Rotavirus RT PCR detecting kit and its detecting method

A detection kit, RT-PCR technology, applied in the biological field, can solve the problems of low sensitivity, virus contamination, strong infectivity, etc.

Inactive Publication Date: 2006-08-09
GUANGDONG INST OF MICROORGANISM +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The current food hygiene standards cannot explain the contamination of viruses in food. Although viruses are strictly intracellular parasites and cannot reproduce in food, a small amount of viruses in food can cause disease
According to foreign data, there are considerable numbers and types of foodborne viruses and the diseases they cause, some of which are widespread, highly contagious, prone to outbreaks, and serious hazards. This is not only a danger in the field of food hygiene. Factors, a major threat to people's public health and nutritional health, but also a serious loss to the food industry and the national economy
[0005] Rotavirus is an important food-borne virus. Conventional det...

Method used

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  • Rotavirus RT PCR detecting kit and its detecting method
  • Rotavirus RT PCR detecting kit and its detecting method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0029] Example 1: Detection of Rotavirus in Feces

[0030] (1) Extract sample nucleic acid with Trizol kit: take 200-300ul sample, add 900ul Trizol reagent, mix repeatedly with a pipette several times and let stand for 5min; then add 200ul chloroform, shake vigorously for 15S, and then let stand for 2- 3min; centrifuge at 12000g at 4°C for 15min, discard the upper liquid, add 500ul isopropanol, mix thoroughly, and place at room temperature for 10min; centrifuge at 12000g at 4°C for 10min, discard the supernatant again; Wash the precipitate; centrifuge at 7500g at 4°C for 5 minutes, carefully discard the ethanol; after fully drying, suspend the RNA precipitate in an appropriate amount of RNase-free water.

[0031] (2) Take MLV reverse transcriptase 1ul, DNA polymerase 1ul, Rnasin 0.5ul, primer 4ul, 2mmol / LdNTP2.5ul, 10× buffer 2.5ul, 25mmol / LMgCl in the PCR kit respectively with a micro-sampler 2 4Ul, add 1ul nucleic acid of the sample to be tested, then make up the volume to ...

Embodiment 2

[0038] Embodiment two: detection of rotavirus in shellfish

[0039] (1) Bioaccumulation of viruses

[0040] Purchase shellfish from the local aquatic market, stock them in a container filled with seawater, and then put rotavirus samples in the container to simulate the environment of a natural water body to naturally enrich shellfish. At the same time, a negative control was set up under the same conditions (no poisoning). Change the water and re-inject the poison every 8 hours, and the whole bioconcentration time is 24 hours.

[0041] (2) Activation and concentration of viruses in shellfish

[0042] Take five shellfish that have undergone bio-enrichment as a sample, shell them, take 1.5g of their stomach and digestive tract, then transfer them to a 50ml Falcon tube, add 15ml of cold sterilized 0.05mol / L Glycine-0.14mol / L sodium chloride (pH7.5), and then homogenized at high speed on ice with a Waring blender. In order to prevent cross-contamination, the Waring blender sho...

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Abstract

Thisa invention relates to a semi-set RT-PCR test reagent box for testing and verifying wheel-like virus including a MLV inverse transcriptase, Rnasin, DNA poly-enzyme, primers, dNTP, MgCl2 and 10 x buffer solution, in which, the upstream primer P1 is 5'-GTA TGG TAT TGA ATA TAC CAC-3', the downstream primer P2 is 5'-GAT CCT GTT GGC CAT CC-3. The RT-PCR test method includes: picking up a due test sample template, adding MLV inverse transcriptases, Rnasin, DNA poly-enzymes, primers, dNTP, MgCl2, 10 x buffer solution, the due test sample template and DECP water to be mixed uniformly to be enlarged on a PRC instrument, then the product is electrophoresis-enlarged in a device to get a result, which is recorded, analyzed and judged.

Description

[Affiliated technical field] [0001] The invention relates to a RT-PCR detection kit for detecting and identifying rotavirus and a use method thereof, belonging to the field of biotechnology. [Background technique] [0002] Human rotaviruses belong to the genus Rotavirus in the Reoviridae family. There are three groups of rotaviruses that can cause diarrhea in humans: A, B, and C. Among them, group A is infantile diarrhea rotavirus, group B and C are adult rotavirus. No matter in developed countries or developing countries, diarrhea caused by group A rotavirus has a relatively high incidence rate, and is the most important cause of morbidity and death in infants and young children. In my country, the situation is even more serious. Diarrhea caused by rotavirus and adult diarrhea are very prevalent. According to the statistics of WHO in 1997, there are 1.4 billion rotavirus infections in the world every year, and 870,000 people in developing countries die from it. There are...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12Q1/04
Inventor 吴清平寇晓霞张菊梅阙绍辉
Owner GUANGDONG INST OF MICROORGANISM
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