic disease caused by the larval stage of the tapeworm Echinococcus granulosus (sensu lato), and infections involve both human and ungulate animals [1]. Several serological examination techniques have been developed for the detection of E. granulosus These methods have low sensitivity and cross-reactivity is likely to outcome with other taeniid cestodes [6, 8]
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