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  • Title: [Preliminary application of recombinase -aided amplification in detection of Clonorchis sinensis metacercariae in freshwater fish].
    Author: Chen J, Wang Z, Huang W, Wang J, Chen L, Sun Y, Zhao L, Zhao Y, Qian Y, Duan J, Zhang Q.
    Journal: Zhongguo Xue Xi Chong Bing Fang Zhi Za Zhi; 2023 Nov 22; 35(5):458-463. PubMed ID: 38148534.
    Abstract:
    OBJECTIVE: To evaluate the performance of recombinase-aided amplification (RAA) assay in detection of Clonorchis sinensis metacercariae in freshwater fish samples, so as to provide insights into standardization and field application of this assay. METHODS: Wild freshwater fish samples were collected in the rivers of administrative villages where C. sinensis-infected residents lived in Jiangyan District, Xinghua County and Taixing County of Taizhou City, Jiangsu Province from June to September 2022. Genomic DNA was extracted from six freshwater fish specimens (5 g each) containing 0, 1, 2, 4, 8 and 16 C. sinensis metacercariae for fluorescent RAA assay, and the diagnostic sensitivity was evaluated. Fluorescent RAA assay was performed with genomic DNA from C. sinensis, Metorchis orientalis, Haplorchis pumilio and Centrocestus formosanus metacercariae as templates to evaluate its cross-reactions. In addition, the detection of fluorescent RAA assay and direct compression method for C. sinensis metacercariae was compared in field-collected freshwater fish samples. RESULTS: Positive amplification was found in fresh-water fish specimens containing different numbers of C. sinensis metacercariae, and fluorescent RAA assay was effective to detect one C. sinensis metacercaria in 5 g freshwater fish specimens within 20 min. Fluorescent RAA assay tested negative for DNA from M. orientalis, H. pumilio and C. formosanus metacercariae. Fluorescent RAA assay and direct compression method showed 5.36% (93/1 735) and 2.88% (50/1 735) detection rates for C. sinensis metacercariae in 1 735 field-collected freshwater fish samples, with a statistically significant difference seen (χ2 = 478.150, P < 0.001). There was a significant difference in the detection of C. sinensis metacercariae in different species of freshwater fish by both the direct compression method (χ2 = 11.20, P < 0.05) and fluorescent RAA assay (χ2 = 20.26, P < 0.001), and the detection of C. sinensis metacercariae was higher in Pseudorasbora parva than in other fish species by both the direct compression method and fluorescent RAA assay (both P values < 0.05). CONCLUSIONS: Fluorescent RAA assay has a high sensitivity for detection of C. sinensis metacercariae in freshwater fish samples, and has no cross-reactions with M. orientalis, H. pumilio or C. formosanus metacercariae. Fluorescent RAA assay shows a higher accuracy for detection of C. sinensis infections in field-collected freshwater fish than the direct compression method. ［摘要］ 目的 评价重组酶介导的核酸等温扩增技术 (recombinase-aided amplification, RAA) 用于淡水鱼肉样本中华支 睾吸虫囊蚴检测效果, 为后续该方法标准化及现场应用提供科学依据。方法 2022 年 6—9 月在江苏省泰州市姜堰区、兴化市、泰兴市华支睾吸虫感染阳性人群所在行政村河道内采集野生淡水鱼样本。取 6 份不含华支睾吸虫囊蚴的鱼肉 组织 (每份 5 g), 分别加入 0、1、2、4、8、16 个华支睾吸虫嚢蚴, 提取鱼肉组织基因组 DNA 后采用荧光 RAA 法进行检测, 评 价该方法检测灵敏度; 分别以华支睾吸虫、东方次睾吸虫、钩棘单睾吸虫、台湾棘带吸虫嚢蚴基因组 DNA 为模板, 采用荧 光 RAA 法检测, 评价其交叉反应; 分别采用荧光 RAA 法和压片法检测现场采集的淡水鱼样本, 比较两种方法检测华支睾 吸虫效果。结果 含 1、2、4、8、16 个华支睾吸虫囊蚴的鱼肉组织样本均出现阳性扩增, 荧光 RAA 法可在 20 min 内对 5 g 鱼 肉组织中含 1 个华支睾吸虫囊蚴的样本实现阳性扩增; 以东方次睾吸虫、钩棘单睾吸虫、台湾棘带吸虫嚢蚴基因组 DNA 为模板进行 RAA 检测, 结果均为阴性。对采集的 1 735 条淡水鱼分别采用荧光 RAA 法和压片法进行检测, 两种方法华支 睾吸虫检出率分别为 5.36% (93/1 735) 和 2.88% (50/1 735), 差异有统计学意义 (χ2 = 478.150, P < 0.001)。经压片法 (χ2 = 11.20, P < 0.05) 和荧光 RAA 法 (χ2 = 20.26, P < 0.001) 检测, 发现不同鱼种华支睾吸虫检出率差异均有统计学意义, 麦穗 鱼华支睾吸虫检出率高于其他鱼种 (P 均< 0.05)。结论 荧光 RAA 法检测淡水鱼肉样本中华支睾吸虫感染的灵敏度较高, 与东方次睾吸虫、钩棘单睾吸虫、台湾棘带吸虫无交叉反应, 检测现场采集淡水鱼华支睾吸虫感染的准确性高于压片法。.
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