| Norovirus (NV) is a major pathogen which causing acute non-bacterial gastroenteritis, and it is becoming a significant public health issue since the increased gradual outbreaks of non-bacterial gastroenteritis in recent years. Because NV can not be cultured in vitro and prone to be mutated, which limit the application of traditional serotyping assays and immunology. However, the molecular biology methods based on PCR is becoming the main researched directions to detect NV at present. This study established the methods of conventional RT-PCR and real-time RT-PCR to detect NV, and studied the pre-treatment of water samples. Using and evaluating the established methods to detect clinical, oysters and water samples collected from some areas of Jiangsu. Investigating the polluted situation of NV, and analyzing the molecular epidemiology characteristics of NV were conducted in some areas of Jiangsu. It provided some data for the risk assessment and molecular epidemiology of NV to our country.1. The establishment and comparison of conventional RT-PCR and real-time RT-PCR to detect NVAccording to the genetic sequence of capsid protein region and ORF1-ORF2 junction in NV, the primers or probes were designed and the reaction system was optimized to establish the conventional RT-PCR and real-time RT-PCR. The two methods can detected NV while with non-crossreactivity to other enteroviruses, which indicated these two methods had a good sepecificity. Detecting different copies of NV, the detection limit of conventional RT-PCR was 103copies/μL, while that of real-time RT-PCR was 102copies/μL. Using real-time RT-PCR to detect different dilution of the standard plasmid of NV GI and GⅡ, the cycle threshold had a good linear relation between102～108 copies/μl of the GI standard plasmid, and the standard curve was Y=40.585-3.301×1gX, which R2 reached 0.997 and Eff was 100.889%; The standard curve of the GⅡ standard plasmid was Y=40.666-3.229×1gX, which R2 reached 0.996 and Eff was 104.039%, and the cycle threshold had a preferable linear relation between 101～108 copies/μL of the GII standard plasmid. The coefficient of variation was 0.18%-0.57% in batches, and the coefficient of variation was 0.71%-2.62% between batches, which indicated real-time RT-PCR had a good reproducibility.Using conventional RT-PCR and real-time RT-PCR to detect 22 clinical samples, comparing with the results of Nantong CDC, the sensitivity and specificity of conventional RT-PCR were 71.43% and 100%, the compliance rate was 90.91%. The results of real-time RT-PCR were fully consistent with Nantong CDC. Then using the two methods to detect 180 clinical samples, the positive rate of conventional RT-PCR was 5.56%, and the compliance rate was 97.22%; while the positive rate of real-time RT-PCR was 8.33%, and the compliance rate was 100%. From the statistical analysis, the detection rate of real-time RT-PCR was significantly higher than conventional RT-PCR(P<0.05), and the positive samples were further conducted the sequencing analysis and it was confirmed that all of them were NV.2. The establishment and optimization of enrich methods for NV from water samplesThis study chose the domestic common MCE membrane, enriching virus from water samples which were polluted by NV. and optimizing the process of adsorption and elution to establish an enrich method for NV. In the process of adsorption, the pH of water samples was adjusted, and the effects of diferent kinds of polyvalent cations to water samples were evaluated, then the concentration of cations was optimized; In the process of elution, the different kinds of eluent to wash the filter membrane were screened, and optimizing the pH and concentration of eluent were also optimized. The results showed that the pH of water samples was 3.5, the average recovery was 28.96%, which indicated the effect of adsorption was better than other pH. Adding Na+, Mg2+or Al3+ to water samples, the average recovery was 48.42%,28.96% and 18.28% respectively. When the concentration of Na+was 0.2mol/L, the membrane had a good effect of absorption, and the average recovery was up to 72.15%. In the process of elution, the effect of eluent NaPP-PB-glycine-0.5%Tween80 was better than other eluents, and the average recovery was up to 107.85%. When the concentration of NaPP was 0.5%, which had a good effect of elution, and the average recovery was 119.94%. The pH of above-mentioned eluent was10.5, the average recovery was 134.90%, which indicated the effect of elution was better than others.Using the MCE adsorption-elution method, it could enrich NV from water samples, which was experimentally polluted 1～103copies/μL NV. All water samples were detected by real-time RT-PCR, it was found that the water samples with 10～103copies/μL NV can be detected after enrich treatment, and without enrich treatment could not be detected NV in these water samples. It was shown that the sensitivity of MCE adsorption-elution method was 1 Ocopies/μL virus in water samples.3. Molecular epidemiology of NV in some areas of JiangsuIn this study we collected 648 clinical samples from diarrhea patients in Yangzhou and Nantong during January to December in 2014,136 oyster samples from Cuiyuanqiao seafood market and Oushang supermarket and 60 water samples from three rivers in Yangzhou, which were detected by real-time RT-PCR. The results showed that 85 positive samples from 648 clinical samples, the positive rate was 13.12%. The positive rate of clinical samples was 19.13%(57/298) in Yangzhou, the positive rate was 8%(28/350) in Nantong, which showed the detective rate of Yangzhou was significantly higher than that of Nantong (P<0.05). The infectious rate of children under 14 years old was 18.04%(57/316), and the infectious rate of children over 14 years old was 8.06%(22/273), which showed the infectious rate of children under 14 years old was significantly higher than the other age groups (P<0.05). The infectious rate of male was 13.01(38/292), while the infectious rate of female was 12.30%(38/309), which showed the infectious rate of male and female had no significant difference (P>0.05).9 positive samples were detected from 136 oyster samples, the positive rate was 6.62%. The positive rate of Cuiyuanqiao seafood market was 7.64%(5/67), and the positive rate of Oushang supermarket was 5.80%(4/69), there was no significant diffrernt between the positive rate of two markets in Yangzhou (P>0.05).13 positive samples were detected from 60 water samples, the positive rate was 21.67%. The positive rate of Erdao river water samples was 10%(2/20), and 20%(4/20) for Andun river,35%(7/20) for Biaodai river. Statistical analysis showed that the pollution rate of three rivers in Yangzhou had no significant difference (P>0.05).According to the genetic sequences of NV capsid protein region,43 positive clinical samples and 1 positive water samples were genotyped by MEGA5.0 software, the results showed that 2 samples belonged to GI NV, including two genotypes GI.2, GI.6; 42 samples belonged to GII NV, including four genotypes GII.4, GII.6, GII.8, GII.17, among them 32 samples were GII.4 subtype, accounting for 72.73% in all the genotyped samples, and belong to the same brach of the world epidemic NV GII.4/2006 variant and GII.4/2010 on the same branch, with 97% of homology. The homology of an NV in 1 water sample and GII.4 subtype NV in the clinical samples were up to 100%, which indicated the human infected by NV may have a close relationship with water. In addition, it was revealed that 1 clinical sample contained GI.2 NV and GII.8, which was the first discovery of mixed infection of GI.2 and GII.8 subtype NV in China. |
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