Culture and immunofluorescence quantitative detection method of human group H rotavirus

A quantitative detection method, immunofluorescence technology, applied in the field of human H group rotavirus culture and immunofluorescence quantitative detection, can solve the problem of quantification of non-viable virus

Pending Publication Date: 2021-06-01
中国疾病预防控制中心病毒病预防控制所
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

PCR technology detects nucleic acid and cannot quantify live virus

Method used

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  • Culture and immunofluorescence quantitative detection method of human group H rotavirus
  • Culture and immunofluorescence quantitative detection method of human group H rotavirus
  • Culture and immunofluorescence quantitative detection method of human group H rotavirus

Examples

Experimental program
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Embodiment 1

[0080] 1. Experimental method

[0081]1.1 Culture MA104 cells

[0082] 1.1.2 Recovery of cells

[0083] (1) Take out the required cell cryopreservation tube from the liquid nitrogen tank, put it into warm water at 37°C and shake it back and forth to melt it quickly, and centrifuge at 800rpm / min for 3min.

[0084] (2) Pour off the cell freezing solution.

[0085] (3) Prepare cell culture medium containing 10% FBS, that is, 1mL FBS+9mL1640, and the actual dosage is calculated according to the specific amount required for each experiment.

[0086] (4) Draw 6mL of cell culture solution into a 25cm 2 of cell flasks.

[0087] (5) at 25cm 2 Pipette 1mL of cell culture medium into the cell flask and blow the cells gently. After blowing, suck the cell suspension back to 25cm 2 Place the cell flask in a carbon dioxide incubator.

[0088] (6) Carbon dioxide culture conditions: the temperature is 37° C., and the carbon dioxide content is 5%.

[0089] (7) Change the medium for the ...

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Abstract

The invention discloses a culture and immunofluorescence quantitative detection method of human group H rotavirus, which comprises the following steps: (1) infecting passage cells with the human group H rotavirus, and carrying out passage amplification culture; (2) preparing an expression vector containing at least part of the VP6 gene sequence and/or at least part of the VP8 gene sequence, expressing a large amount of corresponding proteins in a prokaryotic expression system, and purifying; and (3) preparing an antibody from the purified protein, taking the antibody as a detection antibody, and detecting the human group H rotavirus by using an immunofluorescence detection method. The method is high in specificity, short in period, small in error and good in repeatability. According to the method, the condition of virus infected cells can be intuitively observed, the biological characteristics of the virus can be researched, and a favorable means can be provided for virus quantification.

Description

technical field [0001] The invention belongs to the technical field of microbes, and in particular relates to a method for culturing and immunofluorescence quantitative detection of human group H rotavirus. Background technique [0002] Rotavirus (RV) is the leading cause of severe acute diarrhea in children under 5 years of age worldwide. In low-income countries and high-income countries, the number of infants under 1 year old who are infected accounted for 80% and 65% of the total number respectively. According to WHO estimates, about 420,000 to 494,000 infants and young children worldwide die of RV infection every year. Rotavirus infection has caused more than 2 million hospitalized children with diarrhea. Most of the deaths among infected patients occurred in Asia and Africa. In these developing countries, the increase in mortality may be related to factors such as poor health care, malnutrition, and other diseases. According to statistics, in China, about 40,000 infan...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N7/00G01N33/569C12R1/93
CPCC12N7/00G01N33/56983C12N2720/12351G01N2333/14G01N2469/10
Inventor 段招军庞立丽章青
Owner 中国疾病预防控制中心病毒病预防控制所
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