發(fā)布時(shí)間：2018-07-16 09:24

【摘要】：背景:當(dāng)前,娛樂休閑水質(zhì)的評(píng)估標(biāo)準(zhǔn)主要基于對(duì)糞便污染指示菌(fecal indicator bacteria,FIB)的檢測(cè),然而,這些標(biāo)準(zhǔn)難以準(zhǔn)確預(yù)測(cè)人類水源相關(guān)致病性病毒的存在。在過去二十年中,因娛樂休閑水污染引起的腸道疾病在逐年增加。美國環(huán)境保護(hù)署(EPA)提出,需尋找新的可替換的指示物作為監(jiān)測(cè)水質(zhì)的標(biāo)準(zhǔn)。研究表明人類腺病毒(HAdV)可作為糞便污染的較好候選指示微生物[l],因?yàn)樵谒�環(huán)境中HAdV相較其他腸道病毒更加穩(wěn)定,此外,HAdV也被指出與許多娛樂休閑水相關(guān)疾病的暴發(fā)有關(guān)。由于環(huán)境水中]HAdV的濃度及回收效率偏低,敏感快捷的PCR檢測(cè)又無法鑒別病毒的感染活性,故檢測(cè)水環(huán)境中具有感染活性的腺病毒是目前亟待解決的難題之一。為了克服這一缺陷并探索用腺病毒作為指示物對(duì)水質(zhì)的監(jiān)測(cè)的可能性,本研究以HAdV作為模型,測(cè)試并建立從環(huán)境水中有效濃縮回收有感染活性腺病毒的新方法,評(píng)估了腺病毒在海洋貝類中的富集情況。此外,本研究還嘗試建立有感染力的病毒顆粒與病毒DNA拷貝數(shù)的關(guān)聯(lián)。 方法與結(jié)果：初步的研究結(jié)果顯示,腺病毒空斑形成試驗(yàn)的優(yōu)化條件為：接種0.8x105/ml的A549細(xì)胞于孔板,在PH為6.4-7.6的情況下,感染：HAdV孵育約1.5小時(shí)-2小時(shí),最后每孔加入1.0毫升1.6%的DMEM-瓊脂糖覆蓋,培養(yǎng)感染HAdV的細(xì)胞約9-10天待空斑形成,以結(jié)晶紫-福爾馬林進(jìn)行固定及染色。該優(yōu)化的空斑技術(shù)條件應(yīng)用于整個(gè)研究中,以獲得穩(wěn)定有具有統(tǒng)計(jì)學(xué)意義的數(shù)據(jù)結(jié)果。 為了有效濃縮環(huán)境水中的病毒,我們比較了氯化鋁預(yù)處理或無處理的負(fù)電荷濾膜、五種洗脫緩沖液和濃度、優(yōu)化了洗脫方式和時(shí)間,回收的病毒通過空斑形成數(shù)確定。結(jié)果表明,氯化鋁預(yù)處理HA負(fù)電荷濾膜后,以1.0mM氫氧化鈉溶液磁力攪拌洗脫30分鐘能回收濃縮約80%的腺病毒。而三種從貝類組織中濃縮HAdV的方法中,3%牛肉提取物洗脫-沉淀法可以濃縮回收約90%的腺病毒。 另外,為了評(píng)估腺病毒在水環(huán)境的穩(wěn)定性,等量的HAdV分別接種于海水、水管水、PBS及廢水中。結(jié)果顯示,HAdV在海水中較不穩(wěn)定,且在純廢水中穩(wěn)定度較高。我們還監(jiān)測(cè)了HAdV在海洋軟體生物中的蓄積情況,通過從環(huán)境水中采集的貝類養(yǎng)殖在實(shí)驗(yàn)室水族缸內(nèi),并接種一定量腺病毒,在不同的養(yǎng)殖時(shí)間,取水和貝類,利用空斑形成試驗(yàn)及實(shí)時(shí)熒光定量PCR數(shù)據(jù)觀察腺病毒在海水及軟體生物內(nèi)的濃度變化。研究結(jié)果表明,HAdV可蓄積在貝類胃上皮,并在第5天達(dá)到最高。這一結(jié)果支持了預(yù)計(jì)的假設(shè)：貝類可作為生物指示物以監(jiān)測(cè)水質(zhì)。 結(jié)論：本研究通過一系列實(shí)驗(yàn)與檢測(cè)從而建立了從不同水體環(huán)境中濃縮腺病毒的優(yōu)化方法,并評(píng)估了腺病毒在不同水體環(huán)境中的穩(wěn)定性及蓄積情況,這對(duì)于使用這一病毒作為指示物是極其重要的意義,同時(shí)我們還證實(shí)了,海洋貝類可作為生物指示物監(jiān)測(cè)水質(zhì)的可能。綜上所述,這一有效敏感的濃縮和檢測(cè)HAdV方法的建立,為今后利用人類腺病毒作為可替代的補(bǔ)充指示物加強(qiáng)對(duì)娛樂休閑水源污染的監(jiān)測(cè)和健康風(fēng)險(xiǎn)評(píng)估的可行性奠定了堅(jiān)實(shí)基礎(chǔ)。
[Abstract]:Background: the current assessment criteria for recreational and recreational water quality are mainly based on the detection of fecal indicator bacteria (FIB). However, these standards are difficult to accurately predict the existence of human source related pathogenic viruses. In the past twenty years, intestinal diseases caused by recreational water pollution have increased year by year. The EPA proposed to find new replaceable indicators as a standard for monitoring water quality. Studies have shown that human adenovirus (HAdV) can serve as a preferable candidate indicator for fecal contamination, [l], because HAdV is more stable than other enterovirus in water environment, and HAdV is also pointed out with many recreational and recreational water related diseases. Due to the low concentration and recovery efficiency of]HAdV in environmental water, sensitive and quick PCR detection can not identify the virus infection activity, so it is one of the difficult problems to be solved to detect the infected adenovirus in water environment. In order to overcome this defect and explore the monitoring of water quality by adenovirus as a indicator In this study, we use HAdV as a model to test and establish a new method for effective concentration and recovery of infected activated adenovirus from environmental water, and to evaluate the enrichment of adenovirus in marine shellfish. In addition, this study also attempts to establish the association of infected virus particles with the number of DNA copies of the virus.
