파지_지표성(보고서초안6-원장님보고후수정090122).hwp

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1 NIER NO A study on the bacteriophages as indicators of viruses in water environment

2 : 물에서바이러스에대한대장균파지의지표성에관한연구 장관계바이러스는물환경에서저항성이상대적으로높고, 분석이어려울뿐만아니라비용도많이소요되므로직접적인모니터링에많은어려움이있음따라서장관계바이러스에비해분석이쉽고, 형태학적으로도유사한세균성바이러스의한종류인대장균파지가물에서바이러스의지표로서적합한지를조사하고, 총대장균군및대장균등기존의지표미생물과함께비교 검토하여보다적합한바이러스지표미생물을제시하고자하였음 선진국의대장균파지분석방법검토및최적분석방법선정 phages에대한지표로서의타당성평가및검출방법제시 국내조사지점 (46개소) 선정, 조사및분석 조사항목 : 노로바이러스, 장바이러스, 대장균파지 2항목 (somatic phage, male-specific phage), 총대장균군, 대장균, 수온, ph, 탁도 (9항목) 각미생물항목과바이러스와의검출비율비교, 평가 분원성지표세균및관련수질항목과의상관성검토, 평가 지하수노로바이러스오염실태조사연구용역조사결과평가바이러스지표로서적합한지표미생물제시 미국등여러나라에서대장균파지를바이러스에대한지표로활용하고있으며, 여러문헌에서도지표로서 phages 의장점에보고하고있음

3 대장균파지는장관계바이러스처럼사람이나온혈동물의분변에서유래하며, 수환경에서증식되지않고, 구성과구조및크기에서장관계바이러스와매우유사하므로장관계바이러스에대한좋은지표로제안됨 (EPA, WHO) 그러나, 장관계바이러스때문에발병하는질병에서대장균파지가발견되지않았고, 높은분원성오염물에서는오히려검출되지않는경우도보고됨 미국지하수규정 (Ground Water Rule) 에서는장관계바이러스에대한대장균파지의지표로서의장점을반영하여지표미생물중하나로규정자체조사된 46건시료중노로바이러스가검출된시료의유전자형확인결과 GII-4가대부분검출됨 2007년제주에서처음발견된유전자형 GII-4의 Jeju-4와 14 검출 왕숙천에서새로운유전자형 GII-YURI 검출분석된시료의각미생물항목검출비율은높은비율순으로다음과같음. 자체조사된지하수및하천수 46건시료 : somatic phage> 총대장균군 > male-specific phage> 대장균 > 노로바이러스 > 장바이러스 연구용역에서조사된지하수 300건시료 : 총대장균군 > 노로바이러스 > 대장균 > male- specific phage> somatic phage> 장바이러스바이러스가검출되었을때다른미생물조사항목과동시에검출된비율이비교적높았던항목은대장균, 대장균파지 (male-specific phage) 와대장균 (MP_EC) 및대장균파지 (somatic phage) 와대장균 (SP_EC) 이었음미생물항목들과바이러스간의상관성이나타난항목은대장균파지 (malespecific phage) 와대장균 (MP_EC), 대장균파지 (somatic phage) 와대장균 (SP_EC), 대장균 (EC) 등이었음 (p<0.05, r=0.3이상기준 ) 검출비율조사결과와상관성검토결과및실제실험의용이성등을종합적으로감안할때, 바이러스에대한지표미생물로적합한항목은대장균파지 (male-specific phage) 와대장균, 대장균파지 (somatic phage) 와대장균및 2종류의대장균파지와대장균순으로적합한것으로조사되었음따라서, 물에서바이러스오염에대한지표미생물로는기존의분변오염지표인

4 대장균과함께대장균파아지 (somatic phage 및 male specific phage) 도같이적용하는방안을제시 지하수등물에서바이러스관리방안개선을위한근거자료로활용

