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. 2025 Sep 17;41:e00291. doi: 10.1016/j.fawpar.2025.e00291

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

Wen Li 1, Xuan Xuan Song 1, Si Si Ru 1, Jie Hao 1, Cheng Yue Cao 1, Xi Zhang 1,⁎
PMCID: PMC12508883  PMID: 41079405

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.

Keywords: Spirometra mansoni, Molecular diagnostics, PCR, Quantitative real-time PCR, Loop-mediated isothermal amplification

Highlights

  • •

    Established PCR, qPCR, and LAMP detection methods for Spirometra mansoni in cat and dog faeces.

  • •

    The qPCR sensitivity reached 100 copies/μL, with good reproducibility (CV < 5 %), indicating accurate quantification.

  • •

    The LAMP method exhibited high sensitivity, allowing for visual interpretation, and is suitable for rapid onsite screening.

  • •

    All methods demonstrated strong specificity, with no cross-reaction with common parasites.

1. Introduction

Adult Spirometra mansoni (Cestoda: Diphyllobothriidae) mainly parasitize feline/canine animals, whereas the metacestode plerocercoid can parasitize various vertebrates, including humans, causing a food/water-borne parasitic zoonosis known as sparganosis (Kuchta et al., 2024). It manifests mainly as larva migrans that may involve the whole body, resulting in blindness, limb paralysis, and even death (Liu et al., 2024a, Liu et al., 2024b; Iampreechakul et al., 2025). Sparganosis is highly prevalent in eastern and southeastern Asian countries. To date, more than 1300 cases have been reported in China, accounting for the highest global burden (Kuchta et al., 2021). As sparganosis is mostly sporadic and has not received attention, it is considered a neglected parasitic disease. Consequently, S. mansoni, its causative agent, remains a chronically overlooked zoonotic cestode.

Cats and dogs, as definitive hosts of S. mansoni, play important roles in the transmission of sparganosis. Adult worms exhibit high egg output, and the large number of eggs or segments released can cause significant environmental contamination (Zhang et al., 2020). The extensive roaming behaviour of cats facilitates large-scale egg dissemination. Cats and dogs are among the most important pets worldwide, with large populations and many potential risk sources (Mazzotta et al., 2024). With the acceleration of urbanization, the number of stray cats and dogs is also increasing. These animals typically inhabit unmanaged environments with poor sanitation, which creates great challenges for zoonotic disease surveillance. Usually, cats and dogs infected with S. mansoni are in a subclinical state, and their symptoms are not obvious and can be easily ignored (Yamasaki et al., 2021). On the basis of the concept of “one health”, monitoring susceptible companion animals such as dogs and cats is important for the prevention and treatment of sparganosis (Bertram et al., 2024).

At present, the diagnosis of S. mansoni infection relies mainly on traditional stool microscopy. This method has obvious shortcomings, such as low sensitivity and a low detection rate, making early diagnosis and epidemiological investigation difficult (Alvarado-Hidalgo et al., 2024; Salazar-Grosskelwing et al., 2025). Although immunological methods (e.g., ELISA) are simple and easy to implement, there are problems of cross-reaction and false positivity (Liu et al., 2015). Moreover, there are challenges in sampling definitive hosts such as cats and dogs for serology, and such sampling is not conducive to large-scale epidemiological investigations. A PCR assay has not been developed for S. mansoni in stool samples from definitive hosts. Methods such as qPCR and loop-mediated isothermal amplification (LAMP) have been increasingly used in the diagnosis of parasites. The qPCR assay has high sensitivity and specificity. LAMP assays do not require sophisticated equipment because of their isothermal amplification characteristics and are suitable for rapid onsite detection (Lizarazo-Zuluaga et al., 2022; Chen et al., 2023; Ahmadi et al., 2025). The purpose of our study was to develop three molecular assays using PCR, qPCR and LAMP for the detection of S. mansoni and to evaluate their performance using faecal samples collected from Zhengzhou.

2. Materials and methods

2.1. Sample collection and experimental animals

Plerocercoids used in the study were isolated from infected snakes (Zaocys dhumnades) in Changsha, China (Su et al., 2024). Plerocercoids were identified as S. mansoni on the basis of mitochondrial cytochrome c oxidase subunit 1 (cox1) genotyping (Kuchta et al., 2021). Methods for adult worm acquisition were as described previously (Wang et al., 2024). Eggs can be detected in the faeces of infected cats at 8–12 days postinfection. After that, the number of eggs in faeces increases rapidly and plateaus at approximately 19 days after infection (Zhang et al., 2020). Therefore, the stool samples were divided into 3 groups according to the time of infection: the negative group, early infection group (1–14 days after infection), and mid-late infection group (20 days after infection). Approximately 5 g of faeces from the internal core area and surface area was collected, mixed evenly, packed into 2-ml EP tubes, sealed, and stored at −80 °C. To explore the influence of different sampling sites on the detection results, 200 mg of stool sample was collected from the outside at both ends, inside at both ends, outside at the middle and inside at the middle for testing. The overall experimental workflow is shown in Fig. 1.

