# The Development and Evaluation of a Loop-Mediated Isothermal Amplification (LAMP) Method for the Detection of Spirometra mansoni in Dogs

## TL;DR

A new LAMP method was developed to detect Spirometra mansoni eggs in dog feces, offering a faster and more specific diagnostic tool for sparganosis.

## Contribution

The study introduces a novel LAMP assay for rapid and specific detection of S. mansoni eggs in dog feces.

## Key findings

- The LAMP assay detected S. mansoni eggs in 70.21% of stray dog fecal samples.
- LAMP showed a typical ladder pattern and fluorescent signal only in S. mansoni-infected samples.
- LAMP outperformed PCR in detection rate and could detect eggs at earlier infection stages.

## Abstract

Spirometra mansoni (S. mansoni) is a zoonotic parasite with a global distribution, infecting humans as an intermediate host. After entering the human body, spargana larvae migrate to various body parts. Adult S. mansoni parasitizes the small intestines of dogs and cats. There are over 50 million stray dogs and cats in China, which may accelerate the incidence of sparganosis. Epidemiological examination and clinical diagnosis of S. mansoni are quite limited. Common serum immunological detection methods are not suitable for popularization and low-resource applications. The dot immunogold filtration assay (DIGFA) for sparganosis detection has cross-reactions with lung fluke disease and cysticercosis. Recently, loop-mediated isothermal amplification (LAMP) has been developed. The LAMP assay was successfully employed for the specific and rapid diagnosis of several parasitic infections. However, reports about the analysis for sparganosis using the LAMP technique are limited. Here, a LAMP assay which can detect S. mansoni eggs within 60 min at a constant temperature has been established. The performances of LAMP assays showed clear differences between the negative control and S. mansoni-infected samples.

Spirometra mansoni is a zoonotic parasite that inhabits the intestines of dogs and cats. The plerocercoids (spargana) parasitize several vertebrates, including humans, resulting in a food-borne zoonosis known as sparganosis. In this study, it has been established that a LAMP assay can detect S. mansoni eggs in dog feces. A total of 97 fecal samples were collected from Changsha City, Hunan Province. The fecal DNA was extracted before designing primers for LAMP based on the S. mansoni cox1 gene. The specificity of this method was verified by PCR using LAMP outer primers or inner primers and nested PCR with S. mansoni-specific cox1 primers. DNA samples from five control dog worms were analyzed using the LAMP assay to evaluate the specificity. The detection rate of LAMP for S. mansoni eggs was 70.21% in stray dogs. PCR and nested PCR produced specific bands on agarose gel electrophoresis consistent with the expected length. When the LAMP assay was conducted using S. mansoni-infected samples, negative samples, and genomic DNA from control worms, only the S. mansoni-infected samples showed a typical ladder pattern. The samples were stained with SYBR Green I, and only the S. mansoni-infected samples had a fluorescent signal. In addition, compared with PCR and microscope, LAMP method can detect eggs in the shortest infection days, and its detection rate was higher than that of PCR. These results suggest that the established LAMP method have many advantages in detecting Spirometra mansoni.