Higgins James A, Trout James M, Fayer Ronald, Shelton Daniel, Jenkins Mark C
USDA-ARS, Rm 202, Bldg 173, 10300 Baltimore Blvd, Beltsville, MD 20705, USA.
Water Res. 2003 Sep;37(15):3551-60. doi: 10.1016/S0043-1354(03)00251-3.
Continuous flow centrifugation (CFC) was used in conjunction with immunomagnetic separation (IMS) and immunofluorescence microscopy (IFA) and nested PCR to recover and detect oocysts of Cryptosporidium parvum and cysts of Giardia intestinalis from 10L volumes of source water samples. Using a spiking dose of 100 oocysts, nine of 10 runs were positive by IFA, with a mean recovery of 4.4+/-2.27 oocysts; when another 10 runs were analyzed using nested PCR to the TRAP C-1 and Cp41 genes, nine of 10 were positive with both PCR assays. When the spiking dose was reduced to 10 oocysts in 10L, 10 of 12 runs were positive by IFA, with a mean oocyst recovery of 3.25+/-3.25 oocysts. When 10 cysts of Giardia intestinalis were co-spiked with oocysts into 10L of source water, five of seven runs were positive, with a mean cyst recovery of x=0.85+/-0.7. When 10 oocysts (enumerated using a fluorescence activated cell sorter) were spiked into 10L volumes of tap water, one of 10 runs was positive, with one oocyst detected. For the majority of the source water samples, turbidities of the source water samples ranged from 1.1 to 22 NTU, but exceeded 100 NTU for some samples collected when sediment was disturbed. The turbidities of pellets recovered using CFC and resuspended in 10 mL of water were very high (exceeding 500 NTU for the source water-derived pellets and 100 NTU for the tap water-derived pellets). While not as efficient as existing capsule-filtration based methods (i.e., US EPA methods 1622/1623), CFC and IMS may provide a more rapid and economical alternative for isolation of C. parvum oocysts from highly turbid water samples containing small quantities of oocysts.
连续流动离心法（CFC）与免疫磁珠分离法（IMS）、免疫荧光显微镜法（IFA）和巢式聚合酶链反应（PCR）联合使用，从10升源水样本中回收并检测微小隐孢子虫卵囊和肠贾第鞭毛虫包囊。在加入100个卵囊的加标剂量下，10次运行中有9次IFA检测呈阳性，卵囊平均回收率为4.4±2.27个；当使用巢式PCR对TRAP C-1和Cp41基因进行另外10次运行分析时，两种PCR检测10次中有9次呈阳性。当加标剂量降至10升中10个卵囊时，12次运行中有10次IFA检测呈阳性，卵囊平均回收率为3.25±3.25个。当将10个肠贾第鞭毛虫包囊与卵囊共同加入10升源水中时，7次运行中有5次呈阳性，包囊平均回收率为x = 0.85±0.7个。当将10个卵囊（使用荧光激活细胞分选仪计数）加入10升自来水中时，10次运行中有1次呈阳性，检测到1个卵囊。对于大多数源水样本，源水样本的浊度范围为1.1至22 NTU，但在扰动沉积物时采集的一些样本浊度超过100 NTU。使用CFC回收并重悬于10毫升水中的沉淀浊度非常高（源水沉淀超过500 NTU，自来水沉淀超过100 NTU）。虽然不如现有的基于胶囊过滤的方法（即美国环保署方法1622/1623）高效，但CFC和IMS可能为从含有少量卵囊的高浊度水样中分离微小隐孢子虫卵囊提供一种更快速、经济的替代方法。
