A rapid and visual detection assay for Clonorchis sinensis based on recombinase polymerase amplification and lateral flow dipstick
Xiao-Xiao Ma, Xue Bai, Hongchang Li, Jing Ding, Huiyuan Zhang, Yang‐Yuan Qiu et al. · 2023 · 15 citationsRead the paper
BACKGROUND: Fish-borne zoonotic clonorchiasis, caused by Clonorchis sinensis, is an emerging public health problem in several countries with more than 15 million people infected globally. However, a lack of accurate point-of-care (POC) diagnostic tests in resource-limited areas is still a critical barrier to effective treatment and control of clonorchiasis. The development of the recombinase polymerase amplification(RPA) assay, a POC diagnostic test based on the amplification of pathogen DNA, has provided a new, simple and inexpensive tool for disease detection with high sensitivity and specificity. METHODS: A novel RPA method was developed based on specific primers and probes, and combined with the dipstick, to allow for the rapid and intuitive detection of C. sinensis through the amplification of the mitochondrial cytochrome c oxidase subunit 1 (COX1) gene. The lower limit of detection for the combined RPA/lateral flow dipstick (RPA-LFD) assay was evaluated using dilutions of the target DNA sequence. Cross-reactivity was evaluated using genomic DNA from 10 additional control parasites. Forty human clinical stool samples were tested to verify its performance. RESULTS: The evaluated primers designed from the C. sinensis COX1 region can be used to detect adult worms, metacercariae, and eggs at 39 °C within 20 min, and the results can be visually observed using the LFD. The detection limit of pathogen genomic DNA was as low as 10 fg, and the number of metacercaria(e) in fish and egg(s) in faeces were both as low as one. This improved the sensitivity of low-infection detection tremendously. The test is species-specific, and no other related control parasites were detected. In human stool samples with eggs per gram (EPG) > 50, the RPA-LFD assay was performed consistent with conventional Kato-Katz (KK) and PCR methods. CONCLUSION: The established RPA-LFD assay provides a powerful tool for the diagnosis and epidemiological survey of C. sinensis from human and animal samples, and has important implications for the effective control of clonorchiasis.
What the full text shows
Working prototype- Detection limit for C. sinensis genomic DNA is 10 fg, which is 1000 times more sensitive than conventional PCR. — 10 fg (Purified genomic DNA of adult worms)
- The assay can detect as few as one metacercaria in fish or one egg in faeces. — 1 egg or 1 metacercaria (Purified samples)
- The assay can detect as few as one egg in 200 mg of spiked human faeces, whereas conventional PCR requires more than 25 eggs. — 1 egg per 200 mg stool (Egg-spiked human faeces)
- The assay is species-specific with no cross-reactivity against 10 other control parasites. — 0% cross-reactivity (Genomic DNA from 10 control parasites at 10 ng/μl)
- The assay showed a 35% positive rate in clinical stool samples, consistent with PCR results. — 35% positive rate (40 human stool samples from endemic area (EPG > 50))
- The assay provides visual results within 20 minutes at 39 °C. — 20 min at 39 °C (RPA-LFD reaction)
- Setup
- 40 human clinical stool samples, 10 control parasite DNA samples, and spiked faecal samples (1-5000 eggs).
- How
- The researchers developed an RPA-LFD assay targeting the mitochondrial COX1 gene of C. sinensis using biotin-labeled reverse primers and FAM-labeled probes. The amplified DNA was visualized using a lateral flow dipstick with gold-labeled anti-FAM antibodies.
- Limitations
- Clinical validation was limited to a small sample size of 40 human stool samples. · The study focused on samples with EPG > 50 for clinical consistency. · Requires a metal heater to maintain 39 °C for optimal signal.
Summarized by Gemma 4 from the open-access full text · 8 Oct 2026. Read it