跳到主要內容

臺灣博碩士論文加值系統

(54.92.164.9) 您好!臺灣時間:2022/01/23 05:48
字體大小: 字級放大   字級縮小   預設字形  
回查詢結果 :::

詳目顯示

: 
twitterline
研究生:董文蘭
研究生(外文):Wen-Lan Tung
論文名稱:以聚合酶連鎖反應偵測水中之隱胞子蟲
論文名稱(外文):Primer Design and Detection of Cryptosporidium Oocysts in Taiwan Rivers by Polymerase Chain Reaction
指導教授:胡苔莉胡苔莉引用關係
指導教授(外文):Tai-Lee Hu
學位類別:碩士
校院名稱:逢甲大學
系所名稱:環境工程與科學所
學門:工程學門
學類:環境工程學類
論文種類:學術論文
論文出版年:2005
畢業學年度:93
語文別:中文
論文頁數:88
中文關鍵詞:隱胞子蟲聚合酶連鎖反應
外文關鍵詞:PCRCryptosporidium sp.
相關次數:
  • 被引用被引用:10
  • 點閱點閱:186
  • 評分評分:
  • 下載下載:42
  • 收藏至我的研究室書目清單書目收藏:0
摘 要
隱胞子蟲(Cryptosporidium sp.)是一種常見的腸道寄生性原生動物,所引起的疾病稱為隱胞子蟲症(cryptosporidiosis),其能藉由飲水進入人體。Cryptosporidium之胞囊(oocyst)可在惡劣的環境下存活,且對消毒劑的抗性大,一般的淨水處理程序無法有效地降低oocysts的含量,而無法保障民眾飲用水的安全。目前Cryptosporidium sp.的檢測仍以免疫螢光抗體分析法(immunofluorescence antibody, IFA)為主,但此法較費時且專一性不高。本研究之目的為利用專一性高之聚合酶連鎖反應(polymerase chain reaction, PCR)來檢測水中的Cryptosporidium,主要以引子設計,PCR偵測之最適化條件探討及應用於自然水體之偵測。
Oocysts之胞壁以六次的凍-融(液態氮 5 min, 70℃ 5 min)破裂釋放DNA後,再以20% Chelex® 100純化DNA進行PCR。Primer pr2為針對Cryptosporidium sp.之18S rRNA序列設計,其長度為20 bp,GC含量為50%,forward序列為5’-GCCTTGAATACTCCAGCATG-
3’;reverse序列為5’-ACTACGACGGTATCTGATCG-3’,可合成長度為210 bp之產物。而以pr2進行PCR之最適化條件為煉合溫度55℃、熱循環週期40 cycle時,可以得到最佳的產物量。以保存在福馬林中之標準oocysts溶液(利用106 oocysts/mL)進行PCR偵測Cryptosporidium oocysts之極限為103 oocysts/mL。以pr2可檢測出港尾溪、筏子溪及食水嵙溪水中含有Cryptosporidium sp.的存在,顯示國內的灌溉水可能已被Cryptosporidium sp.污染,對人體健康雖無立即性的危害,但仍需訂定相關議題之規範來防止致病性Cryptosporidium sp.的感染,使其感染風險降至最低。
Abstract
Cryptosporidium sp. is an intestinal parasitical protozoan; usually it can enter the human body through drinking water and cause cryptosporidiosis. Cryptosporidium oocysts could survive in severe environments, and is highly resistant to disinfectant. The conventional water treatment process is unable to reduce the amount of oocysts effectively, thus, unable to ensure the safety of the drinking water. The detection of Cryptosporidium sp. was mainly using immunofluorescence antibody (IFA), which is time-consuming and sometimes unspecific. The objective of this study is to use specific polymerase chain reaction (PCR) to detect Cryptosporidium sp. in water. The main points of this study include primer designing, investigation of the optimal amplification conditions for the PCR and applying the primer to detect the Cryptosporidium in water body.
DNA released from oocysts by disruption with six cycles of freezing in liquid nitrogen for 5 min and thawing at 70ºC water bath for 5 min was extracted with 20% Chelex® 100 to make the DNA available for PCR detection. Primer pr2 was designed from Cryptosporidium 18S rRNA, its length was 20 bp, GC content was 50%, the sequence of the forward primer was 5’-GCCTTGAATACTCCAGCATG-3’, and the reverse primer was 5’-ACTACGACGGTATCTGATCG-3’. The PCR product was 210 bp. The amplification conditions for the PCR were the anneling temperature of 55ºC with 40 cycles; the optimal productivity could be obtained by pr2. The limitation for using pr2 to detect Cryptosporidium oocysts with PCR was 103 oocysts/mL of the standard solution (106 oocysts/mL) which preserved in formaldehyde. The results showed that pr2 could detect Cryptosporidium sp. in Gangwei River, Fazi River and Shishuike River, implying that the irrigation water in Taiwan was contaminated with Cryptosporidium sp. There is no damage to the human health immediately, but still need to stipulate the regulation in order to prevent and lower the risk of infecting protozoan.
目 錄

