Establishing The Methods For Detecting Norovirus Of Foods And Water And Applying In Norovirus Gastroenteritis Outbreaks

ObjectiveThis research mainly aimed at establishing and evaluating a set of methods for detecting Norovirus of several foods with high risks and water. The methods were applied in Norovirus gastroenteritis ourbreaks, for tracing the sources of the outbreaks and applying the powerful laboratory evidences to prevent and control the outbreaks.MethodsA set of methods for detecting Norovirus of foods and water inclueded three parts. The reaction conditions and systems were optimized that the assay for detection Norovirus by Realtime PCR was established. The recovery yields and sensitivity of the MCE-PEG methods (The Mixed Cellulose Esters Membrane was used to concentrate norovirus firstly, and PEG was used to concentrate norovirus secondly) for water specimens were evaluated. The processes were optimized to establish the methods for detecting Norovirus in the shellfishes, vegetables and softfruits. When Norovirus gastroenteritis outbreak, collect the suspicious foods and water to detect the virus, also the epidemiological investigations were conducted to confirm the outbreak sources.Results1. The Realtime PCR assay can detect as few as 1 copy/μl virus. It was showed a high linear dynamic range of quantitaion between 108-10'copy/μl. There were no cross reaction with Rotavirus, Adenovirus, Astrovirus, Enterovirus. Also There were no cross reaction between Norovirus G I and GⅡ.The intra-batch and inter-batch reproducibility were quite well.2. The water specimens which contain norovirus were concentrate 100 times by Mixed Cellulose Esters Membrane, and the mean recovery yields was 56.12%. The water specimens were cpncentrate 1000 times by PEG, and the mean recovery yields was 42.47%. The sensitivity of the Mixed Cellulose Esters Membrane and PEG to concentrate the virus were 10copys/ul and lcopys/μl respectively.3. The processes to detect Norovirus of the foods were optimized as follows: elution with Glycin(0.05mol/L)-Nacl(0.15 mol/L,ph9.5),vortex 5 min, to add Chloroform-butanol after PEG, extraction RNA with QIAGEN. As results of these methods, the recovery yields of the vegetable, shellfishe, softfruit were 24.86%,2.08% and 5.42% respectively. The sensitivity of the vegetable,shellfishe, softfruit were 5.93×104 copy/g,6.11×105 copy/g and 4.91×106 copy/g respectively.4. The methods for concentrating virus of the water specimens were applied in 2 outbreaks. First,the rivulet water that the feces of the villagers evacuated directly was positive for Norovirus. The sequences of the virus detected from the anus swabs of the illness were the same as the rivulet water's. This reminding that the rivulet water was contaminated by the feces, giving supports to disinfect the rivulet. Second, a NoV gastroenteritis outbreak occurred in a school, epidemiological investigations indicated the potable water supply by the well was the suspicious source. The potable water specimens were detected the same sequence virus with the patients, confirming that this was a waterborne Norovirus gastroenteritis outbreak.ConclusionA set of operable methods for detecting Norovirus of foods and water were established firstly in our country. With the application in outbreaks, this set of methods show a certain pratical values.