重组酶介导的等温核酸扩增技术检测淡水鱼华支睾吸虫囊蚴的初步应用

Preliminary application of recombinase⁃aided amplification in detection of Clonorchis sinensis metacercariae in freshwater fish

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1735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%(93/1735)和2.88%(50/1735),差异有统计学意义(χ^(2)=478.150,P<0.001)。经压片法(χ^(2)=11.20,P<0.05)和荧光RAA法(χ^(2)=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。

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 freshwater 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%(93/1735)and 2.88%(50/1735)detections rate for C.sinensis metacercariae in 1735 field⁃collected freshwater fish samples,with a statistically significant difference seen([χ^(2)]=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([χ^(2)]=11.20,P<0.05)and fluorescent RAA assay([χ^(2)]=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 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.