淡水鱼中华支睾吸虫囊蚴PCR检测技术的建立及应用分析

Establishment and application analysis of PCR detection technique for Clonorchis sinensis in freshwater fish

目的建立灵敏度高,特异性强的淡水鱼中华支睾吸虫囊蚴PCR检测方法。方法采用DNeasy组织提取试剂盒提取淡水鱼中华支睾吸虫囊蚴DNA,根据核糖体第二内转录间隔区(ITS2)基因序列合成1对特异性CS1/CS2引物,对模板DNA进行PCR扩增。预试验后优化反应条件,评估反应的特异性和灵敏性。用消化镜检法和PCR法同时检测2015-2016年上海市4区121件淡水鱼样本并比较结果。结果该方法检测含有华支睾吸虫囊蚴麦穗鱼的DNA为阳性,其余对照均为阴性;检测按1∶10倍比稀释阳性样本的灵敏度达到fg水平。应用于上海市4区121件淡水鱼样本中华支睾吸虫囊蚴检测,消化镜检法检出阳性3件,阳性率2.48%;PCR法检出5件(包含消化法检出的3件),阳性率为4.13%,两法基本相当,差异无统计学意义(χ^2=0.52,P>0.05)。结论 PCR法检测淡水鱼中华支睾吸虫囊蚴具有简便快速且特异性敏感性高的特点,具有较好的应用前景。

Objective To establish a PCR method for detecting Clonorchis sinensis in freshwater fish with high sensitivity and specificity.Methods The DNeasy tissue extraction kit was used to extract the DNA of C.sinensis from the freshwater fish,and a pair of specific CS1/CS2 primers were synthesized according to the ribosomal second internal transcribed spacer (ITS2)gene sequence,and the template DNA was amplified by PCR.The reaction conditions were optimized after the preliminary test to evaluate the specificity and sensitivity of the reaction.Simultaneous detection of 121 freshwater fish samples from 4districts of Shanghai in 2015-2016by gastrointestinal microscopy and PCR was performed and the results were compared.Results By using PCR,the detection of metacercariae DNA of Clonorchis sinensis in psendorasbora parva was positive,while the control samples were all negative.The sensitivity detection limit of positive samples (1:10multiple dilution)was fg.The detection results of 121 freshwater fish samples were 3 positive (2.48%)by pepsin digestion,and 5positive (4.13%,including 3 positive samples by pepsin digestion).Conclusion The detection of C.sinensis in freshwater fish by PCR was simple,rapid and high specific sensitivity,and has a good application prospect.