Fig. 1.

Fig. 1

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Flow chart of the study.

2.2. DNA extraction

Adult worm DNA and egg-derived DNA were extracted using the EasyPure® Genomic DNA Kit (TransGen Biotech Co., Ltd., Beijing, China). Faecal DNA was extracted using a Faecal Genomic DNA Extraction Kit (Beijing Solarbio Science & Technology Co., Ltd., Beijing, China). The DNA concentration was determined using a NanoDrop 2000 ultramicro spectrophotometer, and the extracted DNA was stored at −80 °C.

2.3. Development of PCR, qPCR and LAMP amplification systems

A PCR assay was conducted targeting the mitochondrial cox1 gene (GenBank no: KT376494.1) (Zhang et al., 2016). The primers used were as follows: F: 5′-TGTTTAGGTAGCGTGGTTTGG-3′; R: 5′-ACCACAAACCACGTGTCATGC-3′. To explore whether the sampling site influenced the detection results, faeces were sampled at different sites for triplicate assays. Moreover, to investigate the effects of the stool storage temperature and duration on the detection results, five storage temperatures (37 °C, room temperature (25 °C), 4 °C, −20 °C and − 80 °C) and five storage durations (1 week, 2 weeks, 1 month, 3 months, and 6 months) were tested.

For the qPCR, the mitochondrial cytb gene (no: NC_011037.1) was selected as the target gene (Gong et al., 2022). The sequences were as follows: upstream, 5’-TAATAATCTAGCCACCAACACTA-3′; downstream, 5’-TGTCTATAAAGCCTGAGTG ATA-3′; and TaqMan probe, FAM-AGACTCAACAGAACGAAGCATAGCA-BHQ1. In the qPCR, the reaction results were strongly influenced by the concentrations of primers, probes and Mg2+ (Songjaeng et al., 2022; Liu et al., 2024b). Therefore, the reactions were optimized by optimizing the following primer concentrations: 0.1 μΜ, 0.2 μΜ, and 0.4 μΜ; probe concentrations: 0.25 μΜ, 0.5 μΜ, and 1 μΜ; and Mg2+ concentrations: 1.5 mM, 2 mM, and 2.5 mM. Given the effect of the annealing temperature on the specificity and accuracy of the reaction, gradient temperatures (55 °C, 60 °C, and 65 °C) were used to determine the optimal annealing temperature (Chen et al., 2025).

For the LAMP reaction, Primer Explorer V5 (https://primerexplorer.eiken.co.jp/e/) was used to design primers according to the principles in the literature (Panno et al., 2020), and different primer combinations were designed to target the cytb and nad5 genes, as detailed in Supplementary Table S1. By setting different internal and external primer ratios (32: 1, 16: 1, 8: 1, 4: 1, and 2: 1) and different reaction temperatures (60 °C, 61 °C, 62 °C, 63 °C, 64 °C, and 65 °C) for optimization, the optimal system and amplification temperature for LAMP were determined. LAMP reaction tubes (fluorescent dye method) were supplemented with 1.25 μL of EveGreen dye (20 ×) and detected by a QuantStedio5 quantitative real-time PCR instrument. LAMP reaction tubes (pH indicator method) were supplemented with 1.25 μL of neutral red dye (20 ×), and the colour change was observed with the naked eye. All primers were validated using Oligo7, eliminating primer dimers and hairpins, and specificity was validated using NCBI BLAST. The primers used were synthesized by Sangon Biotech (Shanghai) Co., Ltd. (Shanghai, China).

2.4. Determination of sensitivity

To determine the PCR sensitivity and limit of detection (LOD), DNA extracted from eggs and positive faeces was serially diluted twofold prior to the assay. The amplified products were analysed by 1.5 % agarose gel electrophoresis, and the lowest concentration at which a positive band appeared was determined as the LOD for the PCR assay (Liu et al., 2017). Moreover, detection was carried out for infected groups at different time points after infection to determine whether a PCR assay can be used for the early diagnosis of S. mansoni (Luo et al., 2025).

