中国农业大学学报2025，Vol.30Issue(7)：161-173,13.DOI:10.11841/j.issn.1007-4333.2025.07.14
淡水鱼中5种人兽共患吸虫囊蚴多重PCR检测方法的建立
Establishment of multiplex PCR for detection of five species of zoonotic trematode metacercariae in freshwater fish
摘要
Abstract
To rapidly identify the trematode metacercariae species infecting freshwater fish,five pairs of specific primers were designed based on the conserved regions of Clonorchis sinensis,Metorchis orientalis,Metagonimus yokogawai,Echinochasmus japonicus and Echinostoma hortense sequences in GenBank,and a multiplex PCR method was established.The results showed that:1)In the PCR reaction system,the optimal primer concentration was 10 μmol/L,the optimal annealing temperature was 59 ℃,and the specific fragments for each trematode species could be amplified simultaneously.The detected results of Clinostomum complanatum,Holostephanus sp.,Echinostoma revolutum,Fasciola hepatica and Schistosoma turkestanicum were all negative.The minimum detectable concentration of recombinant plasmid DNA for five species trematode metacercariae was 1 ng/μL;Both inter-batch and intra-batch experiments were consistent.2)The multiplex PCR detection results were validated by single PCR,and the coincidence rate was 100％.3)Among the 100 Pseudorasbora parva samples detected by multiplex PCR,and 21(21/100,21％)were positive,eight(8/100,8％)were single pathogen infections,and 13(13/100,13％)were mixed infections involving multiple pathogens.The positive rates for C.sinensis,M.orientalis,M.yokogawai,E.japonicus and E.hortense were 11(11％),nine(9％),four(4％),eight(8％),and eight(8％),respectively,which were consistent with the results obtained by single PCR and morphological methods.In conclusion,this study successfully established a multiplex PCR method for the simultaneous detection of trematode metacercariae in fish,including C.sinensis,M.orientalis,M.yokogawai,E.japonicus and E.hortense.This method has the advantages of strong specificity,high sensitivity,and excllent repeatability,which is expected to be applied in fish disease detection,sudden fish related public health events,and entry-exit inspection and quarantine.关键词
华支睾吸虫/东方次睾吸虫/横川后殖吸虫/日本棘隙吸虫/圆圃棘口吸虫/多重PCRKey words
Clonorchis sinensis/Metorchis orientalis/Metagonimus yokogawai/Echinochasmus japonicus/Echinostoma hortense/multiplex PCR分类
农业科技引用本文复制引用
王雪,高俊峰,王春仁,李犇,李晶,兰卓,侯美如,杨彦,王佳文,刘雪薇,邱鸿宇..淡水鱼中5种人兽共患吸虫囊蚴多重PCR检测方法的建立[J].中国农业大学学报,2025,30(7):161-173,13.基金项目
黑龙江省自然科学基金重点项目(ZD2022C006) （ZD2022C006）
国家自然科学基金项目(31972703) （31972703）
国家重点研发项目(2017YFD0501306) （2017YFD0501306）
