OBJECTIVETo explore a convenient and effective method for norovirus nucleic acid extraction from oysters suitable for long-term viral surveillance.

METHODSTwo methods, namely method A (glycine washing and polyethylene glycol precipitation of the virus followed by silica gel centrifugal column) and method B (protease K digestion followed by application of paramagnetic silicon) were compared for their performance in norovirus nucleic acid extraction from oysters. Real-time RT-PCR was used to detect norovirus in naturally infected oysters and in oysters with induced infection.

RESULTSThe two methods yielded comparable positive detection rates for the samples, but the recovery rate of the virus was higher with method B than with method A.

CONCLUSIONMethod B is a more convenient and rapid method for norovirus nucleic acid extraction from oysters and suitable for long-term surveillance of norovirus.

Animals ; Centrifugation ; methods ; Norovirus ; genetics ; isolation & purification ; Ostreidae ; virology ; RNA, Viral ; isolation & purification

A simple, rapid and sensitive colorimetric reverse transcription loop-mediated isothermal amplification (RT-LAMP) method was established to detect norovirus genotype GII. The method employed a set of six specially designed primers that recognized eight distinct sequences of RNA-dependant RNA polymerase and capsid protein gene for amplification of nucleic acid under isothermal conditions at 65 degrees C for 60 minutes. The amplification process of RT-LAMP was monitored by the addition of HNB (Hydroxy naphthol blue) dye prior to amplification. A positive reaction was indicated by a color change from violet to sky blue and confirmed by agarose electrophoresis. The specificity of the RT-LAMP was validated by detecting several different diarrhea viruses including norovirus genotype GII. The sensitivity was determined by serial dilutions of RNA molecules from in vitro transcription of norovirus genotype GII in parallel with conventional RT-PCR detection. The assay was further evaluated with 93 clinical specimens of diarrhea patients. The results showed that the sensitivity of RT-LAMP was 1 000 copies/microL with a high specificity and the relative sensitivity was at the same level as that of conventional RT-PCR. Positive rate of RT-LAMP in analysis of clinical specimens was approximately the same as that of conventional RT-PCR as well. This colorimetric RT-LAMP assay was potential for rapid detection of norovirus genotype GII on spot due to the observation of visual result with high specificity and sensitivity, time-saving and cost benefit.
Caliciviridae Infections ; diagnosis ; virology ; Colorimetry ; methods ; Feces ; virology ; Genotype ; Humans ; Norovirus ; genetics ; isolation & purification ; Nucleic Acid Amplification Techniques ; methods

Caliciviridae Infections ; epidemiology ; genetics ; virology ; China ; epidemiology ; Gastroenteritis ; virology ; Humans ; Molecular Epidemiology ; Norovirus ; genetics ; RNA, Viral ; isolation & purification

A new viral sampling concentration device was designed which was equipped with a new cationic filter membrane-Nanoceram suitable for field sampling. Norovirus Genegroup II was detected from environmental water with the aid of this device. The effects on virus recovery of prefiltration, various second-concentration methods, and different eluants were investigated through pre-experiment. The concentration optimized process, and the optimal concentration process were then determined. The results showed that the prefiltration had a profound effect on virus recovery, and two second-concentration method: PEG-NaC1 precipitation and celite adsorption, had almost the same concentration effects. The Na2 HPO4 solution of 0.15 mol/L was selected as the final eluant to elute the adsorbed Nuorovirus from the celite. The virus recovery of Nanoceram was determined to be 3.02%. Finally, successful detection of Norovirus GII in sewage from Yangqiao River, Fengtai District, Beijing was acheived. All these data had shown that the Naneceram filter concentration method could concentrate Norovirus from environmental water with a steady effects.
Filtration ; instrumentation ; methods ; Fractional Precipitation ; instrumentation ; methods ; Genotype ; Norovirus ; classification ; genetics ; isolation & purification ; Rivers ; virology ; Water Microbiology

Noroviruses (NoVs) are one of the most important foodborne viral pathogens worldwide. Shellfish are the most common carriers of NoVs as they can concentrate and accumulate large amounts of the virus through filter feeding from seawater. Shellfish may selectively accumulate NoVs with different genotypes, and this bioaccumulation may depend on the season and location. Our previous studies found various histo-blood group antigens (HBGAs) in shellfish tissues. While HBGAs might be the main reason that NoVs are accumulated in shellfish, the detailed mechanism behind NoV concentration and bioaccumulation in shellfish is not clear. Here we review current research into NoV bioaccumulation, tissue distribution, seasonal variation, and binding mechanism in shellfish. This paper may provide insight into controlling NoV transmission and decreasing the risks associated with shellfish consumption.
Animals ; Caliciviridae Infections ; transmission ; virology ; Food Contamination ; analysis ; Foodborne Diseases ; virology ; Humans ; Norovirus ; classification ; genetics ; isolation & purification ; physiology ; Shellfish ; virology

We set up an SYBR Green I real-time RT-PCR method for the detection of genogroup II Norovirus, and this method's primers were encompassed the conservative region of Norovirus II. The limit of the detection was 10(2) copies. The standard curve's linear range was 10(2)-10(6) copies, correlation coefficient was 0.9952, the slope was -2.982, and the intercept was 35.84. This method possessed specificity for genogroup II Norovirus, without any cross-reaction with rotavirus, adenovirus, hepatitis A virus or astrovirus. The coefficients of variation (CV) of the C(t) values of the standard plasmid were 0.95%-1.69% (n = 5) in intra-assay and 0.87%-1.24% (n = 3) in inter-assay. We used this method to detect 30 shellfish samples, and found 3 samples were positive. This method is sensitive, specific and reliable for Norovirus II. It can be used to detect the Norovirus II in the shellfish rapidly.
Animals ; Bivalvia ; virology ; Norovirus ; isolation & purification ; Organic Chemicals ; RNA, Viral ; analysis ; isolation & purification ; Reverse Transcriptase Polymerase Chain Reaction ; methods ; Sensitivity and Specificity ; Shellfish ; virology ; Species Specificity

OBJECTIVETo establish a one-step four multiplex reverse transcription polymerase chain reaction (RT-PCR) method based on Homo-Tag Assisted Non-Dimer System (HAND) system for simultaneous detection of 4 diarrhea viruses of rotavirus, astrovirus, norovirus and sapovirus.