Methods and results: the preliminary results showed that the optimization conditions for the adenovirus plaque formation test were: inoculated 0.8x105/ml A549 cells on the orifice and PH 6.4-7.6, the infection: HAdV was incubated for about 1.5 hours -2 hours, and 1 ml of DMEM- agar was added to each hole, and the cells infected with HAdV were to be empty for about 9-10 days. The plaque is formed and fixed and dyed with crystal violet formalin. The optimized space spot technical conditions are applied to the whole study to obtain stable and statistically significant data.
In order to effectively concentrate the virus in the environmental water, we compared the aluminum chloride pretreated or untreated negative charge filter, five kinds of elution buffer and concentration, optimized the elution mode and time. The recovered virus was determined by the number of plaque formation. The results showed that aluminum chloride was pretreated with HA negative charge filter and 1.0mM sodium hydroxide solution magnetic stirring. 30 minutes of elution can recover about 80% of the adenoviruses concentrated. And three kinds of beef extract elution - precipitation method can concentrate about 90% of the adenoviruses from the three methods of concentrating HAdV from shellfish tissues.
In addition, in order to evaluate the stability of adenovirus in water environment, the equal amount of HAdV was inoculated in seawater, water pipe, PBS and wastewater. The results showed that HAdV was more unstable in the sea water and had higher stability in pure waste water. We also monitored the accumulation of HAdV in marine mollusks, and by the shellfish culture collected from the environmental water. Laboratory aquarium, inoculated with a certain amount of adenovirus, in different breeding time, water and shellfish, using plaque formation test and real-time fluorescence quantitative PCR data to observe the changes in the concentration of adenovirus in seawater and mollusc. The results show that HAdV can accumulate in the shellfish gastric epithelium and reach the highest in fifth days. This result supports the results. The expected hypothesis is that shellfish can be used as biological indicators to monitor water quality.
Conclusion: in this study, a series of experiments and tests have been made to establish the optimization of adenovirus from different water environment and evaluate the stability and accumulation of adenovirus in different water environment. It is very important for the use of the virus as an indicator. The possibility of monitoring water quality as a biological indicator. To sum up, this effective and sensitive enrichment and detection of HAdV method has laid a solid foundation for the future use of human adenovirus as an alternative complementary indicator to strengthen the monitoring of recreational and recreational water pollution and the feasibility of health risk assessment.
【學(xué)位授予單位】：武漢大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2014
【分類號(hào)】：R123.1

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