Study On Norovirus Nucleic Acid Detection Methods In Different Samples

Noroviruses are an important cause of acute gastroenteritis(AGE)worldwide.Due to its low infectious dose,high infectivity,and efficient transmission,norovirus-associated acute gastroenteritis is the infectious disease with the second greatest burden worldwide,affecting humans in all age groups,especially children,the old,and the immunosuppressed.Foodborne virus and waterborne virus are transmitted by food and water to infect human,they cause a major public health concern worldwide,among which norovirus(NoV)account for the first place.Thus,it is important to study the prevention and control of foodborne and waterborne disease through the long monitoring of NoV and HAV in food and water.Because of the variety of food,the diversity of the water source,the complex component of the food and water,the low and uneven distribution of the contaminated virus,and the food and water contain various factors that inhibit virus detection,making the detection of foodborne and waterborne viruses become the difficulties of the global public health.In this study,the NoV detection methods from water and foods were developed and evaluated,which provides a reference standard laboratory testing technique for the detection and monitoring of food-and water-borne viral diseases in China.1.Laboritory evaluation and application of the detection method of norovirus in stool samplesLaboritory evaluation of published the multiple Realtime RT-PCR detection method of Norovirus.Between January 2012 and December 2017,1,863 stool samples from Children Hospitalized for Acute Gastroenteritis were collected at Maternal and Child Health Hospital in Hohhot.All samples were screened for noroviruses by the method.The results showed that:Norovirus was detected in 24.15%of these inpatient cases,ranging from 12.78%to 32.92%in different years.Norovirus was detected throughout the year,with a peak in winter.Based on sequence analysis of the partial VP1 gene,the 306 identified norovirus strains were divided into six genotypes:GII.3(71.24%),GII.4(23.53%),and GII.2,GII.5,GII.6,and GII.13(total 5.23%).Based on further sequence analysis of the RNA-dependent RNA polymerase(RdRp),GII.P12/GII.3,GII.Pe/GII.4,and GII.P4/GII.4 were identified as predominant genotypes,accounting for 92.6%of genotyped strains.The median age of the children with norovirus infection was 8.0(range 0-59)months.However,children infected with GII.3 were younger(median 7.0 months)than GII.4-positive patients(median 10.0 months).2.The development of detection method of norovirus in waterUsing Murine norovirus-1(MNV-1)as a model virus and process control virus,applying viral adsoption-elution(VIRADEL)and realtime RT-PCR method,two norovirus detection methods from water were developed and evaluated based on the sampling ways and volumes.The results showed that:(1)a modified VIRADEL method combined with ultrafiltration was evaluated and applied for recovering viral pathogens from drinking water.The recovery efficiencies of MNV-1 and NoV were compared by various elution buffers with different charged filters in VIRADEL.The virus recovery was best optimal by using 1%Tralk buffer in negatively charged filter HA or positively charged filter BA.The recovery efficiencies of NoV and HAV were 15.25%and 6.76%in artificially contaminated water using the modified VIRADEL.A centrifugal ultrafiltration,as the second concentration,reached a final volume of 0.2mL in 20 min.This method had been confirmed by three different laboratories,and submitted to Standardization Administration of China.(2)Biotroy semi-automatic microbiological concentration system,a virus concentration system suitable for field sampling,was developed independently in this study.In the field,this system can complete to collect 1000L water sample in 2 hours without external power(10L/min of water flowrate).After sampling,the Nanoceram membrane device of this system was transport to laboratory with low temperature,then was eluted by 1%tralk buffer and second concentrated by PEG6000 and ultrafiltration.Finally,the MNV-I elution efficiency was 70.45%,and 1mL final volume of water sample was obtained.(3)The modified VIRADEL were successfully applied to recover the contaminated viral pathogens from drinking water of Jingmi diversion canal in Beijing,China.The viral metagenomics of the water were characterized by high through-put sequencing.The mammals animal viruses in the water can be divided into 5 families,among which NoV GII.17,Enterovirus 71(EV71),HAV and Aichivirus(Aichi 1)were discovered.3.The optimiztion and evaluation of the detection method of norovirus in foodUsing MNV-1 as the model virus and realtime RT-PCR,the detection methods of NoV in various foods(red soft berries,carbohydrate foods,protein and fat foods)were developed.(1)Tralk,TGBE,TPB and NaPP elution buffer were used to assess the elution effect of MNV-1,and two second concentration methods of PEG6000 precipitation and ultrafiltration were used to assess the recovery efficiency of MNV-1.The results showed that using TGBE buffer-PEG6000 precipitation,the NoV detection limit is 103 copies/10g in the strawberry;In the grapes,the NoV detection limit is 104 copies/10g by the TGBE buffer-Ultrafiltration;In lettuces the NoV detection limit is 103 genocopy/10g by the Tralk buffer-PEG6000 precipitation;while in onions,the NoV detection limit is 104 copies/10g by the Tralk buffer-Ultrafiltration.(2)Comparison of elution-concentration method and direct RNA extraction method of MNV-1 detection in cake and luncheon meat,no MNV-1 was detected by elution-concentration method,while the latter method was able to detect MNV-1,with 106 and 104 copies/10g of NoV detection limit,respectively.4.Detection of virus in shellfishIn this study,330 shellfish samples were collected in Beijing,and the virus spectrum in these shellfish samples was developed through high through-put sequencing.A total of 4594270 viral reads were obtained by Hiseq sequencing,among which 21%of the reads were matched to the viruses that infect animals(965185/4594270),and 1.26%(12205/965185)reads were further matched to the viruses that infect mammals,these viruses can be divided into 20 families,including 12 genera of Picomaviridae and the common human enteric viral pathogens,including Enterovirus,HAV,Aichivirus and NoV were detected.GII.1,GII.2,GII.13,GII.14 and GII.17 of NoVs were found,and the NoV GII.2 and GII.17 of this study were similar to current epidemic strains,the amino acid homology of NoV GII.2 and GII.17 were 100%and 98%.In conclusion,laboritory evaluation of published the multiple Realtime RT-PCR detection method of Norovirus,and this method was used to obtain prevalence of noroviruses in children hospitalized for acute gastroenteritis in Hohhot,China,2012-2017.A Biotroy semi-automatic microbiological concentration system suitable for field water sampling was developed.Evaluation of the detection of NoV in stool samples,and promising methods for the detection of NoV from water and food were established.Moreover,the modified methods were successfully applied to recover the contaminated viral pathogens from stool sample,drinking water and shellfish,and the viral metagenomics of the drinking water and shellfish was evaluated.