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

遗传生物技术检测水中低水平病原体的研究进展

• 批准号: 10555189
• 负责人: OHGAKI Shinichiro
• 金额: $ 4.54万
• 依托单位: The University of Tokyo
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (B)
• 财政年份: 1998
• 资助国家: 日本
• 起止时间: 1998 至 1999
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-10555189/
• 关键词: PCR; virus; water microbiology; ozonation; concentration; negatively-charged membrane; disinfection; 検出; ゲノム; 酸洗浄

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.

水体中肠道病毒的管理是保障饮用水安全的重要问题之一。PCR检测时间短于6 h，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。但现有的水体病毒浓缩方法不适用于RT-PCR检测。建立了一种以F特异性RNA大肠杆菌噬菌体QB和脊髓灰质炎病毒1型为模型病毒的酸洗碱洗脱负电荷膜浓缩病毒的方法。采用TaqMan定量RT-PCR或空斑法检测QB和脊髓灰质炎病毒浓度。对不同pH条件下病毒及其基因组RNA的抗性进行了测试，并将其应用于病毒浓缩方法。酸冲洗程序成功地提高了回收率，这也被空斑试验所证实。采用臭氧消毒法对病毒进行消毒，得到假阳性结果。通过对臭氧灭活过程的定量分析，用一个简单的模型解释了臭氧的灭活机理。

项目成果

嶋崎明寛: "水中のポリオウイルス濃縮法の定量的RT-PCR法による回収率の測定"日本水環境学会年会講演集. 34. 321 (2000)

Akihiro Shimazaki：“通过定量 RT-PCR 测量水中脊髓灰质炎病毒浓度的回收率”日本水环境学会年会记录 34. 321 (2000)。

嶋崎明寛: "水中RNAウイルスの陰電荷膜を用いた濃縮法への酸洗浄の導入"環境工学研究フォーラム講演集. 36. 64-66 (1999)

Akihiro Shimazaki：“使用带负电荷的膜对水中 RNA 病毒进行酸清洗的浓缩方法简介”环境工程研究论坛论文集。 36. 64-66 (1999)。

嶋崎 明寛: "水中RNAウイルスの陰電荷膜を用いた濃縮法への酸洗浄工程の導入"第36回環境工学フォーラム講演集. 64-66 (1999)

Akihiro Shimazaki：“使用带负电荷的膜浓缩水中 RNA 病毒的酸洗过程简介”第 36 届环境工程论坛论文集 64-66 (1999)。

Shinichiro Ohggaki: "Current Status of Disinfection"Jour. of Japan Society of Water Environment. 21. 560-565 (1998)

Shinichiro Ohggaki：《消毒现状》杂志。

Akihiro SHIMASAKI: "A Virus Concentration Method of RNA Viruses in Water Using Negatively-Charged Membrane with Acid Rinse Procedure"Proc. of Environmental Research Forum. 36. 64-66 (1999)

Akihiro SHIMASAKI：“使用带负电荷的膜和酸冲洗程序的水中 RNA 病毒的病毒浓缩方法”Proc。