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

IF 3.1 Q2 PARASITOLOGY

Food and Waterborne Parasitology Pub Date : 2025-09-17 DOI:10.1016/j.fawpar.2025.e00291

Wen Li, Xuan Xuan Song, Si Si Ru, Jie Hao, Cheng Yue Cao, Xi Zhang

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引用次数: 0

Abstract

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.

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犬、猫粪便中曼氏螺旋体PCR、qPCR和LAMP检测方法的建立

曼氏肺metra mansoni是一种重要的人畜共患寄生虫绦虫，主要通过猫和狗等最终宿主的粪便传播。然而，目前尚无最终宿主粪便中曼氏链球菌的分子检测方法。本研究建立了一种检测终寄主粪便中的曼氏链球菌的PCR方法，并评估了采样地点、样本保存温度和时间对检测结果的影响。在优化的引物、探针浓度和退火温度下，对cytb基因进行qPCR和LAMP检测。评价PCR、qPCR和LAMP三种检测方法的敏感性和特异性。使用这些已建立的分析方法在现场进行了应用。cox1基因的PCR敏感性分别为0.7 ng/μL（蛋源DNA）和1.4 ng/μL（猫粪DNA）。采样位置对检测结果无显著影响，在37℃、25℃、4℃、- 20℃、- 80℃条件下保存180天后，样品仍能有效检测到目的基因。qPCR检测灵敏度为100拷贝/μL，扩增效率为107.625% (R2 = 0.997)，批内/批间变异系数（CVs）为5%，重复性好，适合定量检测。LAMP检测的灵敏度分别为7.47 pg/μL（猫粪便DNA）和355.5 fg/μL（蛋源DNA）。3种方法均具有良好的特异性，且与猫、狗粪便中其他常见寄生虫的DNA无交叉反应。共对218份粪便样本进行了三种化验，结果均为阴性。本研究成功建立了最终宿主粪便中曼氏梭菌的PCR、qPCR和LAMP检测系统，具有灵敏度高、特异性强、操作简便等优点，适用于最终宿主感染曼氏梭菌的早期诊断和斯巴格纳病外溢风险的流行病学评估。

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来源期刊

Food and Waterborne Parasitology Immunology and Microbiology-Parasitology

CiteScore

5.10

自引率

4.00%

发文量

审稿时长

13 weeks

期刊介绍： Food and Waterborne Parasitology publishes high quality papers containing original research findings, investigative reports, and scientific proceedings on parasites which are transmitted to humans via the consumption of food or water. The relevant parasites include protozoa, nematodes, cestodes and trematodes which are transmitted by food or water and capable of infecting humans. Pertinent food includes products of animal or plant origin which are domestic or wild, and consumed by humans. Animals and plants from both terrestrial and aquatic sources are included, as well as studies related to potable and other types of water which serve to harbor, perpetuate or disseminate food and waterborne parasites. Studies dealing with prevalence, transmission, epidemiology, risk assessment and mitigation, including control measures and test methodologies for parasites in food and water are of particular interest. Evidence of the emergence of such parasites and interactions among domestic animals, wildlife and humans are of interest. The impact of parasites on the health and welfare of humans is viewed as very important and within scope of the journal. Manuscripts with scientifically generated information on associations between food and waterborne parasitic diseases and lifestyle, culture and economies are also welcome. Studies involving animal experiments must meet the International Guiding Principles for Biomedical Research Involving Animals as issued by the Council for International Organizations of Medical Sciences.