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A method for detecting hepatitis E virus in pig liver

A virus and hepatitis E technology, applied in the field of virology, can solve the problems of many PCR inhibitors, low product recovery rate, false negative PCR results, etc.

Active Publication Date: 2022-04-19
CHINA NAT CENT FOR FOOD SAFETY RISK ASSESSMENT
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the recovery rate of the extracted product by this method is low and there are too many PCR inhibitors, resulting in false negative results of PCR.

Method used

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  • A method for detecting hepatitis E virus in pig liver
  • A method for detecting hepatitis E virus in pig liver
  • A method for detecting hepatitis E virus in pig liver

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0043] Embodiment 1: RNA extraction method in pig liver

[0044] (1) Take 1 g of pig liver sample and cut it into pieces, put it in a homogenizing bag, add 2 μL of RNaseOUT, then add 10 μL of MS2 process control control and 7 mL of TRIZOL solution, and homogenize for 5 minutes;

[0045] (2) Take at least 5mL supernatant, centrifuge at 3000rpm at 4°C for 10min, take 4mL of supernatant into a new centrifuge tube, add 1mL chloroform, shake and mix, and centrifuge at 12000rpm at 4°C for 15min. Aspirate 140 μL of supernatant, and extract viral RNA according to the operating instructions of the viral RNA extraction kit;

[0046] (3) Add 1ml of 75% alcohol to 50μL RNA extract, mix well, centrifuge at 12000rpm for 15min, discard the supernatant, place in a vacuum drying oven at 50°C, vacuum dry for 20min, then add 50μL of water to dissolve the product . Take 5 μL of the final product, add 1 μl of the HEV standard control, and make a water control at the same time to ensure that PCR in...

Embodiment 2

[0076] Example 2: Changing detection parameters

[0077] Change step (1) of Example 1 to add 5 μL of RNaseOUT.

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Abstract

This application provides a method for detecting hepatitis E virus in pig liver. The specific steps are: take 1-2 g of pig liver sample and cut it into pieces, add RNaseOUT, then add MS2 process control control and TRIZOL solution, and homogenize with a homogenizer; Take the supernatant, centrifuge, add chloroform, shake and mix, draw the supernatant, and extract the virus RNA; use 75% alcohol to process the RNA extract, centrifuge, dry, add water to dissolve, and obtain the final product, and then use fluorescence HEV detection was performed by reverse transcription PCR method. The recovery efficiency of the method provided by the invention is higher than that of the existing methods in the art; the PCR inhibitor removal effect is better than that of the PCR inhibitor removal kit, and the primers and probes used in the detection are self-designed and have high sensitivity.

Description

technical field [0001] The invention relates to the technical field of virology, in particular to a method for detecting hepatitis E virus in pig liver. Background technique [0002] Hepatitis E virus (HEV) is one of the main pathogens causing acute hepatitis in humans, and has attracted more and more attention from the international community in recent years. The molecular structure of HEV is a single-stranded non-enveloped RNA virus with a diameter of 27-34nm. It belongs to the Hepatitis virus family Hepatitis E virus. The genome length is about 6.6-7.3kb, including 3 open reading frames (ORF1-3), ORF1 It encodes non-structural proteins related to viral transcription and replication, ORF2 encodes viral capsid protein, and ORF3 encodes a small molecule phosphoprotein, which is related to the cytoskeleton and specific immune response of HEV. Some developing countries in Asia and Africa show an epidemic trend, while developed countries show a sporadic trend, and my country i...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/70C12Q1/686C12N15/11C12R1/93
CPCC12Q1/707C12Q1/686C12Q2545/113C12Q2563/107
Inventor 王佳慧李凤琴江涛李楠
Owner CHINA NAT CENT FOR FOOD SAFETY RISK ASSESSMENT