5 Abstract This study aims to determine the suitable indicators of viruses. The detection rate and correlation between candidate microorganisms including coliphages and viruses in water environment were investigated Coliphages stem from faeces of humans or warm-blooded animals and preferably not be able to multiply in water environment. The major advantage of coliphages, compared with viruses, is that they are detectable by simple, inexpensive and rapid techniques. Also they share many fundamental properties and features like structure, composition, morphology, size and site of replication etc. Most of 46 water samples(ground water 32, river 14) were identified as genotype GII-4 norovirus. Specific genotype were GII Jeju-4, GII Jeju-14 and GII YURI. Microbial detection rate of 46 water samples were as follows; somatic phage accounted for the highest rate(31 samples, 67.4%), followed by total coliform(30, 65.2%) male-specific phage(20, 43.5%), E.coli(19, 41.3%), norovirus(9, 19.6%) and enterovirus(5, 10.9%). E.coil(EC), male-specific phage with E.coli(MP_EC) and phage with E.coli(MP_EC) showed the high detection rate simultaneously with viruses. The detection of viruses was correlated with male-specific phage with E.coil(MP_EC), somatic phage with E.coli(SP_EC),and E.coli. These results suggest that suitable virus indicators were male-specific phage with E.coli(MP_EC), followed by somatic phage with E.coli(SM_EC) and two phages with E.coli(P_EC).

6 Ⅰ Ⅱ Ⅲ

7 Ⅳ Ⅴ Ⅵ

21 kgcm2 l kgcm2 l l

22 ᄋ ᄋ ᄋ ᄋ l ᄋ l l kgcm2

23 ml ml μm μl μl μl μl ml ml μl

24 ml μl ml μl

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26 μl

27 ml ml nm

34 ml

35 Water temp. ph Turbidity

37 35 조사지점수 ( 건 ) 하천수등 지하수 S.Phage MS. Phage norovirus enterovirus Total coliforms 14 6 E.coli

38 C) o Water temperatu TC EC SP MP NV N/E ph 6 5 TC EC SP MP NV N/E Turbidity (NTU) 20 0 TC EC SP MP NV N/E

39 C) o ph 10 Water temperatu TC EC SP MP NV N/E 6 5 TC EC SP MP NV N/E Turbidity (NTU) 5 0 TC EC SP MP NV N/E

40 N 3 (mg/l) NO TC EC SP MP NV NV-EV

42 40 100% 조사결과 ( 건 ) 항목별검출건수노로바이러스와동시검출건수 조사결과 ( 건 ) 80% 60% 40% 20% 40% 노로바이러스와동시검출비율 (%) 38.9% 50% 0 TC EC SP MP 2P 2P_EC 2POE 2P_TC SP_EC MP_EC 0% TC EC SP MP 2P 2P_EC 2POE 2P_TC SP_EC MP_EC 조사항목 조사항목 항목별검출건수 NV또는EV와동시검출건수 % 80% 동시검출비율 (%) 조사결과 ( 건 ) 조사결과 ( 건 ) 60% 40% 50% 50% 50% 60% % 0 0% TC EC SP MP 2P 2P_EC 2POE 2P_TC SP_EC MP_EC TC EC SP MP 2P 2P_EC 2POE 2P_TC SP_EC MP_EC 조사항목 조사항목

44 % 조사결과 ( 건 ) 항목별검출건수노로바이러스와동시검출건수 조사결과 ( 건 ) 80% 60% 40% 20% 0% 노로바이러스와동시검출비율 (%) 42.1% 41.7% 50% TC EC SP MP 2P 2P_EC 2POE 2P_TC SP_EC MP_EC TC EC SP MP 2P 2P_EC 2POE 2P_TC SP_EC MP_EC 조사항목 조사항목 항목별검출건수 NV 또는 EV 와동시검출건수 100% 80% 동시검출비율 (%) 조사결과 ( 건 ) 조사결과 ( 건 ) 60% 40% 20% 0% 42.1% 41.7% 50% TC EC SP MP 2P 2P_EC 2POE 2P_TC SP_EC MP_EC TC EC SP MP 2P 2P_EC 2POE 2P_TC SP_EC MP_EC 조사항목 조사항목

