权益分类	功能权益	普通用户	{{item.name}}会员
{{category.name}}	{{benefitItem.name}}

Sandpit contamination with protozoa and bacteria

沙坑被原生动物和细菌污染

基本信息

批准号：
09670256
负责人：
UGA Shoji
金额：
$ 1.79万
依托单位：
Kobe University School of medicine
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
1997
资助国家：
日本
起止时间：
1997 至 1998
项目状态：
已结题

项目摘要

We established a new detection method of Cryptosporidium oocysts from the soil by a centrifugal floatation method using sucrose solution. Because the oocysts in the soil samples were surrounded by many of the particulate sand components, usual centrifugal floatation techniques could not be applied to effective collection of the oocysts. This problem was overcome by adding gelatin to the sucrose solution at a concentration of 0.1%. Although the recovery efficiency of the method varied from 10 to 20%, this technique was used for our survey. Soil samples around the farm, which had been confirmed to be enzootic for cryptosporidiasis, were examined and we found that all of 16 samples were positive. Studies on the contamination with Cryptosporidium in sandpits in public parks are being carried out at present.Different from Cryptosporidium oocyst, Toxoplasma oocyst could not be identified only by morphological approach. Then, we attempted a new detection method using a polymerase chain reaction. Purification method of Toxoca oocysts from soil sample was the same as that used for Cryptosporidium. Purified samples suspended in 400 μl of TNE buffer (containing 1% PVP and 1% SDS) were used for DNA extraction according to the standard procedure and were subjected to the PCR.Escherichia coli was detected from 120 out of 144 (80%) sand samples examined. The contamination rate of Toxocara egg examined at the same sandpits was 30% (43/144). which was different from the results obtained through E.coli study. Average number of E.coli found in 100 g of positive sand was 3400, and the numbers of E.coli in 10 sandpits were over the maximum detection limit of E.coli of this test (160,000). In a case of E.coli examination, some of the unused sea sand samples showed positive for E.coli showing that a E.coli examination of the sand will not be a good indicator for the fecal contamination.

建立了一种新的检测土壤中隐孢子虫卵囊的方法--蔗糖离心漂浮法。由于卵囊周围有许多颗粒状的沙粒成分，通常的离心浮选技术不能有效地收集卵囊。通过将明胶以0.1%的浓度添加到蔗糖溶液中来克服该问题。虽然该方法的回收率从10%到20%不等，但我们的调查使用了这种技术。对已确认为隐孢子虫流行区的农场周围土壤样品进行了检测，发现16个样品均为阳性。公园沙坑中隐孢子虫污染的研究正在进行中，与隐孢子虫卵囊不同，弓形虫卵囊不能仅通过形态学方法鉴定。然后，我们尝试了一种新的检测方法，使用聚合酶链反应。土样中弓形虫卵囊的纯化方法与隐孢子虫相同。用TNE缓冲液（含1%PVP和1%SDS）400 μl悬浮样品，按标准方法提取DNA，进行PCR检测，在144份样品中检出大肠杆菌120份（80%）。在同一个沙池中检测的弓形虫卵污染率为30%（43/144）。这与大肠杆菌研究结果不同。每100 g阳性沙中平均检出大肠杆菌3400个，10个沙坑中大肠杆菌数超过本试验大肠杆菌最高检出限（16万个）。在一宗大肠杆菌检验个案中，部分未使用的海沙样本对大肠杆菌呈阳性反应，显示对海沙进行大肠杆菌检验并不能作为粪便污染的良好指标。