METHODSPrimers were designed according to the conserved genome sequence of the 4 viruses and the homologous tail sequences were added to the 5' end. The multiplex RT-PCR system was constructed by optimizing the PCR parameters such as the concentration of universal tag primer and genome-specific Homo-Tailed primers. The specificity, stability and sensitivity of the method were evaluated systematically.

RESULTSThe 4 multiplex RT-PCR methods based on HAND system was established successfully. Specificity analysis showed no cross reaction between the 4 diarrhea viruses. The sensitivity analysis showed detection limits for rotavirus, astrovirus, norovirus and sapovirus of 48, 1.92, 9.6 and 48 pg per reaction, respectively.

CONCLUSIONThe established HAND system-based multiplex RT-PCR assay allows simple, rapid, specific, sensitive, and stable for detection of the 4 common diarrhea viruses at low costs and is suitable for application in general medical laboratories.

Astroviridae ; genetics ; isolation & purification ; Diarrhea ; virology ; Feces ; virology ; Humans ; Multiplex Polymerase Chain Reaction ; methods ; Norovirus ; genetics ; isolation & purification ; RNA, Viral ; isolation & purification ; Reverse Transcriptase Polymerase Chain Reaction ; methods ; Rotavirus ; genetics ; isolation & purification ; Sapovirus ; genetics ; isolation & purification

Noroviruses are the leading cause of epidemic gastroenteritis, including foodborne outbreak, in Korea. The prevalence of human noroviruses was studied in diarrheal stool samples of patients with acute gastroenteritis by conventional duplex reverse transcription (RT)-PCR. Diarrheal stool samples were collected from 1,685 patients from the local hospitals in Seoul. The prevalence of the noroviruses was 22.8% (222/972 patients) in 2012 and 11.2% (80/713 patients) in 2013, with a total of 17.9% (302/1,685 patients). Genotyping was performed on 302 norovirus-positive stool samples to reveal 5.6% prevalence of genogroup I (GI) (17/302) and 94.4% prevalence of genogroup II (GII) (285/302). The patients with norovirus-associated acute gastroenteritis mostly showed prevalence of GII norovirus, especially GII.4 (64.6%; 195/302).
Acute Disease ; Feces/virology ; Gastroenteritis/*diagnosis/epidemiology/virology ; Genotype ; Humans ; Norovirus/*genetics/isolation & purification ; Prevalence ; RNA, Viral/genetics/metabolism ; Real-Time Polymerase Chain Reaction

To describe epidemiologic characteristics of norovirus infection and its genotype in Shenzhen area of 2010. Stool specimens were collected from four monitoring point and detected by reverse transcription polymerase chain reaction (PT-PCR). Positive PCR products were purified and sequenced, and the sequences were performed by Clustal W and MEGA 4.0 programs, then genotype was identified and phylogenetic tree was constructed. Nucleotide sequence analysis revealed that 79 strains of NV belonged to norovirus genogroup II and 6 belonged to genogroup 1 of all 85 positive products. While 55 strains belonged to G II/4(2006b), 16 strains belonged to G II/4(2008 variant), 2 strains belonged to G II /1, 4 strains belonged to G II/5, 2 strains belonged to G II/11, 1 strain belonged to GII/4, 2 strains belonged to GII/5, 3 strains belonged to GI/6. The main genogroups of norovirus in Shenzhen ware GI and G II. G II /4 was one of the most major genotype of norovirus , while G II /4(2006b) variant was identified as the predominant strain in Shenzhen area.
Adolescent ; Adult ; Caliciviridae Infections ; virology ; Child ; Child, Preschool ; China ; Female ; Genotype ; Humans ; Infant ; Male ; Molecular Sequence Data ; Norovirus ; classification ; genetics ; isolation & purification ; Phylogeny ; Young Adult

OBJECTIVETo investigate the molecular epidemiological characteristics of norovirus in Guangzhou from 2009 to 2011.

METHODSA total of 183 water samples, 1162 seafood samples and 1066 diarrhea stool specimens were collected from January 2010 to May 2011, June 2009 to June 2011 and July 2009 to December 2010 respectively in Guangzhou. Norovirus was detected by real time reverse transcript-PCR (qRT-PCR). The partial polymerase gene was amplified from norovirus positive samples, then sequenced and compared with the sequences of norovirus in GenBank. The phylogenetic tree was created.

RESULTSThe positive rate was 19.67% (36/183), 8.26% (96/1162) and 37.05% (395/1066) in water samples, seafood and diarrhea patients respectively. Noroviruses from positive samples could be divided into 10 representative strains, in which 7 representative strains of genotype of 208 samples was type G2-4. The sequences from water, seafood and stool specimens were highly homologous with the similarity of 94% - 100%.

CONCLUSIONIn Guangzhou, the predominant Norovirus genotype was G2-4 and the positive rate of samples was high.

Base Sequence ; Caliciviridae Infections ; epidemiology ; virology ; China ; epidemiology ; Diarrhea ; virology ; Genotype ; Humans ; Molecular Epidemiology ; Norovirus ; classification ; genetics ; isolation & purification ; Phylogeny ; RNA, Viral ; genetics ; Seafood ; virology ; Water Microbiology