Method for concentrating and detecting norovirus in water based on aminated silica gel

An aminoated silica gel and virus technology, applied in the field of enrichment and detection of norovirus, can solve the problems of difficulty in accurate measurement, difficult virus culture, time-consuming operation, etc. The effect of improving the removal efficiency

Active Publication Date: 2016-12-07
ZHEJIANG CENT FOR DISEASE CONTROL & PREVENTION
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] The difficulty in the detection of norovirus in water is that the content is too low and the basic components that interfere with the determination are complex, which has caused great difficulties for accurate determination
Most water bodies need to pass through the concentration of viruses in water samples before they can be detected, but unnecessary pollutants in the water are also concentrated and adsorbed, thereby interfering with the test results
For example, the flocculation and sedimentation method is usually used to concentrate water samples, but the residual organic substances may affect the PCR results
In addition, traditional virus concentration methods, such as cell culture virus identification, are time-consuming in operation and expensive in terms of technical costs, and it is difficult to culture viruses in vitro

Method used

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  • Method for concentrating and detecting norovirus in water based on aminated silica gel
  • Method for concentrating and detecting norovirus in water based on aminated silica gel
  • Method for concentrating and detecting norovirus in water based on aminated silica gel

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0040] Embodiment 1, the efficiency of norovirus in the enrichment water body of polyethylenimine silica gel material

[0041] (1) Preparation of polyethyleneimine silica gel material

[0042] Silica gel activation: move 10g of silica gel to the Erlenmeyer flask, add 50ml, 1mol / L double-distilled aqueous solution of nitric acid, heat to a slight boiling state, and keep stirring for 4 hours. After the reaction, the product is washed with distilled water and methanol to neutral , and then vacuum-dried at 110°C to prepare 10 g of activated silica gel.

[0043] Physical packaging: Weigh 1g of activated silica gel and add it to 10mL of polyethyleneimine methanol solution (1.0mg / mL), mix well, keep shaking at 60°C for 20 hours, centrifuge at 4000×g for 1min, and discard the supernatant , the precipitation was followed by 0.5mol / LH 2 SO 4 Suction filter the aqueous solution, deionized water and soak in 1mol / L ammonia water for 5 minutes, centrifuge to remove the supernatant, and t...

Embodiment 2

[0067] Example 2, 3-aminopropyltriethoxysilane-silica gel material enrichment efficiency of norovirus in water

[0068] (1) Preparation of 3-aminopropyltriethoxysilane-silica gel material

[0069] Put the silica gel in a muffle furnace and dry overnight at 150°C. Add 1 g of dried silica gel to 10 mL of 3-aminopropyltriethoxysilane in toluene mixture (0.03 mg / mL) and mix well, then shake continuously for 20 hours at 56 ° C, centrifuge at 4000 × g for 1 min, The supernatant was discarded, the precipitate was washed successively with toluene, acetone and methanol, and dried at 105° C. for 5 hours to obtain 1 g of 3-aminopropyltriethoxysilane-silica gel.

[0070] (2) The effect of pH value on the enrichment of Norovirus by 3-aminopropyltriethoxysilane-silica gel ( figure 2 )

[0071] Add 1g of 3-aminopropyltriethoxysilane-silica gel prepared in step (1) to 10mL norovirus concentration of 4.39×10 7 (copy / mL) water samples were incubated at room temperature for 2 hours at diffe...

Embodiment 3

[0080] Example 3, the efficiency of N-aminoethyl-γ-aminopropyltrimethoxysilane-silica gel material enrichment of Norovirus in water

[0081] (1) Preparation of N-aminoethyl-γ-aminopropyltrimethoxysilane-silica gel material

[0082] Put the silica gel in a muffle furnace and dry overnight at 150°C. Add 1 g of dried silica gel to 10 mL of N-aminoethyl-γ-aminopropyltrimethoxysilane in toluene mixture (0.04 mg / mL) and mix well, then shake continuously for 12 hours at 26 ° C. Centrifuge at 4000×g for 1 min at 40°C, discard the supernatant, wash the precipitate with toluene, acetone and methanol in turn, dry at 105°C for 5 hours, and then dry at 60°C for 6 hours to obtain N-aminoethyl-γ-ammonia Propyltrimethoxysilane-silica gel 1 g.

[0083] (2) The effect of pH value on the enrichment of Norovirus by N-aminoethyl-γ-aminopropyltrimethoxysilane-silica gel ( figure 2 )

[0084] Add 1 g of the prepared N-aminoethyl-γ-aminopropyltrimethoxysilane-silica gel to 10 mL of norovirus at ...

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Abstract

The invention discloses a method for concentrating and detecting norovirus in water based on aminated silica gel. The method comprises the following steps: mixing a norovirus containing polluted water sample to be detected with amination silica gel, carrying out oscillation incubation for 2 hours at a temperature of 20 to 30 DEG C under a pH value of 5 to 10, after incubation, performing centrifugation, soaking the precipitate in a BGT eluent for 15 to 30 minutes, wherein during the soaking period, an ultrasonic treatment (20-25 KHz) is performed for 5 minutes at a time interval of 45 seconds; carrying out centrifugation, and obtaining the supernate, namely the norovirus concentrate of the polluted water sample. The concentration efficiency is 7 to 8 times higher than that of non-aminated original silica gel and is also prominently higher than that of 3-aminopropyl triethoxysilane amino silica gel (60.65-65.91%) and N-aminoethyl-gamma-trimethoxysilane amino silica gel (72.65-85.76%).

Description

(1) Technical field [0001] The invention relates to an enrichment detection method for norovirus, in particular to a method for concentrated detection of norovirus in water based on aminated silica gel. (2) Background technology [0002] Norovirus (NoV) belongs to calicivirus, which is a non-enveloped single-stranded positive-sense RNA virus with a genome size of 7624 bases, including three open reading frames (ORF1, ORF2, and ORF3). According to the entire capsid gene sequence of NoV, the virus can be divided into 5 genomes (GI-GV); currently, 29 genotypes are known. Among them, the NoVs causing human infection are mainly 8 genotypes in the G I genome and 17 genotypes in the G II genome. Through mutation and recombination, the genome of norovirus undergoes rapid mutation, showing gene diversification, which leads to the generation of new mutant strains and the outbreak of acute gastroenteritis disease. NoV is highly infectious, even less than 10 2 Copies / mL of NoV partic...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N7/00C12Q1/70C12Q1/68C01B33/146
CPCC01B33/146C12N7/00C12N2770/16051C12Q1/686C12Q1/70
Inventor 廖宁波章荣华张严峻陈江
Owner ZHEJIANG CENT FOR DISEASE CONTROL & PREVENTION
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