To determine the sensitivity of qPCR and ensure qualitative detection, amplification products from qPCR were purified using a DNA Gel Extraction Kit (Sangon Biotech (Shanghai) Co., Ltd., Shanghai, China) and ligated to the cloning vector pUC57 to construct a recombinant plasmid. The identified positive recombinant plasmids were subjected to tenfold serial dilutions ranging from 3.33 × 102 to 3.33 × 10−8 ng/μL, and 1 μL of plasmid at different concentrations was added to each reaction tube as a standard template, followed by amplification under optimal reaction conditions. After reaction was complete, a standard curve was plotted with the logarithm of the starting template copy number as the x-axis and the cycle threshold (Ct) value as the y-axis. The lowest plasmid concentration that could be detected was considered the LOD of the qPCR assay. In addition, to ensure the reliability of the assay data, three groups were established to validate the intrabatch and interbatch repeatability: (1) The same sample was processed in 6 replicates per run over 6 consecutive runs under optimal reaction conditions, and the intra- and interassay CVs for Ct values were calculated. (2) Three positive plasmids at high, medium and low concentrations were processed, and the intra-assay CVs for Ct values were calculated. (3) Three positive plasmid samples at different concentrations were processed for five consecutive runs, and the intra-assay and interassay CVs for Ct values were calculated.

To determine the sensitivity of the LAMP reaction, LAMP amplification was performed for positive faecal DNA and plerocercoid DNA in 10-fold serial dilutions under optimal reaction conditions, with nine serial dilutions (101–109) prepared, and ddH2O served as the negative control. The reaction was carried out on a QuantStudio 5 quantitative real-time PCR system, and amplification was performed at a constant temperature for 60 min at the optimal temperature, with fluorescence signals collected at 1-min intervals. The lowest DNA concentration at which a positive result was detected was the LOD.

2.5. Determination of specificity

To validate the specificity of the detection methods, parasites commonly found in cat and dog faeces, including Echinococcus granulosus, Hymenolepis diminuta, Schistosoma japonicum, Clonorchis sinensis, Ancylostoma caninum, Trichinella spiralis, Toxoplasma gondii, Giardia duodenalis and Cryptosporidium sp., were selected as controls for specificity (Table 1). DNA from control parasites was extracted for PCR, qPCR and LAMP assays, and whether cross-reactions occurred in each molecular detection method was determined according to the amplification results.

Table 1.

Parasites used as specificity controls.

Parasites infecting cats and dogs with faecal shedding Excretion Type References
Schistosoma japonicum eggs Carabin et al., 2005
Clonorchis sinensis eggs Li et al., 2024
Echinococcus granulosus eggs or gravid proglottids Wang et al., 2024
Hymenolepis diminuta gravid proglottid Rojekittikhun et al., 2014
Ancylostoma caninum eggs Venkatesan et al., 2023
Trichinella spiralis parasite fragment Omeragic et al., 2024
Giardia lamblia cyst Ryan et al., 2021
Cryptosporidium sp. oocyst Mukbel et al., 2024
Toxoplasma gondii oocyst Liang et al., 2024
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2.6. Epidemiological investigation

To investigate S. mansoni infection in cats and dogs (both pet and stray) and evaluate the spillover risk of sparganosis, we collected 55 stray dog faeces samples from Zhengzhou Stray Animal Center and 21 stray cat faeces samples from urban suburbs, as well as samples from 28 pet cats and 50 pet dogs in different residential areas, and detected them using three established assays. In addition, for other felids and canids, 22 tiger faeces samples, 21 lion faeces samples, 17 Vulpes zerda faeces samples and 4 leopard faeces samples were collected at the zoo for testing (Supplementary Fig. S1 and Table S2).

3. Results

3.1. PCR assay

On the basis of the genomic sequence of S. mansoni (Supplementary Table S3), 6 sets of PCR primers were designed, among which cox1F1R1 was chosen for subsequent experiments because of its high specificity and 99.64 % identity to the reference gene, as demonstrated by sequencing, whereas the other primer combinations resulted in nonspecific bands when negative faecal samples were tested (Supplementary Fig. S2).