中文摘要 Ⅰ
英文摘要 Ⅲ
目錄 Ⅴ
圖目錄 Ⅷ
表目錄 Ⅸ
第一章 緒論 1
1-1 前言 1
1-2 研究內容 3
第二章 文獻回顧 4
2-1 致病性原生動物-Cryptosporidium 4
2-2 檢測Cryptosporidium之方法 5
2-2-1 免疫螢光抗體分析法(immunofluorescence antibody, IFA) 6
2-2-2 酵素免疫分析法(enzyme immunoassay, EIA) 8
2-2-3 DAPI (4’-6-diamidino-2-phenylindole)/PI (propidium iodide)染色法
9
2-2-4 流式細胞儀(flow cytometry) 11
2-2-5 螢光原位雜交法(fluorescent in situ hybridization, FISH) 12
2-2-6 聚合酶連鎖反應(polymerase chain reaction, PCR) 13
2-3 PCR檢測Cryptosporidium之限制條件 16
2-3-1 Oocysts之破裂 16
2-3-2 DNA之萃取純化 17
2-3-3 引子的設計 18
2-3-4 煉合溫度 19
2-3-5 反應週期數 19
第三章 材料與方法 20
3-1 菌種來源 20
3-2 清洗oocysts 20
3-3 Oocysts之破裂 20
3-3-1 凍-融(freeze-thaw) 20
3-3-2 Oocysts之計數 21
3-4 DNA萃取純化 21
3-4-1 InstaGene™ Matrix試劑 21
3-4-2 Phenol-chloroform溶液 22
3-4-3 Chelex® 100萃取純化 22
3-5 Primer的設計 23
3-6 聚合酶連鎖反應 25
3-7 PCR產物之檢測 26
3-8 PCR之偵測極限 26
3-9 PCR產物定序 27
3-10 環境水樣之採集、濃縮及純化 27
3-10-1 以Envirochek濾管採集水樣 27
3-10-2 水樣濃縮 28
3-10-3 以Percoll-Sucrose梯度澄清法純化水樣 28
第四章 結果與討論 29
4-1 評估不同凍-融對oocysts之破壞效率 29
4-2 DNA之純化效率 31
4-3 分析Cryptosporidium sp.之PCR最佳操作條件 34
4-4 PCR之偵測極限 44
4-5 環境水樣之檢測 47
第五章 結論 50
第六章 參考文獻 51
附錄一 由GeneFisher網站軟體設計之Cryptosporidium sp.引子組 65
附錄二 序列比對圖 67


圖 目 錄

圖一 NCBI網站搜尋之關鍵字與結果 23
圖二 Cryptosporidium sp.之基因序列 23
圖三 Primer設計參數 24
圖四 以文獻資料對pr1 primer進行PCR測試 36
圖五 煉合溫度55℃下,循環週期數對pr1 primer進行PCR之影響 37
圖六 39個循環週期數下,煉合溫度對pr1 primer進行PCR之影響 38
圖七 Oocysts之前處理對pr2 primer進行PCR之影響 41
圖八 煉合溫度對pr2為引子之Cryptosporidium PCR產物影響 42
圖九 煉合溫度與循環週期數對PCR產物量的影響 43
圖十 以PCR方法偵測Cryptosporidium之偵測極限 46
圖十一 環境水樣以pr2為引子進行PCR放大 49


