Establishment And Optimization Of Noroviruses Detection Methods In Shellfish

Noroviruses (NVs) are now the most widely recognized viral agents associated with food and waterborne outbreaks of non-bacterial gastroenteritis world-wide. Epidemiological investigation indicated that mollusk was the most important vehicle of NVs. Outbreaks of the viral diseases have been occasionally related to the consumption of bivalve shellfish harvested in faecal-polluted areas in recent years, which caused health risk and huge losses to consumers .The amount of shellfish production in China was the biggest all over the world, So accurate detection of NVs in fishery products and investigation of the NVs pollution of shellfish were the effective ways of preventing outbreaks of endemic situation and reducing economic loss of fishery.The identification and detection of the NVs have been difficult prior to development of molecular methods because they have not yet been cultured in vitro, and they show great genetic diversity. These factors limit the use of traditional immunology and serotyping assays. Reverse transcription-polymerase chain reaction (RT-PCR) assay was the principal diagnostic tool for NVs. The applications of Real-time RT-PCR method and real-time Nucleic acid sequence-based amplification (real-time NASBA) to the detection of viruses and pathogens have become more and more popular.The Reaction Systems with four primers of NVs were optimized according to conserved sequence in RNA polymerase region of NVs genomes. JV13I/JV12Y was the best primer set to detect NVs in shellfish. Nine methods were designed by orthogonal experiment, the main factors that affecting the enrichment of NVs and these interaction factors were considered adequately. The optimum of virus enrichment were selected by artificial contamination and RT-PCR detection .The optimum of virus enrichment was firstly absorbed with threonine buffer (PH7.5) twice, then precipitated twice with mixture of 9% PEG6 000 and 7% PEG8 000. The recovery rate of NVs was as high as 23%.A real-time RT-PCR method was developed for detection of NVs in shellfish using the SYBR Green I and the standard high pure plasmid DNA which contain the target gene fragment. The methods release most Interference of non-specific amplification by the way of changing the temperature of signals collection. There were good linear relationships over a range of 108-103copies per reaction, the sensitivity was as high as 10-5 dilutions RNA of NVs, which was 100 times high of routine RT-PCR. The methods were convenient, rapid and low cost.One step real-time RT-PCR assay with TaqMan probe was developed based on the copy RNA which was transcribed using SP6 RNA polymerase in vitro. The method was simple, accurate and eliminate the results difference of RT efficiency and processing method. There were good linear relationship over a range of 108-102 copies per reaction, the sensitivity can reach 10-6 dilutions RNA of NVs.The primers and molecule beacon of real-time NASBA were designed, and a real-time NASBA method was developed based on LightCycler2.0 system. The sensitivity of this method was higher than real-time RT-PCR methods. There was good linear relationship over a range of 108-100 copies per reaction. To our knowledge, however, no study has described that NVs RNA with the application of the real-time NASBA assay using the LightCycler as the real-time instrument. Overall, this study demonstrated that the LightCycler can be used successfully as the real-time platform to perform NASBA, thus avoiding the requirement for additional specialized laboratory equipment.The 186 shellfish samples were collected in eight different areas between September 2007 and August 2008, and these samples were detected by routine RT-PCR and real-time RT-PCR. The results preliminarily reveal that the Noroviruses pollution status of shellfish in the Area of Shandong Peninsula area at molecular level. The results showed that the NVs in shellfish over the Area of Shandong Peninsula is mainly GGII-4 type, 5 positive NVs were detected in shellfish which collected in winter. To some extent, this reflects that the NVs Pollution in Shellfish in Shandong coastal area,and supplies the data of NVs pollution in shellfish. This study provides good technical support for further risk assessment and molecular epidemiological survey.