Following a series of dilutions of the template DNA (Supplementary Table S4), ova-derived DNA was detectable at a dilution of 1: 128 (sensitivity of 0.7 ng/μL), and faecal DNA was detectable at a dilution of 1: 64 (sensitivity of 1.4 ng/μL) (Fig. 2A, B). Moreover, compared with the direct smear method, the positive faecal DNA method yielded positive results as early as 9 days after infection, 2 days earlier (Fig. 2C). DNA was extracted and detected from samples obtained from different regions of the faeces (lateral, medial, middle-lateral, and middle-medial), with positive bands observed at all sampling sites, demonstrating no significant differences in intensity (Fig. 2D, E). One-way ANOVA based on the grey value of the gel electrophoresis band brightness analysis indicated that there was no significant difference between the different sampling sites (F = 0.599, p = 0.633). The variance homogeneity test also confirmed this hypothesis (p = 0.669). Samples stored under different temperature conditions were tested. At 37 °C, the band brightness decreased with prolonged storage time, whereas samples stored at 25 °C, 4 °C, −20 °C, and − 80 °C for 180 days maintained high brightness (Supplementary Fig. S3).

Fig. 2.

Fig. 2

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Determination of the sensitivity of the PCR assay. Lane M is the 2000 Marker. A. Agarose gel electrophoresis diagram for PCR products of egg-derived DNA samples in serial dilutions and the corresponding histograms of grey value analysis of the bands. Lanes 1–8: DNA samples diluted 1:2–1:256. Lane 9: blank control. B. Agarose gel electrophoresis diagram of PCR products of faecal DNA in serial dilutions. Lanes 1–7: DNA samples diluted 1:2–1:128. Lane 8: blank control. C. Lanes 1–14: amplification results of cat faecal DNA 1–14 days after infection, respectively. Lanes 15–17: results of positive, negative and blank controls, respectively. D. Pattern diagram of sampling sites in faeces; the sampling sites corresponding to 1, 2, 3 and 4 in the figure are outside at both ends, inside at both ends, and outside at the middle and inside at the middle, respectively. Created with BioGDP.com (Jiang et al., 2025). E. Electrophoretograms of amplified products from different sampling sites. Agarose gel electrophoresis diagrams in Lanes 1–3, 4–6, 7–9 and 10–12 correspond to PCR-amplified faecal DNA collected from the outside at both ends, inside at both ends, outside at the middle and inside at the middle, respectively. Lanes 13–15 represent the results of the positive control, negative control and blank control, respectively.

3.2. qPCR assay

The results of primer concentration optimization revealed that the amplification effect was optimal at 0.4 μM, where a smooth amplification curve, stable Ct value and high signal intensity were observed (Fig. 3A). The probe concentration optimization results revealed that the fluorescence signal was stable at 0.5 μM without amplification curve jitter; thus, 0.5 μM was considered the optimal probe concentration (Fig. 3B). According to the Mg2+ concentration optimization results, 2.0 mM was determined to be the optimal Mg2+ concentration (Fig. 3C). The annealing temperature optimization suggested that the amplification curve was smooth and that the Ct value was small at 60 °C and 55 °C. Considering that the higher the annealing temperature was, the higher the specificity of the reaction was, 60 °C was selected as the optimal annealing temperature (Fig. 3D). Finally, the qPCR procedure involved predenaturation at 95 °C for 30 s, followed by 40 cycles of denaturation at 95 °C for 5 s and annealing at 60 °C for 30 s.

Fig. 3.

Fig. 3

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Amplification curves of qPCR under different reaction conditions. A. The primer concentrations of 1, 2, and 3 were 0.1 μM, 0.2 μM, and 0.4 μM, respectively. B. The probe concentrations of 1, 2, and 3 were 0.25 μM, 0.5 μM, and 1 μM, respectively. C. The Mg2+ concentrations of 1, 2, and 3 were 1.5 mM, 2.0 mM, and 2.5 mM, respectively. D. The annealing temperatures of 1, 2, and 3 were 55 °C, 60 °C, and 65 °C, respectively. The amplification curve and Ct values revealed that the optimal concentrations of primer, probe, and Mg2+ and temperature were 0.2 μM, 0.5 μM, 2.0 mM and 60 °C, respectively.

