人粪便中华支睾吸虫虫卵PCR快速检测方法的建立及评价  

Establishment and evaluation of polymerase chain reaction for detection of Clonorchis sinensis eggs in human feces

作　　者：马文晨 李文君 于欣 刘晓晖[2] 魏春艳 史艳宇 MA Wen-chen;LI Wen-jun;YU Xin;LIU Xiao-hui;WEI Chun-yan;SHI Yan-yu(Ji'lin International Travel Health Care Center,Changchun,Jilin 130062,China;不详)

机构地区：[1]吉林国际旅行卫生保健中心,吉林长春130062 [2]吉林省产品质量监督检验院 [3]长春海关技术中心 [4]长春中医药大学创新实践中心

出　　处：《中国卫生检验杂志》2023年第23期2845-2848,共4页Chinese Journal of Health Laboratory Technology

基　　金：长春海关科研项目(CCHG2023KY06)。

摘　　要：目的建立一种快速、特异、灵敏的人粪便中华支睾吸虫虫卵检测方法,并对方法进行评价,为快速、准确、有效诊断人是否患有华支睾吸虫病提供依据。方法以华支睾吸虫细胞色素c氧化酶亚基1基因为靶位点,用Primer Express3.0软件设计引物,经Blast软件对相似序列搜索后,筛选出一套最优与常见寄生虫无交叉反应的引物;分别用猫后睾吸虫、粪类圆线虫、日本血吸虫、细粒棘球虫、卫氏并殖吸虫等可感染人类、虫卵形态易混淆、种属关系较近的寄生虫的DNA进行特异性实验;将含有目的片段的阳性质粒梯度稀释后进行PCR扩增,确定检测方法的灵敏性;利用建立的方法对吉林省白城市周边人粪样品进行检测,验证方法的可行性。结果利用细胞色素c氧化酶亚基1基因设计的引物仅对华支睾吸虫(或虫卵)DNA有扩增条带,而其他对照以及阴性对照均未有明显扩增条带,对华支睾吸虫虫卵阳性质粒DNA检测敏感性可达到2.47×10^(2)copies/μl,建立的方法能够对实际粪便中的华支睾吸虫虫卵进行有效扩增,与传统图片镜检法比较二者检测结果一致。结论PCR方法特异性强,灵敏度高,可以快速、准确检测人粪便中的华支睾吸虫虫卵,可以为华支睾吸虫病的快速诊断提供技术支持。Objective This paper aims to establish a rapid,specific and sensitive method for the detection of Clonorchis sinensis eggs in human feces,evaluate the method,and provide basis for the rapid,accurate and effective diagnosis of Clonorchis sinen-sis.Methods The cytochrome c oxidase subunit 1 gene of Clonorchis sinensis was used as the target site,and Primer Express 3.0 software was used to design primers.After searching similar sequences by Blast software,an optimal set of primers without cross-reaction with common parasites was selected.The DNA of Feline opisthorchis,Strongylus faecalis,Schistosoma japoni-cum,Echinococcus granulosus,Paragonimus welsoni and other parasites that can infect human beings and whose egg morphology is easily confused and the species relationship is relatively close were used for specific experiments.The positive plasmid contai-ning the target fragment was diluted and amplified by PCR to determine the sensitivity of the detection method.The method was used to detect fecal samples around Baicheng City,Jilin Province,and verify the feasibility of the method.Results The prim-ers designed by using the cytochrome c oxidase subunit 1 gene only had amplified bands on the DNA of Clonorchis sinensis or eggs,while other controls and negative controls did not have obvious amplified bands.The sensitivity of the plasmid DNA of Clonorchis sinensis egg reached 2.47×10^(2) copies/μl.The established method can effectively amplify the eggs of Clonorchis sinensis in the actual feces,and the results are consistent with the traditional picture microscopy method.Conclusion PCR method has strong specificity and sensitivity,which can detect Clonorchis sinensis eggs in human feces quickly and accurately,and can provide technical support for the rapid diagnosis of Clonorchis sinensis.

关 键 词：华支睾吸虫虫卵 细胞色素c氧化酶亚基1基因 PCR 人粪便 

分 类 号：R446[医药卫生—诊断学]