表 目 錄

表一 Oocyst之染色特徵 10
表二 染劑之染色結果與原蟲oocysts活性之相關性 10
表三 PCR方法檢測水體中Cryptosporidium oocysts之比較 14
表四 檢測方法之比較 15
表五 本研究使用之Cryptosporidium 引子特性 25
表六 Cryptosporidium oocysts之前處理與次數對oocysts破裂之影響 30
表七 DNA純化方法與純化效率 32
表八 三條河川水樣之水質 48
表九 環境水樣之PCR產物定序結果 48
第六章 參考文獻
黃志彬、胥直利、許昺慕、江國瑛、李奇翰,1999,飲用水水源中致病性微生物-梨形鞭毛蟲及隱孢子蟲調查及管制評估期末報告,行政院環境保護署EPA-88-J1-02-03-008。
Alles A. J., Waldron M. A., Sierra L. S. and Mattia A. R., 1995, Prospective comparison of direct immunofluorescence and conventional staining methods for detection of Giardia and Cryptosporidium spp. in human fecal specimens, J. Clin. Microbiol., 33 (6), 1632-1634.
Anusz K. Z., Mason P. H., Riggs M. W. and Perryman L. E., 1990, Detection of Cryptosporidium parvum oocysts in bovine feces by monoclonal antibody capture enzyme-linked immunosorbent assay, J. Clin. Microbiol., 28 (12), 2770-2774.
Armon R., Gold D., Brodsky M. and Oron G., 2002, Surface and subsurface irrigation with effluents of different qualities and presence of Cryptosporidium oocysts in soil and on crops, Wat. Sci. Technol., 46 (3), 115-122.
Arrowood M. J. and Sterling C. R., 1987, Isolation of Cryptosporidium oocysts and sporozoites using discontinuous sucrose and isopycnic percoll gradients, J. Parasitol., 73 (2), 314-319.
Bio-Rad, 2000, InstaGene™ Matrix, Bio-Rad, C. A.
Campbell A. T., Robertson L. J. and Smith H. V., 1992, Viability of Cryptosporidium parvum oocysts: correlation of in vitro excystation with inclusion or exclusion of fluorogenic vital dyes, Appl. Environ. Microbiol., 58 (11), 3488-3493.
Clontech, 2003, Advantage® 2 PCR kit user manual, BD Biosciences, C. A.
Current W. L. and Garcia L. S., 1991, Cryptosporidiosis, Clin. Microbiol. Rev., 4 (3), 325-358.
Dagan R., Fraser D., El-On J., Kassis I., Deckelbaum R. and Turner S., 1995, Evaluation of an enzyme immunoassay for the detection of Cryptosporidium spp. in stool specimens from infants and young children in field studies, Am. J. Trop. Med. Hyg., 52 (2), 134-138.
Davies C. M., Kaucner C., Deere D. and Ashbolt N. J., 2003, Recovery and enumeration of Cryptosporidium parvum from animal fecal matrices, Appl. Environ. Microbiol., 69 (5), 2842-2847.
Deere D., Vesey G., Milner M., Williams K., Ashbolt N. and Veal D., 1998, Rapid method for fluorescent in situ ribosomal RNA labelling of Cryptosporidium parvum, J. Appl. Microbiol., 85 (5), 807-818.
Deng M. Q. and Cliver D. O., 2000, Comparative detection of Cryptosporidium parvum oocysts from apple juice, Int. J. Food Microbiol., 54 (3), 155-162.
Deng M. Q., Lam K. M. and Cliver D. O., 2000, Immunomagnetic separation of Cryptosporidium parvum oocysts using MACS MicroBeads and high gradient separation columns, J. Microbiol. Methods, 40 (1), 11-17.
Eeles R. A. and Stamps A. C., 1993, Polymerase chain reaction (PCR): the technique and its applications, R. G. Landes Co., U. S. A.
Elwin K., Chalmers R. M., Roberts R., Guy E. C. and Casemore D. P., 2001, Modification of a rapid method for the identification of gene-specific polymorphisms in Cryptosporidium parvum and its application to clinical and epidemiological investigations, Appl. Environ. Microbiol., 67 (12), 5581-5584.
Fayer R., Speer C. A. and Dubey J. P., 1997, The general biology of Cryptosporidium, In: Fayer R. (ed.), Cryptosporidium and Cryptosporidiosis, CRC Press, Boca Raton, F. L., 1-41.
Fayer R., Trout J. M., Graczyk T. K. and Lewis E. J., 2000, Prevalence of Cryptosporidium, Giardia and Eimeria infections in post-weaned and adult cattle on three Maryland farms, Vet. Parasitol., 93 (2), 103-112.