46 % 216 조사결과 ( 건 ) 항목별검출건수 노로바이러스와동시검출건수 조사결과 ( 건 ) 80% 60% 40% 40.5% 42.9% 45.5% % 0% 노로바이러스와동시검출비율 (%) TC EC SP MP 2P 2P_EC 2POE 2P_TC SP_EC MP_EC TC EC SP MP 2P 2P_EC 2POE 2P_TC SP_EC MP_EC 조사항목 조사항목 항목별검출건수 NV 또는 EV 와동시검출건수 100% 80% 동시검출비율 (%) 45.9% 조사결과 ( 건 ) 조사결과 ( 건 ) 60% 40% 50% % 0 0% TC EC SP MP 2P 2P_EC 2POE 2P_TC SP_EC MP_EC TC EC SP MP 2P 2P_EC 2POE 2P_TC SP_EC MP_EC 조사항목 조사항목

47 δ δ

50 0.4 SP_EC P_EC NV 0.2 츋 1 P_TC Dimension POE TC EC MP NV_EV MP_EC -0.4 P SP Dimension 1

51 0.3 MP 0.2 POE P MP_EC Dimension NV_EV NV -0.1 TC P_TC -0.2 SP EC SP_EC P_EC Dimension 1

53 POE Dimension SP_EC -0.2 MP_EC -0.4 NV_EV P NV EC MP P_TC SP P_EC TC Dimension 1

65 Ⅰ Ⅱ Ⅱ

66 Ⅱ Ⅰ Ⅱ

79 β

81 ) EPA 1602 : Single agar layer(sal) 방법으로써지하수및다른수환경에서의 somatic coliphages 와 male specific(f+) 를계수하고검출하는방법이다.

82 Sterile dilution tubes with screw caps Test tube rack ) F-pilus : ertility code 를포함하고있는 pili( 섬모 ) 로써 male-specific phages 가 host bacteria 를감염시킬때경유하는부분 3) E. coli Famp : Streptomycin and ampicillin 에저항성을가지는 Famp plasmid 를가지는 antibiotic-resistant host-culture strains 4) E. coli CN-13 : Nalidixic acid 에저항성을가지는 antibiotic-resistant host-culture strains

83 μ μ μ

84 Reagent water %(w/v) Sodium thiosulfate

85 Magnesium chloride(80 X, 4 M) Nalidixic acid 5) μ Ampicillin/Streptomycin μ Single Agar Layer(SAL) 6) 5) Nalidixic acid sodium salt 는독성물질이므로, 반드시후드에서작업하고, 보호복, 보호장갑, 보호안경을착용한다. 6) 상용화된제품을사용할수도있으며, 이때배지의조제는제품의설명서를따른다.

86 Tryptic(trypticase) soy broth(tsb) TSB with ampicillin/streptomycin for growth of E. coli F amp TSB with nalidixic acid for growth of E. coli CN X Tryptic(trypticase) soy Agar(TSA) X TSA with ampicillin/streptomycin for growth of E. coli F amp

87 X TSA with nalidixic acid for growth of E. coli CN

88 6.1 Coliphage stock from sewage filtrate General QC requirements Initial precision and recovery(ipr)

89 Method blanks 7)

90 6.4 Matrix spikes(ms) Ongoing precision and recovery(orp) 8)

91 Record maintenance 6.7 Enumeration of coliphage QC spiking suspensions )

92 Preparation of plates for enumeration of somatic coliphage μ μ )

93 Preparation of plates for enumeration of male-specific(f+) coliphage 6.10 Preparation of plates of somatic coliphage method blank μ μ

94 Preparation of male-specific(f+) coliphage method blank Log-phase host bacteria stock cultures

95 Preparation of media for testing

96 을수행하기위한시료와숙주세균의혼합 가유지되는항온수조에 분간둔다 (plating)

97 SAL IPR, OSP, MS positive control spikes spiking volume

98 Method 1602 QC

( )Kju225.hwp

( )Kju225.hwp 비임균성비뇨생식기감염에서 의진단적효용성 Usefulness of the Mycofast Test (MYCOFAST Evolution 2) for the Diagnosis of Nongonococcal Genitourinary Infections Hang Ro Park, Yang Hyun Kim, Ho Jae Lee, Jea Sang Oh, Hyoung Jin