Key Laboratory of Zoonosis Research, Ministry of Education, Institute of Zoonosis, College of Veterinary Medicine, Jilin University, Changchun, 130012, Jilin, China.
Qingdao Special Servicemen Recuperation Center of PLA Navy, Qingdao, 266071, Shandong, China.
Parasit Vectors. 2023 May 19;16(1):165. doi: 10.1186/s13071-023-05774-5.
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.
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.
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.
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.
由华支睾吸虫引起的食源性人畜共患肝吸虫病是一些国家正在出现的公共卫生问题，全球有超过 1500 万人感染。然而，在资源有限的地区缺乏准确的即时检测（POC）诊断测试仍然是有效治疗和控制肝吸虫病的关键障碍。基于病原体 DNA 扩增的即时检测诊断测试——重组酶聚合酶扩增（RPA）检测方法的发展，为疾病检测提供了一种新的、简单和廉价的工具，具有高灵敏度和特异性。
本研究基于特异性引物和探针，开发了一种新的 RPA 方法，并与试纸条相结合，通过扩增线粒体细胞色素 c 氧化酶亚单位 1（COX1）基因，实现了对华支睾吸虫的快速、直观检测。通过对靶 DNA 序列的稀释，评估了联合 RPA/侧流试纸条（RPA-LFD）检测方法的最低检测限。用 10 种额外的对照寄生虫的基因组 DNA 评估交叉反应性。对 40 份人类临床粪便样本进行了测试，以验证其性能。
从华支睾吸虫 COX1 区设计的评估引物可用于在 39°C 下在 20 分钟内检测成虫、囊蚴和虫卵，结果可通过试纸条进行直观观察。病原体基因组 DNA 的检测限低至 10 fg，鱼中的囊蚴和粪便中的虫卵数量均低至一个。这极大地提高了低感染检测的灵敏度。该测试具有物种特异性，没有检测到其他相关的对照寄生虫。在每克粪便虫卵数（EPG）>50 的人类粪便样本中，RPA-LFD 检测与传统加藤厚涂片（KK）和 PCR 方法一致。
本研究建立的 RPA-LFD 检测方法为人类和动物样本中肝吸虫的诊断和流行病学调查提供了有力工具，对肝吸虫病的有效控制具有重要意义。
