Development of Detection low level of pthogens in water by genetic biotechnology

水中肠道病毒的管理是饮用水安全的重要问题之一。PCR检测时间不超过6小时，无需培养致病性病毒，灵敏度高，是水环境中病毒监测的可行方法之一。但已建立的水中病毒浓度方法不适合RT-PCR检测。建立了一种带负电荷膜酸冲洗碱性洗脱的病毒浓缩方法，并应用于F特异性RNA噬菌体QB和脊髓灰质炎病毒1作为模型病毒。采用定量TaqMan RT-PCR或空斑法检测QB和脊髓灰质炎病毒的浓度。试验了不同pH条件下病毒及其基因组RNA的抗性，并将其应用于病毒浓缩法。酸冲洗法成功地提高了回收率，这也证实了空斑试验。采用臭氧法对病毒进行消毒，结果为假阳性。从灭活的定量分析出发，用简单的模型解释了臭氧化灭活的机理。

Management of enteric viruses in water is one of the important issues for the safety of drinking water. PCR is one of the feasible monitoring methods of viruses in water environment, because it requires for detection less than 6 hours and no cultivation of pathogenic viruses with high sensitivity. However established virus concentration method from water are not suitable for detection by RT-PCR. A Virus concentration method with negatively-charged membrane using acid rinse and alkaline elution was developed and applied to F specific RNA coliphage QB and poliovirus 1 as model viruses. The concentration of QB and poliovirus were determined by quantitative TaqMan RT-PCR or plaque assay. Various pH condition were tested for resistance of virus and its genomic RNA, and applied to the virus concentration method. Acid rinse procedure was successfully increased the recovery yields, which was also confirmed by plaque assay. Ozonation was applied as disinfection of viruses and false positive result was obtained. From the quantitative analysis of inactivation, mechanism of inactivation by ozonation was explained by a simple model.