Rapid detection method and kit for dairy cow cryptosporidia

A detection kit and technology for cryptosporidium, applied in biochemical equipment and methods, microbe measurement/inspection, resistance to vector-borne diseases, etc., can solve problems such as no treatment methods, and achieve containment of transmission, simple operation, and specificity strong effect

Inactive Publication Date: 2016-07-06
LANZHOU INST OF VETERINARY SCI CHINESE ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Once infected with Cryptosporidium, there is no effective treatment

Method used

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  • Rapid detection method and kit for dairy cow cryptosporidia
  • Rapid detection method and kit for dairy cow cryptosporidia

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0032] Dairy cattle Cryptosporidium rapid detection kit of the present invention comprises:

[0033] Sodium lauroyl sarcosinate aqueous solution, Tween-20 aqueous solution, primer-probe mixture, PBST solution, flow chromatography test strips (MileniaHybridtech1strips, Wintron Bio), RPA kit (TwistAmpnfokit, Wintron Bio) provided Freeze-dried enzyme and RPA reaction master mix (29.5μl RehydrationBuffer and 2.5μl 280mM MagnesiumAcetate mixture).

[0034] The specific preparation method is:

[0035] (1) Fecal sample treatment solution: ① 0.1% aqueous solution of sodium lauroyl sarcosinate with a mass percentage of 400 μl / tube was divided into 2 ml centrifuge tubes for later use; ② 10% mass percentage of Tween-20 The aqueous solution was divided into 2ml centrifuge tubes according to 500μl / tube and set aside.

[0036] (2) Primer-probe mixture: This application aims at a conserved sequence of the small ribosomal 18S gene of Cryptosporidium parvum, Cryptosporidium bovis, Cryptospor...

Embodiment 3

[0053] The sensitivity experiment of embodiment 3 kits of the present invention

[0054] The experimental process is as follows: the purified Cryptosporidium eggs are serially diluted with pure water, and each sample of the serially diluted Cryptosporidium is mixed with the feces of dairy cows not infected with Cryptosporidium, and after mixing, DNA is extracted from them according to the above method, and Carry out RPA amplification reaction, the amount of the DNA of the Cryptosporidium egg added in each amplification reaction is respectively 10 4 , 10 3 , 10 2 , 10, 5, 1, 0.5, 0.1. After the reaction, flow chromatography test strip detection was carried out according to the aforementioned method, and the results showed that the method could detect the DNA of 0.5 Cryptosporidium eggs added to the reaction system.

[0055] For experimental results, see figure 1 (Note: The appearance of the control band is to show that the function of the test strip is normal, and the app...

Embodiment 4

[0056] The specificity experiment of embodiment 4 kit of the present invention

[0057] The experimental process is: Toxoplasma gondii, Giardia lamblia, Eimeria, Microsporidium and non-infected Cryptosporidium cow feces DNA and four Cryptosporidium DNA for specific analysis, it was found that the kit Only Cryptosporidium parvum, Cryptosporidium amnesii, Cryptosporidium bovis, and Cryptosporidium wrightei can be detected in cows.

[0058] For experimental results, see figure 2 (Note: The appearance of the control strip is to show that the test strip is functioning normally, and the appearance of the detection strip indicates positive).

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PUM

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Abstract

The invention provides a rapid detection kit for dairy cow cryptosporidia.The kit comprises a sodium lauroyl sarcosine water solution, a Twain-20 water solution, a primer and probe mixed solution, a PBST solution, a flow measuring chromatography test strip, RPA lyophozyme and RPA reaction premix.The invention further provides a rapid detection method for the dairy cow cryptosporidia.The kit has the advantages of being high in sensitivity and specificity, rapid in reaction, easy to operate and the like.No DNA extraction kit needs to be used for extracting sample DNA, a laboratory environment can be completely separated through the advantages of RPA-flow measuring chromatography, and therefore whether daily cows are infected with the cryptosporidia or not can be rapidly detected in a daily cow farm on site, losses are reduced by taking corresponding measures, and propagation is restrained.

Description

technical field [0001] The invention relates to a rapid detection method and a detection kit for dairy cow cryptosporidium. Background technique [0002] Cryptosporidium eggs are widely present in the environment and can infect a variety of vertebrates, causing diarrhea and even death in varying degrees. For the dairy farming industry, cryptosporidiosis is an important parasitic disease, which can cause calf diarrhea, inhibit the growth and development of young animals, reduce the output and quantity of livestock products, and cause a lot of waste of feed. At the same time, cow manure is an important source of infection, threatening human health. Once infected with Cryptosporidium, there is no effective treatment. Therefore, it is particularly important to establish a quick and easy diagnostic method for Cryptosporidium in dairy cows. Contents of the invention [0003] The object of the invention is to address the deficiencies of the prior art, and propose a quick and e...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12Q1/04C12N15/10
CPCC12N15/1003C12Q1/6844C12Q1/6893C12Q2521/507C12Q2537/1376C12Q2522/101C12Q2565/625C12Q2527/125Y02A50/30
Inventor 周东辉吴耀东徐民俊郑文斌宋慧群朱兴全
Owner LANZHOU INST OF VETERINARY SCI CHINESE ACAD OF AGRI SCI
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