Feng Y. Y., Ong S. L., Hu J. Y., Song L. F., Tan X. L. and Ng W. J., 2003, Effect of particles on the recovery of Cryptosporidium oocysts from source water samples of various turbidities, Appl. Environ. Microbiol., 69 (4), 1898-1903.
Ferrari B. C., Vesey G., Weir C., Williams K. L. and Veal D. A., 1999, Comparison of Cryptosporidium-specific and Giardia-specific monoclonal antibodies for monitoring water samples, Wat. Res., 33 (7), 1611-1617.
Garcia L. S. and Shimizu R. Y., 1997, Evaluation of nine immunoassay kits (enzyme immunoassay and direct fluorescence) for detection of Giardia lamblia and Cryptosporidium parvum in human fecal specimens, J. Clin. Microbiol., 35 (6), 1526-1529.
Gasser R. B. and O’Donoghue P., 1999, Isolation, propagation and characterization of Cryptosporidium, Int. J. Parasitol., 29 (9), 1379-1413.
Graczyk T. K., Fayer R., Knight R., Mhangami-Ruwende B., Trout J. M., Da Silva A. J. and Pieniazek N. J., 2000, Mechanical transport and transmission of Cryptosporidium parvum oocysts by wild filth flies, Am. J. Trop. Med. Hyg., 63 (3, 4), 178-183.
Graczyk T. K., Grimes B. H., Knight R., Da Silva A. J., Pieniazek N. J. and Veal D. A., 2003, Detection of Cryptosporidium parvum and Giardia lamblia carried by synanthropic flies by combined fluorescent in situ hybridization and a monoclonal antibody, Am. J. Trop. Med. Hyg., 68 (2), 228-232.
Guyot K., Follet-Dumoulin A., Recourt C., Lelièvre E., Cailliez J. C. and Dei-Cas E., 2002, PCR-restriction fragment length polymorphism analysis of a diagnostic 452-base-pair DNA fragment discriminates between Cryptosporidium parvum and C. meleagridis and between C. parvum isolates of human and animal origin, Appl. Environ. Microbiol., 68 (4), 2071-2076.
Haas C. N. and Rose J. B., 1994, Reconciliation of microbial risk models and outbreak epidemiology: the case of the Milwaukee outbreak, In: Proc. Am. Water Works Assoc., New York, U. S. A., 517-523.
Hallier-Soulier S. and Guillot E., 1999, An immunomagnetic separation polymerase chain reaction assay for rapid and ultra-sensitive detection of Cryptosporidium parvum in drinking water, FEMS Microbiol. Lett., 176 (2), 285-289.
Hancock C. M., Rose J. B. and Callahan M., 1998, Crypto and Giardia in US groundwater, J. Am. Water Works Assoc., 90 (3), 58-61.
Hashimoto A., Kunikane S. and Hirata T., 2002, Prevalence of Cryptosporidium oocysts and Giardia cysts in the drinking water supply in Japan, Wat. Res., 36 (3), 519-526.
Held P. G., 2003, Nucleic acid purity assessment using A260/A280 ratios, Application Note, Bio-Tek Instruments, Winooski, VT.
Higgins J. A., Fayer R., Trout J. M., Xiao L., Lal A. A., Kerby S. and Jenkins M. C., 2001, Real-time PCR for the detection of Cryptosporidium parvum, J. Microbiol. Methods, 47 (3), 323-337.
Higgins J. A., Trout J. M., Fayer R., Shelton D. and Jenkins M. C., 2003, Recovery and detection of Cryptosporidium parvum oocysts from water samples using continuous flow centrifugation, Wat. Res., 37 (15), 3551-3560.
Hoffman R. M., Standridge J. H., Prieve A. F., Cucunato J. C. and Bernhardt M., 1997, Using flow cytometry to detect protozoa, J. Am. Water Works Assoc., 89 (9), 104-111.
Jellison K. L., Hemond H. F. and Schauer D. B., 2002, Sources and species of Cryptosporidium oocysts in the Wachusett Reservoir watershed, Appl. Environ. Microbiol., 68 (2), 569-575.
Jenkins M. B., Anguish L. J., Bowman D. D., Walker M. J. and Ghiorse W. C., 1997, Assessment of a dye permeability assay for determination of inactivation rates of Cryptosporidium parvum oocysts, Appl. Environ. Microbiol., 63 (10), 3844-3850.
