Method for detecting norovirus in food based on negatively charged membrane concentration

A membrane concentration and negative charge technology, applied in the field of food norovirus elution and adsorption, can solve the problems of different concentration methods, different results, different physical and chemical properties, and incomparability, etc., to achieve the virus elution rate and detection. The effect of improving the detection rate, good repeatability and improving detection sensitivity

Pending Publication Date: 2017-03-22
JIANGXI AGRICULTURAL UNIVERSITY
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0010] In summary, although there have been many international reports on the concentration detection methods of norovirus in different foods, due to the different physical and chemical properties of materials and materials, adsorption-elution conditions, and operating procedures in various concentration methods, As a result, the results of different enrichment methods are also very different, and countries are not comparable

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  • Method for detecting norovirus in food based on negatively charged membrane concentration
  • Method for detecting norovirus in food based on negatively charged membrane concentration
  • Method for detecting norovirus in food based on negatively charged membrane concentration

Examples

Experimental program
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Embodiment 1

[0038] Example 1: Norovirus concentration and detection method establishment in lettuce samples

[0039] Before concentrating the virus in the lettuce sample, first carry out disinfection treatment (high temperature and high pressure sterilization treatment) to the virus concentration container and the like.

[0040] 1. Use BE eluent to elute norovirus in lettuce samples

[0041] Fresh vegetables (lettuce) are packed in 15g per serving. Add 140 μL norovirus-positive feces supernatant to each vegetable, spread it on the vegetable leaves (5 μl per spot), and place it in a secondary biological safety cabinet for 30 minutes. After air-drying, chop the vegetable leaves, put them into a 50ml centrifuge tube, add 30ml of eluent BE (tris 12.11g, glycine 3.75g, beef extract 10g, ddH 2 (01 L, pH=9.5), vortex at room temperature for 30 min, centrifuge at 10000 rpm for 15 min, and transfer the supernatant to another 50 ml centrifuge tube.

[0042] 2. Concentration of Norovirus in the E...

Embodiment 2

[0060] Example 2: Establishment of norovirus concentration and detection method in blueberry samples

[0061] Before concentrating the virus in the blueberry sample, the virus concentrating container and the like were sterilized (high temperature and high pressure sterilization).

[0062] 1. Use BE eluent to elute norovirus in blueberry samples

[0063] Fruit (blueberry) is divided into 15g per serving. Add 140 μl of norovirus NoV GI and NoV GII positive fecal supernatant to each blueberry, apply dots on the surface of blueberries (5 μl per dot), and place in a secondary biological safety cabinet for 30-60 minutes. After being air-dried, put it into a 50ml centrifuge tube, add 30ml of eluent BE (Tris 12.11g, Glycine 3.75g, Beef extract 20g, ddH 2 (O 1L, pH=9.5), add 150 μl each of pectinase (30000U / g, 0.02g / mL) and cellulase (10000U / g, 0.01g / mL), vortex at room temperature for 30min, centrifuge at 10000rpm for 15min, and Transfer the clear to another 50ml centrifuge tube. ...

Embodiment 3

[0079] Embodiment 3: Norovirus concentration and establishment of detection method in ham samples

[0080] Before concentrating the virus in the ham sample, first carry out disinfection treatment (high temperature and high pressure sterilization treatment) to the virus concentration container and the like.

[0081] 1. Use BE eluent to elute norovirus in ham samples

[0082] Meat and other instant cooked food (ham) is packed in 15g per serving. Add 140 μL of norovirus NoV GI and NoV GII positive fecal supernatant to each ham, spread it on the surface of the ham in spots (5 μl per spot), and place it in a secondary biological safety cabinet for 30-60 minutes. After being air-dried, cut the ham into pieces, put it into a 50ml centrifuge tube, and add 30ml of eluent ALK (KH 2 PO 4 6.8g, NaCl 58.5g, Trinton X-100 1mL, ddH 2 O 1L, pH=9.2), adjust the pH value of the eluent between 9.2-9.5, vortex at room temperature for 30min, centrifuge at 10,000rpm for 15min, transfer the sup...

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Abstract

The invention discloses a method for detecting norovirus in a food based on negatively charged membrane concentration. The method comprises the following steps: immersing a sample to be detected in an eluant, carrying out oscillation elution at room temperature, and centrifuging the above obtained solution to obtain a supernatant; adjusting the pH value of the supernatant to 2-4, and carrying out suction filtration by using a negatively charged filter membrane with the aperture of 0.45 [mu]m to obtain suction-filtered filter paper; and shearing the suction-filtered filter paper, extracting RNA, carrying out a real-time quantitative PCR to obtain the Ct value of the sample to be detected, and finally obtaining the content of the norovirus in the sample to be detected according to a norovirus NoV GI standard curve and a norovirus NoV GII standard curve. Compared with existing local standard (DBS13/001-2015), the method has the advantages of time saving, high efficiency, and increase of the virus enrichment rate by 5-10%.

Description

[0001] (1) Technical field [0002] The invention relates to a method for detecting Norovirus in food, in particular to three methods for elution and adsorption of Norovirus in food, and at the same time, real-time fluorescent quantitative PCR is used to detect the amount of concentrated virus (PCRU / mL) to improve the detection rate of Norovirus in food. Sensitivity to viral contamination. [0003] (2) Background technology [0004] In recent years, the frequent occurrence of global food-borne diseases and vicious food contamination incidents has made food safety a serious and ever-expanding world health problem, and has increasingly become a hot spot of global concern. With the current rapid growth of international trade and international tourism, the globalization of the food market has led to high risks of food, and has made food-borne diseases tend to spread globally. As the number one problem of food safety, more than half of foodborne diseases are caused by viruses. Nor...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/68C12R1/93
CPCC12Q1/6851C12Q2531/113C12Q2545/113
Inventor 廖宁波章荣华张严峻陈江
Owner JIANGXI AGRICULTURAL UNIVERSITY
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