To determine the sensitivity of the qPCR assay, the constructed pUC-57-Amp plasmid standard was first validated (Supplementary Figs. S4 and S5). Under optimal conditions, qPCR was performed on a 10-fold serially diluted positive plasmid standard (concentration range: 1 × 1010 to 1 copies/μL), and a standard curve was constructed (Fig. 4A). The Ct value is linearly related to the logarithmic concentration of the template (R2 = 0.997), with an amplification efficiency of 107.625 %, which meets the requirements for quantification. The detection limit of this method is 1 × 102 copies/μL, and the dynamic range spans from 1 × 100 to 1 copy/μL (Fig. 4B). Furthermore, to validate the repeatability of the detection effect, the same sample was first processed for 6 consecutive runs. Despite the fact that, according to the MIQE guidelines, the use of copy numbers derived from standard curves is considered the gold standard for reproducibility analysis, as an important preliminary assessment indicator, we calculated the coefficient of variation (CV) of Ct values to indirectly reflect the precision and stability of the experimental procedures. The results revealed that the coefficients of variation were 1.55 %, 0.69 %, 0.72 %, 1.25 %, 0.56 % and 0.79 %, indicating high intraassay repeatability, and the interassay CV for the 6 assays was 1.29 %, indicating stable interassay repeatability (Table 2). Variance analysis was performed on the results of 6 randomly repeated assays, and no significant difference was detected (p > 0.05), indicating that the qPCR assay is stable and reliable (Fig. 4C). With respect to reproducibility, three positive recombinant plasmids at high, medium and low concentrations (109, 106, and 103 copies/μL, respectively) were subjected to real-time PCR, and the amplification curves are shown in Fig. 4D. The intraassay CVs of the Ct values were calculated to be 1.30 %, 1.12 %, and 0.52 %, indicating that the intraassay repeatability of samples with different concentrations is good (Fig. 4E). The interlaboratory variability coefficients for the continuous testing of the same batch of samples over a five-day period were all less than 5 %. Such a low level of Ct variability typically signifies exceptional reproducibility and reliability of the detection method.

Fig. 4.

Fig. 4

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Establishment of a standard curve for qPCR and results of sensitivity and repeatability tests. A. Standard curve. The coefficient of determination of the standard curve R2 = 0.997, with an amplification efficiency of 107.625 %. B. qPCR amplification curves for positive plasmid standards in 10-fold serial dilutions. Lanes 1–11 correspond to plasmids with copy numbers of 1 × 1010 to 1 copy/μL. C. Amplification diagram of the same sample in replicates in an assay. Lane 12 is a negative control with ddH2O as the template. Plasmids with 1 × 102 copies/μL were detectable. D. Amplification curves of qPCR for positive plasmid samples with high, medium and low concentrations. E. Histogram of the results of the intra-assay repeatability tests for positive recombinant plasmids at high, medium and low concentrations. F. Histogram of the results of interassay repeatability tests for positive recombinant plasmids at high, medium and low concentrations.

Table 2.

Repeatability analysis: intra- and inter-assay variability coefficients for replicate measurements.

 Intra-assay
 Inter-assay
1 2 3 4 5 6
Mean 16.539 16.229 16.200 16.130 16.229 16.176 16.250
SD 0.256 0.112 0.117 0.201 0.090 0.128 0.209
CV 1.55 % 0.69 % 0.72 % 1.25 % 0.56 % 0.79 % 1.29 %
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3.3. LAMP assay

By employing fluorescence dye-based real-time monitoring, the primers targeting the cytb gene were determined to be the optimal primers among the four sets of primers for amplification (Supplementary Fig. S6). The amplification temperature was analysed, and 62 °C was determined to be optimal, as the results of LAMP amplification revealed high fluorescence intensity, a short reaction time, and no false-positive results in the negative control at this temperature (Fig. 5A). Five sets at different ratios of internal primer to external primer were used for optimization. The results revealed that ratios of 16: 1 and 8: 1 resulted in higher fluorescence intensity, earlier amplification onset, and clearer colour changes in the neutral red visualization assay (Fig. 5B). Considering the amplification efficiency and economic benefit, 8: 1 was selected as the optimal ratio of internal primer to external primer. The final optimal reaction conditions were established on the basis of the optimization results, as shown in Supplementary Table S5. A sensitivity test was conducted using a 10-fold serial dilution of faecal DNA (74.7 ng/μL) and cysticercus DNA (355.5 ng/μL). The test results revealed that faecal DNA was detectable in samples diluted up to 104-fold, with an LDL of 7.47 pg/μL (Fig. 5C), while plerocercoid DNA was detectable in samples diluted up to 106-fold, with an LDL limit of 355.5 fg/μL (Fig. 5D). No amplification occurred in the negative control.

Fig. 5.

