华支睾吸虫ICT和PCR检测方法的建立及其应用研究

Establishment and application of ICT and PCR detection for Clonorchis sinensis

目的建立华支睾吸虫的免疫胶体金(ICT)和实时荧光定量PCR检测体系,比较两者检测华支睾吸虫的效果。方法以华支睾吸虫成虫抗原包被胶体金,检测血液样本中的相应抗体,建立华支睾吸虫ICT检测方法;以华支睾吸虫18S r RNA基因作为靶基因,设计RT-PCR的特异性引物和探针,检测粪便样本中的核酸实时荧光定量PCR方法;以两种检测体系与商品化的酶联免疫(ELISA)检测试剂盒平行测定比较,考核检测体系的应用价值。结果对200份华支睾吸虫重点人群样本进行检测,ELISA法检出的华支睾吸虫Ig G抗体阳性例数为20例,阳性率为10.0%;ICT法为16例,阳性率为8.0%,灵敏度为80.0%,特异度为100.0%,符合率为98.0%,ICT和ELISA两种检测方法差异无统计学意义(χ2=2.25,P>0.05);RT-PCR法检出的粪便样本中华支睾吸虫核酸阳性例数为18例,阳性率为9.0%,灵敏度为90.0%,特异度为100.0%,符合率为99.0%,RT-PCR和ELISA两种检测方法差异无统计学意义(χ2=0.05,P>0.05)。说明ICT、RT-PCR两种检测方法与ELISA的测定结果比较一致。结论建立的ICT及RT-PCR两种检测方法特异性好、灵敏度高,两种检测方法均适用于华支睾吸虫现场的快速检测及定量分析。

Objective To establish an immune colloidal gold technique（ICT） for detecting the IgG antibody of Clonorchis sinensis（C.s）, and a real- time fluorescent quantitative PCR（RT- PCR） for detecting the DNA of C.s, and compare their detection effects. Methods The colloidal gold particles were peridiumed by adult worm antigen of C.s to establish the ICT to detect Ig G antibody of C.s. According to the specific sequence of 18 S rRNA of C.s, the primers and the fluorogenic probe of RTPCR were designed, and then the RT-PCR was build to detect fecal samples. The 2 methods we established were parallelly compared with commercialized enzyme-linked immunosorbent assay（ELISA） kit to assess their values in clinical application.Results A total of 200 serum samples from volunteers who had exposure history were used for testing. ELISA verified there were 20 cases positive to the IgG antibody of C.s, with a positive rate of 10.0%. ICT detected 16 cases, with a positive rate of8.0%, a sensitivity of 80.0%, and a specificity of 100.0%. The coincidence rate of the 2 methods was 98.0%, with no significant difference between them（χ^2=2.25, P〉0.05）. The results of RT-PCR indicated that the fecal samples of 18 cases were positive to nucleic acid of C.s, with a positive rate of 9.0%, sensitivity of 90.0%, and specificity of 100.0%. The coincidence rate between the RT-PCR and ELISA was 99.0%, and there was no obvious difference between them（χ^2=0.05, P〉0.05）. Our study showed that the results of our ICT and RT-PCR were the same with those of ELISA. Conclusion The 2 established methods have high sensitivities and specificities, and are suitable for rapid detection and quantitative analysis of C.s.Clonorchis sinensis