新疆水源性隐孢子虫的检测

Detection of Waterborne Cryptosporidium in Xinjiang

目的检测新疆15个城市自来水和河水隐孢子虫污染情况。方法采集水样,分别应用改良美国环保局(EPA)1622法及巢式PCR(nested-PCR)方法进行检测。1)改良EPA1622法:水样经微孔滤膜抽滤、淘洗,磁抗体分离法分离纯化后免疫荧光染色鉴定。2)Nested-PCR法:用试剂盒提取纯化的隐孢子虫卵囊基因组DNA,针对隐孢子虫小亚单位核糖体RNA(18SrRNA)部分基因,依据文献设计并合成引物,用巢式PCR扩增,产物纯化后经SspⅠ及VspⅠ单酶切,并进行RFLP分析。结果2种方法检测新疆15个地区的自来水隐孢子虫卵囊均为阴性,改良EPA1622法检测乌鲁木齐市、昌吉市、伊宁市和吐鲁番市的河水隐孢子虫卵囊阳性。巢式PCR检测乌鲁木齐市和伊宁市水样,均扩增出约830 bp的特异片段,RFLP初步鉴定为小鼠隐孢子虫基因;昌吉市和吐鲁番市河水水样PCR检测阴性。结论新疆乌鲁木齐市、伊宁市河水检出隐孢子虫,初步鉴定为小鼠隐孢子虫。而当地的饮用水未受污染。

Objective To detect Cryptosporidium contamination in tap water and rivers in fifteen cities of Xinjiang.Methods Water samples were collected from fifteen cities in Xinjiang.Samples were tested by a modified form of EPA method 1622 and nested PCR assay,respectively.The modified form of EPA method 1622 consists of several steps,such as filtration with a microporous membrane,elution,immunomagnetic separation,and immunofluorescence assay.Nested PCR was conducted as follows.First,genomic DNA of purified oocysts was extracted with a QIAamp DNA minikit.Two sets of special primers were synthesized based on the sequence of 18S rRNA as described in the literature.After DNA extraction,18SrRNA was amplified by nested PCR.Amplified products were then purified and digested with restriction enzymes SspⅠ and VspⅠ,respectively.Results Cryptosporidium parvum was not detected in tap water from any of the cities.The river water of Urumqi,Changji,Yining,and Tulufan was positive according to the modified form of EPA method 1622.Samples from Urumqi and Yining yielded fragments of about 830 bp according to nested PCR.However,samples from Tulufan and Changji were negative according to PCR.The species of Cryptosporidium isolated from Urumqi and Yining were C.muris,which was preliminarily identified by nested PCR-restriction fragment length polymorphism（nested PCR-RFLP）.Conclusion Rivers in Urumqi and Yining were contaminated with Cryptosporidium that was preliminarily identified as C.muris.Sources of contamination must be traced.There was no Cryptosporidium in tap water,and this may be the result of advanced technology and effective management.