Fig. 5

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Condition optimization and sensitivity determination for the LAMP assay. A. Optimization of amplification temperature. Amplification curve (EvaGreen fluorescent dye method) and neutral red visualization results. The amplification temperatures of Lanes 1–6 were 60 °C, 61 °C, 62 °C, 63 °C, 64 °C, and 65 °C, respectively. Lane 7: negative control with ddH2O as the template. The solution changed from pale yellow to orange-red as the amplification progressed. The results of fluorescence amplification were consistent with those of the visualization. B. Optimization of the ratio of internal primer to external primer. Amplification curve (EvaGreen fluorescent dye method) and neutral red visualization results. Ratios of Lanes 1–5: 32:1, 16:1, 8:1, 4:1, and 2:1, respectively. Lane 6: negative control. The amplification curve revealed that when the ratio of internal primer to external primer was 16:1 or 8:1, the amplification effects were better, with 8:1 selected as the best ratio. C. Sensitivity test of positive faecal DNA for the LAMP assay. DNA dilutions of positive faeces from Lanes 1–9: 101, 102, 103, 104, 105, 106, 107, 108, and 109, respectively. Lane 10: negative control with ddH2O as the template. The amplification curve revealed that 7.47 pg/μL of positive faecal DNA could be detected. D. Sensitivity test of positive plerocercoid DNA for the LAMP assay. Dilutions of Lanes 1–9: 101, 102, 103, 104, 105, 106, 107, 108, and 109, respectively. Lane 10: negative control with ddH2O as the template. The amplification curve revealed that 355.5 fg/μL of plerocercoid genomic DNA could be detected. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

3.4. Determination of specificity

DNA from common parasites in cat and dog faeces (including H. diminuta, C. sinensis, A. caninum, S. japonicum, T. spiralis, E. granulosus, T. gondii, G. duodenalis and Cryptosporidium sp.) was extracted and used for PCR, qPCR and LAMP amplification. S. mansoni DNA was amplified specifically in all three assays without cross-reaction with other parasites (Fig. 6).

Fig. 6.

Fig. 6

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Determination of specificity. A. Determination of specificity of the PCR system. Lane M is the 2000 Marker. Lane 1: positive control with positive faecal DNA as the template. The templates in Lanes 2–10 were the extracted genomic DNA of Hymenolepis diminuta, Clonorchis sinensis, Ancylostoma caninum, Schistosoma japonicum, Trichinella spiralis, Echinococcus granulosus, Toxoplasma gondii, Giardia duodenalis and Cryptosporidium sp., respectively. B. Validation of the specificity of qPCR. Genomic DNA was extracted from S. mansoni (1), H. diminuta (2), C. sinensis (3), A. caninum (4), S. japonicum (5), T. spiralis (6), E. granulosus (7), T. gondii (8), G. duodenalis (9), and Cryptosporidium sp. (10) and used as a template. There were no positive results for other parasites except S. mansoni and no amplification curve for the nontemplate negative control (11), which proved that the qPCR assay is specific. C. and D. LAMP specificity validation results. Lane 1: positive control with egg-derived DNA from positive faeces; Lane 11: negative control with ddH2O as the template; Lanes 2–10: template DNAs extracted from H. diminuta, C. sinensis, A. caninum, S. japonicum, T. spiralis, E. granulosus, T. gondii, G. duodenalis and Cryptosporidium sp., respectively. From the LAMP amplification results of the fluorescent dye method in C and the visualization results in D, it was noted that only when positive faecal DNA was used as the template was the “S” amplification curve consistent with the neutral red visualization results, and only positive samples had colour changes from pale yellow to orange-red. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

3.5. Epidemiological investigation

The samples collected were tested using the three molecular detection methods established, but none of them detected S. mansoni DNA. The results of the PCR assays are shown in Supplementary Figs. S7-S9, the results of the qPCR assays are shown in Supplementary Fig. S10, and the results of the LAMP assays are shown in Supplementary Fig. S11.

4. Discussion

Effective monitoring of the definitive host infection with S. mansoni is critical for the prevention of sparganosis and the safeguarding of public health security (Hong et al., 2016). PCR is a well-established technology characterized by user-friendliness, high sensitivity and specificity, rendering it ideal for detecting low-abundance pathogens, particularly in early infection stages where traditional methods may fail to detect pathogens (Reagan and Sykes, 2019). In our study, six sets of PCR primers were designed, targeting the mitochondrial cox1, cytb and nad5 genes and the ribosomal rrnS and 28S genes of S. mansoni. When negative stools were detected, all the primers except the primer set targeting the cox1 gene showed nonspecific amplification; thus, we selected cox1 for subsequent assays. The PCR method established in our study detected egg-derived DNA and positive faecal DNA at the lowest detection limits of 0.7 ng/μL and 1.4 ng/μL, respectively, with positive results detectable as early as 9 days postinfection, which is 2 days earlier than direct observation. With respect to the faecal DNA extracted using the Faecal Genomic DNA Extraction Kit, the amplified bands were found to be shallow, possibly because of the high defensiveness of the eggs (Krucken et al., 2024). Therefore, we added a grinding step before extraction to improve the eggshell disruption efficiency and DNA extraction performance. Specificity validation revealed that the PCR assay was highly specific, as demonstrated by the lack of cross-reaction with nine common parasites (H. diminuta, C. sinensis, A. caninum, S. japonicum, T. spiralis, E. granulosus, T. gondii, G. duodenalis and Cryptosporidium sp.) in cats and dogs. In addition, we evaluated the impact of different sampling sites on the results of the PCR assay of faeces, confirming the homogeneity of egg distribution throughout the faeces, and assessed the preservation stability of the faecal samples.

