两种检测方法应用于城市水源中蓝氏贾第鞭毛虫和隐孢子虫污染的比较研究

Comparison of two approaches in detection of Giardia lamblia and Cryptosporidium spp.contamination in various water of urban area

目的用PCR和免疫荧光染色镜检法检测城市现场水源中蓝氏贾第鞭毛虫（简称贾第虫）和隐孢子虫．了解上海市水源地贾第虫和隐孢子虫污染状况。方法采集上海市8个区的自来水和3个区的环境水，PCR检测水样本中的贾第虫磷酸丙糖异构酶（triosephosphate isomerase，Tim）基因和隐孢子虫18S rRNA的基因；按《生活饮用水标准检验方法》（GB5750．12-2006）Filta-Max Xpress快速方法进行免疫荧光染色镜检。计算比较两种方法检测贾第虫和隐孢子虫的阳性率。结果共采集200份水样：其中自来水出厂水48份．PCR和荧光镜检法均未检出贾第虫和隐孢子虫；检测原水、动物饲养场周边池水、污水处理厂出水、游泳池水和餐馆养渔池等水样分别为62、25、29、20、16份，PCR法贾第虫阳性率分别为8．1％、36．0％、17．2％、0和0．总阳性率为12．5％；隐孢子虫阳性率分别为6．5％、40．0％、13．8％、0和0，总阳性率为11．8％。免疫荧光染色镜检法贾第虫阳性率分别为9．7％、40．0％、24．1％、0和0，总阳性率为15．1％；隐孢子虫阳性率分别为8．1％、44．0％、17．2％、0和0．总阳性率为13．8％。两种方法检测贾第虫和隐孢子虫一致性分别为96．1％和95.4％，经Kappa检验．两种方法一致性较好（Kappa=0．83和0．79，Kappa〉0．75），PCR与荧光镜检结果无显著性差异（χ^2=0．44和0．26．P〈0．05）。结论两种方法均可用于城市现场水源中贾第虫和隐孢子虫污染的调查。自来水未检出贾第虫和隐孢子虫．但在原水和环境水中检测到贾第虫和隐孢子虫污染，需加强监测。

Objective To understand the contamination status with Giardia lamblia and Cryptospordium spp. in different sources of water in urban area of Shanghai, by application of two tests including PCR and fluorescence microscopy. Methods The effluent water samples from water plants were collected from 8 districts and environmental water from 3 districts. The PCR was performed to detect the DNA extracted from the water samples targeting the triosephosphate isomerase （Tim） gene for Giardia spp. and 18S rRNA gene for Cryptosporidium spp., respectively. The immune-fluorescence microscopy assay（IFA） was performed following Fiha- Max Xpress methods based on procedures described in ＂The National Standard of Detection for Drinking Water （GB5750.12-2006）＂. The positive rates of detecting contamination with Giardia spp. and Cryptosporidium spp. were compared. Results A total of 200 water samples were collected. No positive sample was found contaminated with Giardia spp. and Cryptosporidium spp. in 48 samples of effluent water from water plants. In water samples, collected from source water of water plant（62 samples）, pool water close to animal farms （25 samples）, outdoor water of sewage treatment plant（29 samples）, swimming pool water（20 samples）, and water of fish pond in restaurant （16 samples）, the general positive rates of Giardia spp. by PCR was 12.5% with 8.1%, 36.0%, 17.2%, 0 and 0, and the general positive rates of Cryptosporidium spp. by PCR was 11.8% with 6.5%, 40.0%,13.8%, 0 and 0, in above samples respectively. The general positive rate of Giardia spp. by IFA was 15.1% with 9.7%, 40.0%, 24.1%, 0 and 0, and the general positive rate of Cryptosporidium spp. by IFA was 13.8% with 8.1%, 44.0%, 17.2%, 0 and 0, in above samples respectively. In Kappa test, the identity of two approaches was good with Kappa≥ 0.75 （Kappa=0.83 and 0.79 ）. The positive rates of water samples in PCR and IFA had no significantly difference （χ^2=0.44 and 0.26, P 〈 0.05）. Conclusion The two established approaches, including PCR and IFA, are feasible to be used in epidemiologieal survey in urban areas. No parasites were detected in the effluent water samples from water plants, but the source water of water plant and environmental water were contaminated with Giardia spp. and Cryptosporidium spp. which need to strengthen the surveillance in future.