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Method for detecting infective rotavirus

A virus and rotavirus technology, which is applied in the direction of biochemical equipment and methods, microbial measurement/inspection, etc., can solve the problem of inability to provide infectivity, prevent the outbreak of rotavirus disease, improve the detection range, accuracy and sensitivity high effect

Inactive Publication Date: 2008-02-20
TSINGHUA UNIV
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AI Technical Summary

Problems solved by technology

However, using these methods alone can only determine whether there is rotavirus nucleic acid in water environmental samples, and cannot provide information such as whether it is infectious

Method used

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  • Method for detecting infective rotavirus
  • Method for detecting infective rotavirus

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Embodiment 1

[0023] Embodiment 1, the method for detecting infectious rotavirus

[0024] 1. Rotavirus Titer Determination

[0025] Median tissue culture infection dose (TCID) 50 ) titration.

[0026] 1) Cultivation of rotavirus host cell MA-104 cell line

[0027] Inoculate the macaque kidney cell line MA-104 in 5ml of DMEM high-glucose cell culture medium containing 10% FBS at 37°C in 5% CO 2 Cultured in an incubator and subcultured every 2-3 days. Take the monolayer-covered cells, discard the old cell culture medium, wash the cell surface once with a small amount of PBS solution, add a small amount of trypsin digestion solution (just enough to cover the cell surface), and digest at 37°C. Observe under a microscope, when most of the cells are swollen and rounded, add DMEM (high glucose) cell culture medium containing 10% fetal bovine serum, repeatedly pipette several times with a pipette to form a uniform cell suspension, centrifuge at 900rpm for 5min, discard Supernatant, DMEM cell c...

Embodiment 2

[0114] Embodiment 2, ICC-RT-PCR method detects infectious rotavirus in drinking water

[0115] 1. Add 100, 10, 1, 0.1 μl to 1L drinking water respectively, and the titer is 10 4.5 TCID 50 / ml of rotavirus, so that the titer of rotavirus in the water sample is 10 0.5 、10 -0.5 、10 -1.5 、10 -2.5 TCID 50 / ml, stirred at room temperature for 30min.

[0116] 2. Add aluminum chloride to the water samples respectively to make the final concentration 0.05mol / L, and stir.

[0117] 3. Filter the water sample with a cellulose acetate filter membrane with a pore size of 0.45 μm and a diameter of 47 mm at a filtration rate of 100 ml / min. The virus is adsorbed on the filter surface.

[0118] 4. Use 50ml of H at pH 3.0 2 SO 4 The solution passes through the filter membrane at a rate of 100ml / min to wash away the cations on the surface of the filter membrane.

[0119] 5. Remove the filter membrane, cut the filter membrane into pieces with sterilized scissors, put it into a 50ml cent...

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Abstract

The invention discloses a method used for detecting infectious rotavirus. The method is that under-detect specimen and host cell of the rotavirus are cultured together, then total RNA of the host cell is extracted. Primer pair is composed of the nucleotide segment of which the nucleotide sequence is sequence NO1 in the sequence table and the nucleotide segment of which the nucleotide sequence is sequence NO2 in the sequence table, then the primer pair process RT-PCR. And if the under-detect sample obtains 300-400bp PCR product, the under-detect sample has the infectious rotavirus .The invention not only has good specificity, high sensitivity and strong capacity of resisting impurity and disturbance, but also provides information like potential infection effect which is in the rotavirus of ambient medium, thereby providing strong support for fleetly detecting infectious rotavirus contained in the ambient medium, evaluating infectious risk of the rotavirus in the environment, and taking precautions against the break-out and prevalence of rotavirus diseases.

Description

technical field [0001] The invention relates to a method for detecting infectious rotavirus. Background technique [0002] Pathogenic microorganisms transmitted through water are a major hidden danger to human health and ecological safety, and about 1 / 4 of the diseases in the world are directly or indirectly caused by pathogenic microorganisms in water (Gerba, C.P., Pathogens in the environment. In: Pepper, I.L. , Gerba, C.P., Brusseau, M.L. (Eds.), PollutionScience. Academic Press, New York, 1996. p.279-299). Rotavirus is one of the pathogenic microorganisms widely present in the water environment, mainly from human and animal excreta (Morteza Abbaszadegan, Peter Stewart, 1 and Mark LeChevallier A Strategy for Detection of Viruses in Groundwater by PCR Applied and Environmental Microbiology, February1999, 65(2):444-449). Globally, rotavirus is the most important cause of diarrhea, and the number of people with diarrhea due to water-borne rotavirus is about 111 million and...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/68
Inventor 何苗李丹胡秀华施汉昌刘丽
Owner TSINGHUA UNIV