The qPCR assay enables quantitative analysis, allowing for the determination of egg burdens in samples, assessment of the severity of infection, and monitoring of disease transmission dynamics (Siqueira et al., 2020). Unlike PCR, qPCR can monitor reactions in real time and does not require agarose gel electrophoresis, thus simplifying the reaction workflow. The lowest detection limit of the established qPCR assay in our study was 100 copies/μL. This sensitivity is notably greater than that for Entamoeba histolytica (500 copies/μL) (Zhang et al., 2022a, Zhang et al., 2022b) and comparable to that for T. gondii (100 copies/μL) (Truong and Slapeta, 2023) but lower than that for E. granulosus (10 copies/μL) (Zhang et al., 2022a, Zhang et al., 2022b) and Plasmodium spp. (1 copy/μL) (Cordier et al., 2025). The qPCR assay also revealed high reliability and sensitivity even in low-burden samples. The standard curve established using a plasmid standard in serial dilutions demonstrated excellent linearity, with an amplification efficiency of 107.625 %, confirming robust quantitative ability across a wide concentration range. The intrabatch and interbatch repeatability test results revealed that the CVs were less than 5 %, indicating good stability of the qPCR assay (Ren et al., 2024). However, we also acknowledge the limitations of this study. In assessing reproducibility, we utilized the coefficient of variation (CV) of the Ct values as the primary indicator. In accordance with the latest MIQE guidelines, the use of the CV calculated from the starting template copies derived from the standard curve, after logarithmic transformation, constitutes a more rigorous statistical approach. Although a low CV of the Ct value is typically highly correlated with good reproducibility, we acknowledge the shortcomings in the current analytical method. In future research and applications, we will strictly adhere to international guidelines, employing copy number data to conduct a more standardized assessment of method reproducibility, thereby enhancing the rigor of data analysis. Furthermore, future reproducibility validation could consider the context of negative faecal matrices to more comprehensively simulate the real detection environment. The specificity test results revealed that there was no cross-reaction with 9 common canine/feline parasites. Therefore, the qPCR assay established in our study fulfils all the requirements for high sensitivity, high specificity and high stability.

The advantage of LAMP technology is that it can achieve isothermal amplification without sophisticated equipment and is thus especially suitable for rapid field diagnosis in resource-limited settings. To establish the LAMP assay, four primers targeting at least six different regions on the target gene must be designed (Ramos et al., 2017). Owing to the long primer sequence and multiplex primer sets used for the assay, inherent challenges in LAMP primer design are introduced, e.g., primer dimers and secondary structures (hairpins). Primer dimers deplete primers in the reaction system and reduce the amplification efficiency, whereas secondary structures of the primer or target sequence may hinder the binding of the primer to the template and affect amplification (Jang and Kim, 2022; Kim et al., 2022; Kim et al., 2023). Thus, primer design is critical for the establishment of a LAMP detection method, which directly affects the sensitivity and specificity of the assay (Soroka et al., 2021). In our study, the LAMP assay achieved robust amplification within 35 min, with no false-positive results for the negative control or specificity control, confirming its high specificity and amplification efficiency. Moreover, we optimized the reaction conditions of the primers and determined that 62 °C was the optimal reaction temperature through a gradient temperature test. By adjusting the ratio of the inner and outer primers, the amplification effect was best at a ratio of 8: 1. Under the optimal reaction conditions, we validated the sensitivity of the LAMP assay by the fluorescent dye method. The lowest detection limit for egg-derived DNA in positive faeces was 7.47 pg/μL, which was more than 100-fold greater than that of the traditional PCR method (1.4 ng/μL), and the lowest detection limit for plerocercoid DNA was 355.5 fg/μL. In addition, the LAMP assay established in our study makes it possible to interpret results by colour change using pH indicators (such as neutral red) instead of detection via complex electrophoresis or instruments. Therefore, the LAMP assay is ideal for rapid screening in field settings with limited resources.

