Optimization And Application Of A Novel Method Of Human Noroviruses Based On Receptors Capturing

Human noroviruses(HuNoVs)are one of the major causes worldwide for non-bacterial acute gastroenteritis outbreaks.Reverse transcription polymerase chain reaction(RT-PCR)and real-time quantitative reverse transcription polymerase chain reaction(RT-qPCR)are the most commonly used methods to detect HuNoVs.However,neither method is able to differentiate whether the detected signals are derived from intact virus particles,or residual RNA from degraded virus particles.Moreover,due to low titer virus particles and complex matrix,it is difficult to detect HuNoVs in environmental and food samples.How to solve the above-mentioned technical problems becomes the key of detection,monitoring and prevention of HuNoVs.To discriminate between infectious and non-infectious viruses,in situ capture real-time quantitative reverse transcription polymerase chain reaction(ISC-RT-qPCR)was established based on repceptors capturing and applied to evaluate inactivation efficiencies of Tulane virus(TV)and HuNoVs.However,this method has not been optimized and applied to detect food samples.Based on the perious work,ISC-RT-qPCR was optimized through these conditions including coating concentration of porcine gastric mucin(PGM),incubation time and incubation pH The results indicated that the optimum conditions were: 1.0 mg/mL for type III PGM coating concentration and 56 days for its shelf life in solution,30 min for incubation time(considering time cost),and incubation pH 3.0 for GI and pH 7.0 for GII.ISC-RT-qPCR was compared with RT-qPCR.ISC-RT-qPCR showed a 10-fold and 100-fold increase over that of the RT-qPCR assay for genotype I(GI)and GII,respectively.Ten HuNoVs RT-PCR positive and 5 negative clinical samples from gastroenteritis patients provided by the Beijing center for disease control and prevention(CDC)were used to compare specificity and sensitivity of ISC-RT-qPCR against that of the RT-qPCR assay.The results showed that 10 samples of diarrhea were HuNoVs positive,5 samples of diarrhea were HuNoVs negative.This was 100% consistent with the results of RT-PCR comfirmations.In summary,the ISC-RT-qPCR method is a sensitive and specific method for detection of HuNoVs.The ISC-RT-qPCR was applied to detect HuNoVs in artificially inoculated oysters and retail oysters compared with the conventional RT-qPCR.The results showed that HuNoVs were detected in gills(G),digestive glands(D)and residual tissues(O)of artificially contaminated oyster samples,and the virus titer in gills was the highest.The detection rate of HuNoVs in 36 retail oyster samples(3 samples per month for 12 months)by ISC-RT-qPCR was significantly higher than that by conventional RT-qPCR(p <0.05),and oyster gills could be a better tissue for detecting HuNoVs.ISC-RT-qPCR was also applied to detect HuNoVs in 40 retail shellfish samples(clam,astarte,razor clam,scallop,and abalone).The total and multiple detection rates of HuNoVs were 22.5% and 5.0%,respectively;detection rates of GI and GII HuNoVs were 17.5% and 10.0%,respectively.Overall,ISC-RT-qPCR is a good candidate method on detecting infectious HuNoVs in retail food samples.In addition,ISC-RT-qPCR method is a simple and rapid,low cost,environment-friendly,method without complex procedures such as nucleic acid extraction,thus has a promising prospect.