| 徐焰,彭道荣,熊鸿燕,张小宁,苏明权,孙怡群,郝晓柯.一株宽宿主谱大肠埃希菌噬菌体的RNA比较分析及其环境微生物杀灭效应观察[J].Chinese journal of Epidemiology,2005,26(5):356-360 |
| 一株宽宿主谱大肠埃希菌噬菌体的RNA比较分析及其环境微生物杀灭效应观察 |
| Study on the nucleic acid of E.coli bacteriophage with broad host range and its sterilization effect to sewage samples from the environment |
| Received:June 24, 2004 |
| DOI： |
| KeyWord: 宿主谱 大肠埃希菌噬菌体 微生物杀灭试验 |
| English Key Word: Host range Escherichia coli bacteriophages Germicidal test |
| FundProject:教育部高等学校骨干教师资助计划基金资助项目(2000265266) |
| Author Name | Affiliation | E-mail | | XU Yan | Department of Clinical Laboratories, Xijing Hospital, Fourth Military Medical University, Xi’an 710032, China | | | PENG Dao-rong | Department of Clinical Laboratories, Xijing Hospital, Fourth Military Medical University, Xi’an 710032, China | | | XIONG Hong-yan | 第三军医大学预防医学院军队流行病学教研室 | | | ZHANG Xiao-ning | Department of Clinical Laboratories, Xijing Hospital, Fourth Military Medical University, Xi’an 710032, China | | | SU Ming-quan | Department of Clinical Laboratories, Xijing Hospital, Fourth Military Medical University, Xi’an 710032, China | | | SUN Yi-qun | Department of Clinical Laboratories, Xijing Hospital, Fourth Military Medical University, Xi’an 710032, China | | | HAO Xiao-ke | Department of Clinical Laboratories, Xijing Hospital, Fourth Military Medical University, Xi’an 710032, China | haoxkg@163.com |
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| Abstract: |
| 目的 通过比较研究一株分离自医院污水的宽宿主范围大肠埃希菌(E. coli)噬菌体与野生型单一宿主谱E. coli噬菌体的核酸序列和微生物杀灭效应等生物学特性的改变探讨噬菌体识别特异性机制和将噬菌体作为生物消毒剂应用于环境污水净化的可行性. 方法 采用抗体沉淀盐酸胍一步法分别提取单一宿主谱E. coli噬菌体f2株及宽宿主谱E. coli噬菌体株的RNA, 通过琼脂糖凝胶电泳鉴定其纯度;采用简并引物逆转录聚合酶链反应(RTPCR)及随机引物随机扩增多态性DNA(RAPD)PCR比较分析噬菌体宿主谱改变时核酸序列组成的变化;通过以上两噬菌体对环境样本中活菌和E. coli的杀灭效果的观察, 比较分析宿主谱改变对噬菌体的微生物杀灭效应这一生物学特性的影响. 结果 噬菌体核酸分析试验证实, f2噬菌体及宽噬噬菌体均为6000bp左右的单链RNA噬菌体. RAPDPCR结果显示, 两噬菌体基因cDNA扩增出的RAPDDNA片段有明显的差异, 其中26条为可区分的DNA带型. 利用简并引物进行的RTPCR反应显示, 噬菌体f2cDNA在450bp附近出现重复性较好的扩增片段, 而宽噬噬菌体cDNA在相同条件下未出现扩增产物. 环境水样本微生物杀灭试验结果显示, 宽宿主谱株噬菌体对环境水样本中的E. coli杀灭率为36. 75%～56. 28%, 对水样本中活菌杀灭率为30. 84%～47.96%;而噬菌体f2对环境水样本中的E.coli杀灭率则为19.19%～35.06%,对水样本中活菌杀灭率为13.05%～27.85%.结论 宽宿主谱噬菌体微生物杀灭率明显高于野生型单一宿主谱噬菌体f2(P=0.000);核酸分析证实两噬菌体宿主特异性裂解效应已从基因水平发生了变化. |
| English Abstract: |
| Objective To study the change of nucleic acid sequence and the germicidal effect of an E.coli bacteriophage with broad host range isolated from hospital sewage as well as to study the mechanism of phage host specificity and the effect of killed bacteria by phage-disinfectant to the samples from sewage water. Methods To extract the nucleic acid from phage f_2 and phage with broad host range using anti-serum-carbamidine hydrochloride assay. Purity with agarose gel electrophoresis was then evaluated. Differences of nucleic acid sequence between phage f_2 and phage with broad host range with reverse transcription-polymerase chain reaction(RT-PCR)and random amplified polymorphic DNA(RAPD)-PCR were also comparing and analysed. Through observing the germicidal test of phage f_2 and phage with broad host range to samples from environment, differeent sterilization effects between the two phages were compared. Results Analystic test for nucleic acid revealed that the two phages both belonged to 6000 bp, single-stranded RNA bacteriophage. Significant differences in their specificity of RAPD-PCR and RT-PCR were found during the changed of host range;with26RAPD-cDNA differential fragments found that in two phages RAPD-PCR products. The RT-PCR product of phage f_2 was 450 bp cDNA fragment, but the phage with broad host range did not show PCR product. Treating the sewage water with phage under broad host range, the germicidal test showed that the cleaning rate of E.coli bacteria and phage f_2 in water samples from environment could reach 36.75%- 56.28 %, 30.84%- 47.96 %, 19.19%- 35.06 % and 13.05%- 27.85%, respectively. Conclusion The cleaning rates to E.coli and bacteria by phage with broad host range were obviously higher than phage f_2(P = 0.000). Analytic test for nucleic acid indicated that host-specific lytic effect of phage with broad host range had been changed at genetic level. |
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