Finally, on the basis of the developed systems with PCR, qPCR and LAMP, we tested the faeces of pet cats/dogs and stray cats/dogs around Zhengzhou city. Owing to the limited sample size, no positive samples were detected. Notably, although our study successfully established PCR, qPCR, and LAMP detection systems for S. mansoni, certain limitations remain. First, the field validation samples in our study were all collected from a single geographical region (Zhengzhou and its surrounding areas), and all sample test results were negative. This outcome may genuinely reflect the low infection rate of S. mansoni in the current cat and dog populations in this region. However, this could also be attributed to the relatively limited sample size (n = 218) and insufficiently broad sample sources. Consequently, the current research data are insufficient to comprehensively evaluate the actual detection efficacy of these methods in regions with varying infection rates. Future studies should collect larger-scale, more representative samples from endemic areas for further validation to more accurately assess their epidemiological applicability. With respect to the practicality of the methods, the three techniques each have optimal application scenarios. Conventional PCR methods are relatively common and are suitable for confirmatory testing of batch samples in basic laboratories (Liow et al., 2018); the qPCR method is characterized by high sensitivity and quantifiability, allowing for precise assessment of the infectious load. However, it relies on expensive fluorescent quantitative PCR instruments, with higher operational costs, making it more appropriate for in-depth research on pathogenic mechanisms or for accurate efficacy evaluation (Ngo et al., 2023). In contrast to the high costs associated with PCR and qPCR, the LAMP method is the fastest and has the least stringent equipment requirements (especially the visually interpretable version), offering unparalleled rapid screening advantages in grassroots settings and areas with limited resources (Prado et al., 2024). Nevertheless, primer design for LAMP is more complex, and its actual “field applicability,” such as the use of portable heating blocks, still requires further development and testing. Furthermore, while the specificity validation of this study included a variety of common parasites found in feline and canine faeces, it failed to include other Cestoda of the Pseudophyllidea class as well as Taenia spp. This theoretically presents the possibility of cross-reactivity, and constitutes a limitation of the current study. In future endeavours, we will prioritize the acquisition of DNA samples from these species to conduct a more comprehensive validation of the specificity of this detection system, thereby ensuring its absolute reliability in complex epidemiological environments.

5. Conclusion

In our study, three molecular detection systems for S. mansoni were successfully developed: PCR, qPCR and LAMP. PCR detected egg-derived DNA at concentrations as low as 0.7 ng/μL, demonstrating high sensitivity and thus providing a basic molecular tool for epidemiological investigation. qPCR achieved the lowest detection limit of 100 copies/μL for S. mansoni DNA in low-burden samples, with excellent repeatability and stability, confirming its suitability for quantitative detection. The lowest detection limit of LAMP for egg-derived DNA from positive faeces was 7.47 pg/μL, which was more than 100 times more sensitive than that of the traditional PCR method. LAMP is easy to perform and suitable for rapid field detection. The development of these three molecular detection methods provides strong technical support for early diagnosis, epidemiological surveillance and comprehensive prevention and control of sparganosis.

Funding

This work was supported by Intergovernmental international scientific and technological innovation cooperation projects of National Key Research and Development Program (2024YFE0199100), Fundamental Research Project of key scientific research in Henan Province (24ZX003) and National Natural Science Foundation of China (81971956).

Ethics approval and consent to participate

The animal experiments in our study were carried out on the basis of National Guidelines for Experimental Animal Welfare of China. All animal experimental protocols were approved by the Life Science Ethics Committee of Zhengzhou University (No. ZZUIRB GZR 2023–1102).

CRediT authorship contribution statement

Wen Li: Writing – original draft, Methodology, Formal analysis, Data curation. Xuan Xuan Song: Software, Methodology, Formal analysis. Si Si Ru: Software, Methodology. Jie Hao: Software, Methodology. Cheng Yue Cao: Validation, Methodology. Xi Zhang: Writing – review & editing, Writing – original draft, Visualization, Supervision, Resources, Project administration, Methodology, Investigation, Funding acquisition, Formal analysis.

Declaration of competing interest

The authors declare that they have no competing interests.

Footnotes

Appendix A

Supplementary data to this article can be found online at https://doi.org/10.1016/j.fawpar.2025.e00291.

Appendix A. Supplementary data

Supplementary material

mmc1.docx (3.9MB, docx)

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