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Method for improving specificity of cryptosporidium hominis in water and method for evaluating activity of cryptosporidium hominis in water

A cryptosporidium-specific technology, applied in the field of evaluating human-derived Cryptosporidium activity in water, can solve the problems of secondary pollution, low efficiency, and high cost, and achieve the effects of reducing toxicity and harmfulness, overcoming false positives, and low cost

Inactive Publication Date: 2014-03-12
SHENZHEN POLYTECHNIC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] The further technical problem to be solved by the present invention is to provide a method for evaluating the activity of human-derived Cryptosporidium in water, which is simple and effective, and has high sensitivity, and solves problems such as high cost, low efficiency, and secondary pollution caused by Cryptosporidium evaluation

Method used

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  • Method for improving specificity of cryptosporidium hominis in water and method for evaluating activity of cryptosporidium hominis in water
  • Method for improving specificity of cryptosporidium hominis in water and method for evaluating activity of cryptosporidium hominis in water
  • Method for improving specificity of cryptosporidium hominis in water and method for evaluating activity of cryptosporidium hominis in water

Examples

Experimental program
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Effect test

example 1

[0047] In the present embodiment, the above-mentioned method is used to detect the activity of Cryptosporidium hominis in water, and the specific steps are as follows:

[0048] (1) Provide a water sample containing a certain concentration of active Cryptosporidium to be tested, and the concentration of Cryptosporidium should be controlled at 1 to 5×10 5 within the range of / L;

[0049] (2) Dissolve the PMA dye in 20% DMSO to prepare a 50 mmol / L stock solution, and store it at -20°C in the dark. PMA dye was added dropwise to the sample during detection, and the final concentration in the detection sample was 50 μmol / L. Shake up and down 3 to 4 times, avoid light for 5 to 10 minutes, shake the centrifuge tube from time to time during the reaction, so that the sample can fully contact and react with the PMA dye;

[0050] (3) Place the PMA-stained sample reaction tube on ice or in a low-temperature environment, and irradiate it with a halogen lamp with a power of 500-800W for 2-...

example 2

[0057] According to the method described in Example 1, in order to evaluate the detection sensitivity of the PMA-qPCR method, the Escherichia coli standard was added into the water sample to be tested containing a certain concentration of active Cryptosporidium and the active Cryptosporidium without adding the E. coli standard The water samples to be tested for sporozoites were compared and tested, and the same experimental steps were carried out, PMA staining, strong light irradiation treatment, the results are shown in figure 2 , it can be seen from the figure that the PMA-qPCR method can still accurately and quantitatively detect the activity of Cryptosporidium hominis in the water containing other miscellaneous bacteria.

example 3

[0059] According to the method of Example 1, the comparative test of PMA-qPCR and qPCR in detecting the activity of human-derived Cryptosporidium in water under different sample concentrations is compared. (1) Provide a water sample containing a certain concentration of human-derived active Cryptosporidium to be tested, Cryptosporidium The concentration of insects is controlled at 1~5×10 3 , 1~5×10 4 pcs / L and 1~5×10 5 within three orders of magnitude per unit / L; (2) Extract the DNA in the sample; the process is: after the sample is centrifuged at 10,000rpm for 15min, remove the supernatant, take the precipitated part into a 1.5mL sterilized centrifuge tube, add 1mL sterilized Distill water and shake to mix, centrifuge at 10000rpm for 10min, discard the supernatant, and repeat twice. The obtained pretreated samples were extracted with a DNA extraction kit (Shanghai Huashun Biotechnology Co., Ltd. DNA extraction kit), and finally 80 μL of DNA solution was obtained and stored ...

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Abstract

The invention relates to a method for improving the specificity of cryptosporidium hominis in water and a method for evaluating the activity of cryptosporidium hominis in water. The method is used for evaluating the activity of cryptosporidium hominis in water by utilizing the fluorescent quantitative PCR (polymerase chain reaction) (qPCR) technology in combination with an azide colorant propidium monoazide (PMA), and comprises the steps of irradiating a sample to which a PMA dye is added by strong light, constructing a target gene and primers, carrying out qPCR reaction and detecting cryptosporidium hominis with activity. By adopting the method, the sensitivity and specificity of detection are improved, the operating steps are simplified, the detection time is shortened, and the detection cost is reduced.

Description

technical field [0001] The invention relates to a method for improving the specificity of Cryptosporidium hominis and a method for evaluating the activity of Cryptosporidium hominis in water. Background technique [0002] Cryptosporidium hominis is an important class of pathogenic protozoa in human hygiene. It can parasitize human intestinal epithelial cells and cause symptoms such as gastroenteritis and diarrhea, especially immune dysfunction People such as infants, the elderly and AIDS patients, etc., will cause diarrhea after being infected, and even endanger their lives. The contamination of drinking water by anthropogenic Cryptosporidium has posed a serious threat to the health of citizens, and Cryptosporidium oocysts have become one of the water-borne biological risk factors that have attracted much attention. In my country's newly promulgated "Drinking Water Sanitation Standards", Cryptosporidium and Giardia have been used as unconventional indicators and their limit...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12Q1/04C12R1/90
CPCC12Q1/686C12Q2563/173C12Q2523/313
Inventor 李绍峰冉治霖胡健龙曾辉
Owner SHENZHEN POLYTECHNIC
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