Detection Method For Giardia And Cryptosporidium In Source Water And Investigation Of Pollution In Some Areas In China

The spread of water-borne giardiasis and cryptosporidiosis is an important public health issue. The impairment to drinking water should not be ignored because the conventional chlorinated disinfectant can not completely to disinfect all the Giardia and Cryptosporidium in water. In 2007,our country implement GB 5749-2006"drinking water health standards" and it is the first time to bring Giardia and Cryptosporidium into national standard. In GB 5749-2006, Giardia and Cryptosporidium should be <1/10L in water. With high cost of this detection method,it is not easy to use in most laboratories in our country. So it is necessary to explore a new method with low cost to examine Giardia and Cryptosporidium. At the same time,it has positive significance to understand the pollution in typical regions and to prevent disease to protect people.Objective1.To Understand pollution situation of Giardia and Cryptosporidium in parts of our country, and provide data support for the relevant regulators.2. To establish a method for Giardia and Cryptosporidium detection with low cost and easy operation to make it easy to use in most laboratories in our country. To lay the foundation for revision of the standard test method.3. Explore DNA extraction methods and PCR reaction conditions of Cryptosporidium, and lay the foundation for identification method of Cryptosporidium.Methods1. This paper studied the source water and tap water of water source and water supply plant in northeast, central and north China with a total of 120 samples.The samples were collected in August to October(wet season) and in November to December(dry season),respectively. Detect Giardia and Cryptosporidium with standard method of Filta - Max Xpress and simultaneous detect aerobic bacterial count.total coliforms, Escherichia coli and heat-resistant coliform group to determine the present pollution situation in parts of our country. Then explore the relationship between Cryptosporidium , Giardia and other microbial indicators in water.2. Establish detection method for Cryptosporidium and Giardia through laboratory spiked recovery experiments and detection research of water samples from environment. Then determine the key steps that affect the total recovery rate by the recovery rates at different steps and optimize each step of the operation method.After the method was determined, the Xpress Filta-Max method and this method were used to detect the water source water at the same time to compare the effect of these two methods for the detection of Giardia and Cryptosporidium in source water.3. To determine the experimental conditions of Cryptosporidium DNA extraction and PCR process. Based on some experimental data in some literatures,use the genotype of- Cryptosporidium muris as the research object to judge if freeze-thaw af-fect DNA extraction of Cryptosporidium oocysts and the amount of template to add in PCR process. Then determine the sensitivity of the test method at different concentrations. Collect 192 animal feces in northeast and central China,identify the species of cryptosporidium with this method and judge the f’easibility of the method.Results1.Pollution investigationThe study showed that Cryptosporidium and Giardia are polluted to some degree in these three palaces. In the 60 source water samples, Giardia was detected in 10 samples, the positive rate was 16.7% (10) and exceeding rate was 10% (6 / 60);Cryptosporidium was detected in 26 water samples, the positive rate was 43.3% (26 /60) and exceeding rate was 26.7% respectively. Only in a city in central China, a sample of tap water of Cryptosporidium is detected in dry season.The positive rate and exceeding rate of source water are both higher than that of tap water. Microbial indicators showed that pollution exist in all these regions,especially in the city in central China. The plants here have poor facilities and unsanitary conditions. There is statistical correlation between Cryptosporidium and thermotolerant coliform bacteria.Cryptosporidium and turbidity is positively correlated,it suggested that turbidity can be used as a removal index of Cryptosporidium.2. Establishment of the detection method for Cryptosporidium and GiardiaTwo methods were applied in this research. (1)Method of membrane dissolution- density gradient centrifugation with percoll and sucrose-immunofluorescence assay: impurities and Giardia and Cryptosporidium can not be completely separated, so the membrane clogged in the step of immunofluorescence assay and the test has to be halted.So this method can not be used for dection of Cryptosporidium and Giardia. (2)Method of membrane dissolution-immunomagnetic separation -immunofluorescence assay: The recovery rates at different steps of membrane dissolution-immunomagnetic separation-immunofluorescence assay shows that the step of membrane filtration is the key step that affects the total recovery rate and it is affected by many factors. The average recovery rate of Giardia lamblia is 36.58%,and RSD is 38.44%; The average recovery rate of Cryptosporidium is 19.58% and RSD is 34.32%. They are all in line with the requirements of relevant standards. There are no differences of statistics between the method of membrane dissolution-immunomagnetic separation-immunofluorescence assay and the method of Filta-Max Xpress to examine Giardia and Cryptosporidium.It can be applied to examine Giardia and Cryptosporidium.3. Study on identification of CryptosporidiumIn order to improve the sensitivity of experiments, experiments were carried out using nested PCR method. The study showed that during the PCR process, when the first PCR template volume is 9.5μL, the second PCR template volume is 5μL, the test can get better results. When extracting DNA of Oocysts, freezing and thawing oocysts should be repeated 3 times, and then extract DNA in accordance with the DNA Extraction Kit. When the amout of oocysts reach is 90, 14 positive results of 15 samples, the detection rate is 93.3%. In fecal samples, the cryptosporidium from duck were the C.baileyi, which indicated that the method could be applied to the identification of cryptosporidium.ConclusionsInvestigation and experimental results show that Cryptosporidium and Giardia pollution in China can not be ignored,both of them are positively related with thermotolerant coliform bacteria.Through addition standard recovery test, method for detection of Cryptosporidium and Giardia meet the standard requirements, the recovery rate had no significant difference compared with the standard method. It can greatly reduced the cost of testing and can be used in practical detection. Detection of Cryptosporidium identification method can be effectively applied to the environment in stool samples.