Establishment And Application Of Pretreatment Methods For Efficient Detection Of Norovirus In Oysters

Norovirus(No V)has become the leading cause of non-bacterial gastroenteritis in the world,and the intake of contaminated food or water is a common way to cause viral infections.Shellfish especially oysters have been reported as the important No V transmission vector.In recent years,large-scale gastroenteritis outbreaks caused by consuming raw No V contaminated oysters have been reported,which not only seriously threatened the health of consumers,but also caused huge losses to the aquaculture industry.However,since low the contaminated No V concentration in oysters and complex detection interference substances in oysters.The establishment of a sensitive and efficient No V detection technology system in oysters,especially the pre-processing technology is of great significance for the prevention of shellfish-sourced No V gastroenteritis outbreaks.The purpose of this paper is to establish an efficient pretreatment method for No V detection in oysters.On the basis of grasping the distribution of No V in oysters,a small system proteinase K treatment method and a biotin and tree branch-based method were established.The second-stage amplified immunomagnetic enrichment technology of the molecular molecules,and finally conducted a one-year monitoring of the status of No V pollution in oysters sold in Guangzhou,the main results of this paper are as follows:1.In order to understand the accumulation law and tissue distribution of No V in oysters,laboratory-cultured oysters were artificially contaminated with human No V and Murine norovirus(MNV),and real-time fluorescence quantitative RT-PCR was used to detect the content and distribution of viruses in different oyster tissues.The results showed that the two viruses could both be detected in the gill,intestine and digestive gland tissues of oysters,among which four were detected in the digestive glandtissues.This provide a reference for the study of No V distribution and specific binding mechanism in oysters.2.Improved and established a pre-processing method according to the existing(International Organization for Standardization/Technical Specifications,ISO/TS)15216-2:2013 standard method.Compared with ISO/TS 15216-2: 2013 standard method,the recovery rate was increased from 1.66±0.27% to 11.07±0.09%,and the PCR amplification efficiency was increased from 92.31±9.01% to 124.12±5.99%.A new pretreatment method was used to detect oysters on the market in Guangzhou,and the detection rate was as high as 34.21%.The method has the advantages of short operation time,low cost and high recovery rate.3.In this chapter,three virus enrichment methods were established,including conventional immunomagnetic beads,biotin-amplified immunomagnetic beads,and biotin-coupled dendrimer immunomagnetic beads.In the conventional immunomagnetic bead method,the results showed that when the coupling time was 1.5 h,the amount of magnetic beads was 0.1 mg,the enrichment time was 60 min,and the separation time was 7 min,the virus recovery rate was 15.63±0.43%.Based on the biotin-amplified immunomagnetic bead method,the results showed that when the biotinylated antibody amount was 40 ?g,the immunization time was 25 min,the immunomagnetic beads amount was 80 ?g,and the incubation time was 30 min,the virus recovery rate was37.48±0.20%.In the magnetic enrichment separation method based on polyamide-amine(PAMAM)dendrimer-mediated biotin amplification,when the amount of biotinylated antibody was 40 ?g,the immunization time was 15 min,the amount of immunomagnetic beads were 60 ?g,and the incubation time was 20 min,the virus recovery rate was44.26±1.45%.In the evaluation of oyster samples,the two immunomagnetic bead methods of biotin amplification achieved the purpose of effective virus enrichment,and the detection rates was 18.80%(8/44)and 25%(11/44),respectively.However,thepolyamide-amine(PAMAM)dendrimer magnetic bead method had a higher recovery rate,with the recovery rate is increased by 7% compared with the biotin-amplified immunomagnetic bead method,the use of magnetic beads had reduced by 20 ?g.4.Investigation of virus pollution in oysters sold in Guangzhou area.From December 2018 to December 2019,a total of 52 oyster sold samples in the Guangzhou area were collected,and a total of 19 viruses were detected with a detection rate of36.53%.There was no significant difference in the virus detection rate in oyster samples in different seasons,indicating that oysters could be contaminated by viruses in any season.In summary,the two efficient pretreatment methods were established for No V detection in oysters and were successfully applied them to No V monitoring in oysters.The results will help researchers overcome the bottleneck problem of sample preparation in virus detection.Also,this paper work as an important technical support to prevent and control of No V pollution in oysters in China.