Development of PCR, qPCR and LAMP methods for the detection of Spirometra mansoni (Cestoda: Diphyllobothriidae) in the faeces of dogs and cats.

Spirometra mansoni is an important zoonotic parasitic tapeworm that is transmitted mainly through the faeces of definitive hosts such as cats and dogs. However, there is currently no molecular detection method for S. mansoni in the faeces of definitive hosts. Here, a PCR assay for S. mansoni in the faeces of definitive hosts was developed, and the effects of the sampling site, sample storage temperature and duration on the detection results were evaluated. qPCR assays and LAMP assays targeting the cytb gene were performed with optimized primers, probe concentrations and annealing temperatures. The sensitivity and specificity of three assays, namely, PCR, qPCR and LAMP, were evaluated. Applications in the field were conducted using these established assays. The sensitivity of the cox1 gene to PCR was 0.7 ng/μL (egg-derived DNA) and 1.4 ng/μL (cat faecal DNA). The sampling site had no notable effect on the detection results, and target genes could still be effectively detected in samples after 180 days of storage at 37 °C, 25 °C, 4 °C, -20 °C and - 80 °C. The qPCR assay demonstrated a sensitivity of 100 copies/μL, with an amplification efficiency of 107.625 % (R2 = 0.997), and the intrabatch/interbatch coefficients of variation (CVs) were < 5 %, indicating good repeatability and suitability for quantitative detection. The sensitivity of the LAMP assay was 7.47 pg/μL (cat faecal DNA) and 355.5 fg/μL (egg-derived DNA). All three assays showed good specificity and no cross-reaction with the DNA of other common parasites in cat and dog faeces. A total of 218 stool samples were tested using three assays, all of which were negative. Our study successfully established PCR, qPCR and LAMP detection systems for S. mansoni in the faeces of definitive hosts, with the advantages of high sensitivity, strong specificity and operational simplicity, which are suitable for early diagnosis of infection of definitive hosts with S. mansoni and for epidemiological assessment of the spillover risk of sparganosis.