C. andersoni PCR detecting kit

A detection kit, a technology for Cryptosporidium ansii, is applied in the field of diagnosis and detection of cryptosporidiosis, and can solve the problems of confusion between non-specific dyed particles and oocysts, affecting the accuracy of inspection results, and low detection rate of stool dyeing , to achieve the effect of objective and accurate result judgment, optimized PCR reaction conditions, and high sensitivity

Inactive Publication Date: 2006-08-09
SOUTH CHINA AGRI UNIV
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Problems solved by technology

[0004] So far, techniques such as indirect hemagglutination assay (IHA), immunofluorescence assay (IFA), enzyme-linked immunosorbent assay (ELISA), monoclonal antibody (McAb), and polymerase chain reaction (PCR) have been used for the diagnosis of this disease. or detection and related research, but there is no PCR test kit for the diagnosis or detection of Cryptosporidium amnesii infection in dairy cows
However, conventional detection methods such as saturated sucrose flotation method and improved acid-fast staining method all have many deficiencies: when the sample volume is large, it may cause eye fatigue of the microscopic examination personnel and affect the accuracy of the inspection results; when the oocyst content in the sample is small, The detection rate of stool staining is low, it is easy to miss, and often contains more non-specific staining particles mixed with oocysts, which is difficult for non-professional microscopic examination personnel to distinguish

Method used

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  • C. andersoni PCR detecting kit
  • C. andersoni PCR detecting kit

Examples

Experimental program
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Effect test

Embodiment 1

[0050] The composition of embodiment 1 kit

[0051] The kit contains 15ml of DNA lysate, which contains NaCl 100mM, Tris-HCl (pH8.0) 10mM, EDTA (pH8.0) 25mM, SDS 1% (w / v) and proteinase K 4μg / μl; PCR reaction solution 5 Tube (25μl / reaction×20), the final concentration of dATP, dTTP, dGTP and dCTP is 200μM each, the final concentration of primers ZRQF and ZR is 0.2μM each, Mg 2+ The final concentration is 3.0mM; 2.0g agarose; 100μl ethidium bromide (10μg / μl); 0.25% bromophenol blue spotting buffer 100μl; 5μl Taq enzyme (5U / μl); positive control.

Embodiment 2

[0052] The composition of embodiment 2 test kits

[0053] The kit contains 15ml of DNA lysate, which contains NaCl 100mM, Tris-HCl (pH8.0) 10mM, EDTA (pH8.0) 25mM, SDS 1% (w / v) and proteinase K 4μg / μl; PCR reaction solution 5 Tube (25μl / reaction×20), the final concentration of dATP, dTTP, dGTP and dCTP is 400μM each, the final concentration of primers ZRQF and ZR is 1.0μM each, Mg 2+ The final concentration is 5.0mM; 1 positive control of Cryptosporidium ahnii genomic DNA.

Embodiment 3

[0054] The specificity test of embodiment 3 kits

[0055] Take Cryptosporidium amerii and Cryptosporidium parvum, Cryptosporidium bellinii, Eimeria bovis, Toxoplasma gondii, Eimeria suis, Ascaris suum, Cyclosporidium, Escherichia coli and other 8 controls Each 1 μL of sample DNA was used as a template for specific PCR amplification of the kit, and a blank control was set at the same time.

[0056] PCR amplification conditions:

[0057] Pre-denaturation at 94°C for 5 minutes

[0058]

[0059] Extend at 72°C for 7 minutes

[0060] The amplified products were analyzed by electrophoresis on 1.0% agarose gel to determine their specificity.

[0061] The results of electrophoresis showed that only the DNA of the Cryptosporidium amneri sample amplified a specific fragment of 504bp, while the samples of Cryptosporidium parvum, Cryptosporidium bellini, Eimeria bovis, Toxoplasma gondii, and A. The amplified bands did not appear in the DNA of coccidia, Ascaris suum, Cyclospora, and...

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Abstract

This invention provides a PCR test reagent box for diagnosing and testing An- masked sporidiosis, which is prepared by designing specific primers according to the ITS-1 gene sequence of the masked sporidiosis, setting up PCR test method and optimizing the PCR reaction condition. This invention also provides a method for using the box.

Description

technical field [0001] The invention relates to the technical field of diagnosis and detection of cryptosporidiosis, and more specifically relates to a PCR detection kit and detection method of Cryptosporidium ahnii. Background technique [0002] Cryptosporidiosis (Cryptosporidiosis) is an important zoonotic parasitic disease, which can cause severe diarrhea in humans and cattle, and is one of the main causes of death in AIDS patients. The disease is mainly caused by contamination of water sources with oocysts excreted by animals infected with Cryptosporidium (mainly dairy cows). Cryptosporidium andersoni and C.parvum are the main Cryptosporidium infection in dairy cows. Some data show that in most surface water (rivers, rivers, lakes, reservoirs, etc.), shallow wells polluted by surface water and deep well water with poor protection conditions, poorly treated tap water, treated or untreated sewage, Cryptosporidium oocysts can be detected in filtered swimming pool water (S...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68
Inventor 李国清周荣琼肖淑敏
Owner SOUTH CHINA AGRI UNIV
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