Method for detecting cryptosporidium parvum and detection kit

A technology of Cryptosporidium parvum and detection method, which is applied in the directions of microorganism-based methods, microbial determination/inspection, biochemical equipment and methods, etc., and can solve the problems of inability to detect Cryptosporidium parvum quickly and sensitively.

Active Publication Date: 2014-07-02
INST OF ZOOLOGY CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] Therefore, the object of the present invention is to provide a detection method and a detection kit for Cryptosporidium parvum, which can quickly and effectively detect samples to be tested such as water samples, biological samples, etc. Cryptosporidium parvum in fresh food and fecal samples

Method used

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  • Method for detecting cryptosporidium parvum and detection kit
  • Method for detecting cryptosporidium parvum and detection kit
  • Method for detecting cryptosporidium parvum and detection kit

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0171] Example 1: Sample preparation of Cryptosporidium parvum immunomagnetic separation and purification system (IMS)

[0172] 1. Materials: Streptavidin Particles Plus-DM was purchased from BD Company; Cryptosporidium parvum suspension was preserved by the Institute of Zoology, Chinese Academy of Sciences.

[0173] Preparation of other reagents:

[0174] Preparation of phosphate buffered saline (PBS) (pH=7.4) containing 3% bovine serum albumin (BSA):

[0175] Potassium dihydrogen phosphate (KH 2 PO 4 ): 0.27g;

[0176] Disodium hydrogen phosphate (Na2 HPO 4 ): 1.42g;

[0177] Sodium chloride (NaCl): 8g;

[0178] Potassium chloride (KCl): 0.2g;

[0179] Bovine serum albumin (BSA): 3g;

[0180] Add about 800 μl of deionized water and stir well to dissolve, then add concentrated hydrochloric acid to adjust the pH to 7.4, and finally adjust the volume to 1L. Store at room temperature after high temperature and high pressure sterilization.

[0181] 2. Preparation proc...

Embodiment 2

[0194] Example 2: Establishment of Taqman probe method fluorescent quantitative PCR detection system

[0195] 1. Materials: Cryptosporidium parvum suspension (separated according to the method described in Example 1) and Escherichia coli (purchased from the American Type Culture Collection Center, with the preservation number ATCC8739), both the Peasy-T1 vector and Trans1T1 competent cells were purchased From TransGenBiotech.

[0196] 2. Tool enzyme: Taqmix was purchased from TaKaRa Company.

[0197] 3. Reagents:

[0198] Primers and Taqman probe fluorescent quantitative PCR kits were purchased from TaKaRa Company; IPTG, X-gal, DNA marker (marker), genomic DNA extraction kits, agarose DNA gel recovery kits and plasmid mini-prep kits were purchased from TIANGEN company.

[0199] Preparation of other reagents:

[0200] Preparation of phosphate buffered saline (PBS) (pH=7.4) containing 3% bovine serum albumin (BSA):

[0201] Potassium dihydrogen phosphate (KH 2 PO 4 ): 0...

Embodiment 3

[0248] Example 3: Optimal detection conditions, sensitivity and specificity of the detection method of Cryptosporidium parvum verification of

[0249] 1. Materials: Streptavidin Particles Plus-DM and CellSeparation Magnet were purchased from BD Company; Cryptosporidium parvum suspension (isolated according to the method described in Example 1) and Escherichia coli ( Purchased from the American Type Culture Collection, the preservation number is ATCC8739); biotinylated Cryptosporidium parvum Cp23 monoclonal antibody (Anti-Immunodominantantigen Cp23) was purchased from Huada Protein Company.

[0250] 2. Tool enzyme: 2×Taqmix was purchased from TaKaRa Company.

[0251] 3. Primers and Taqman probe method Fluorescence quantitative PCR kit was purchased from TaKaRa Company; agarose, DNA marker (marker), and genomic DNA extraction kit were purchased from TIANGEN Company.

[0252] 4. Validation of the optimal final concentration of streptomycin magnetic beads coated with biotinyl...

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Abstract

The invention provides a method for detecting cryptosporidium parvum and a detection kit. The detection method comprises the following steps: (1) separating and purifying a sample to be detected by immunomagnetic beads to prepare suspension containing cryptosporidium parvum; and (2) detecting the suspension containing cryptosporidium parvum prepared in the step (1) by fluorogenic quantitative PCR (polymerase chain reaction). The detection kit comprises a reaction system containing biotinylated cryptosporidium parvum Cp23 monoclonal antibody coated streptomycin magnetic beads in the detection method, a specific forward primer of cryptosporidium parvum, a specific reverse primer and a Taqman fluorogenic quantitative probe primer. According to the cryptosporidium parvum detection method, the sensitivity is 100 times higher than that of a traditional method, and is far beyond that of the traditional method.

Description

technical field [0001] The invention relates to the fields of environmental monitoring and immunodiagnosis, and relates to a detection method and a detection kit, in particular to a detection method and a detection kit of Cryptosporidium parvum. Background technique [0002] Cryptosporidium parvum (Cryptosporidium Tyzzer, 1907) widely exists in animals, and is also an important parasitic parasite in humans, which can cause Cryptosporidiosis (Cryptosporidiosis). The species that parasitizes the human body is mainly Cryptosporidium parvum (C.parvum), which is an opportunistic protozoan, but it is also an important pathogen of diarrhea. The clinical symptoms and severity of the disease depend on the immune function and nutritional status of the host. After infection, people with normal immune function mainly manifest as acute watery diarrhea, generally without pus and blood, and defecate more than 2 to 20 times a day. Children with severe infection may have jet watery diarrhea...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12Q1/04C12R1/90
CPCC12Q1/6806C12Q1/686C12Q2561/101C12Q2523/308
Inventor 何宏轩高姗姗王承民罗静
Owner INST OF ZOOLOGY CHINESE ACAD OF SCI
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