Jenkins M. C., O’Brien C., Trout J., Guidry A. and Fayer R., 1998, Hyperimmune bovine colostrum specific for recombinant Cryptosporidium parvum antigen confers partial protection against cryptosporidiosis in immunosuppressed adult mice, Vaccine, 17 (19), 2453-2460.
Johnson D. W., Pieniazek N. J., Griffin D. W., Misener L. and Rose J. B., 1995, Development of a PCR protocol for sensitive detection of Cryptosporidium oocysts in water samples, Appl. Environ. Microbiol., 61 (11), 3849-3855.
Kehl K. S. C., Cicirello H. and Havens P. L., 1995, Comparison of four different methods for the detection of Cryptosporidium species, J. Clin. Microbiol., 33 (2), 416-418.
Kostrzynska M., Sankey M., Haack E., Power C., Aldom J. E., Chagla A. H., Unger S., Palmateer G., Lee H., Trevors J. T. and De Grandis S. A., 1999, Three sample preparation protocols for polymerase chain reaction based detection of Cryptosporidium parvum in environmental samples, J. Microbiol. Methods, 35 (1), 65-71.
Kuczynska E. and Shelton D. R., 1999, Method for detection and enumeration of Cryptosporidium parvum oocysts in feces, manures, and soils, Appl. Environ. Microbiol., 65 (7), 2820-2826.
Kuhn R. C., Rock C. M. and Oshima K. H., 2002, Occurrence of Cryptosporidium and Giardia in wild ducks along the Rio Grande River valley in Southern New Mexico, Appl. Environ. Microbiol., 68 (1), 161-165.
LeChevallier M. W., Norton W. D. and Lee R. G., 1991, Occurrence of Giardia and Cryptosporidium spp. in surface water supplies, Appl. Environ. Microbiol., 57 (9), 2610-2616.
LeChevallier M. W., Di Giovanni G. D., Clancy J. L., Bukhari Z., Bukhari S., Rosen J. S., Sobrinho J. and Frey M. M., 2003, Comparison of method 1623 and cell culture-PCR for detection of Cryptosporidium spp. in source waters, Appl. Environ. Microbiol., 69 (2), 971-979.
Leland D., Mcanulty J., Keene W. and Stevens G., 1993, A cryptosporidiosis outbreak in a filtered public water supply, J. Am. Water Works Assoc., 85, 34-42.
Leng X., Mosier D. A. and Oberst R. D., 1996, Differentiation of Cryptosporidium parvum, C. muris, and C. baileyi by PCR-RFLP analysis of the 18S rRNA gene, Vet. Parasitol., 62 (1-2), 1-7.
Lindquist H. D. A., 1997, Probes for the specific detection of Cryptosporidium parvum, Wat. Res., 31 (10), 2668-2671.
MacKenzie W. R., Hoxie N. J., Proctor M. E., Gradus M. S., Blair K. A., Peterson D. E., Kazmierczak J. J., Addiss D. G., Fox K. R., Rose J. B. and Davis J. P., 1994, A massive outbreak in Milwaukee of Cryptosporidium infection transmitted through the public water supply, N. Engl. J. Med., 331 (3), 161-167.
Madore M. S., Rose J. B., Gerba C. P., Arrowood M. J. and Sterling C. R., 1987, Occurrence of Cryptosporidium oocysts in sewage effluents and selected surface waters, J. Parasitol., 73 (4), 702-705.
Monis P. T. and Saint C. P., 2001, Development of a nested-PCR assay for the detection of Cryptosporidium parvum in finished water, Wat. Res., 35 (7), 1641-1648.
Morgan U. M. and Thompson R. C. A., 1998, Molecular detection of parasitic protozoa, Parasitology, 117 (7), S73-S85.
Morgan U., Weber R., Xiao L., Sulaiman I., Thompson R. C. A., Ndiritu W., Lal A., Moore A. and Deplazes P., 2000, Molecular characterization of Cryptosporidium isolates obtained from human immunodeficiency virus-infected individuals living in Switzerland, Kenya, and the United States, J. Clin. Microbiol., 38 (3), 1180-1183.
Mtambo M. M. A., Nash A. S., Wright S. E., Smith H. V., Blewett D. A. and Jarrett O., 1995, Prevalence of specific anti-Cryptosporidium IgG, IgM and IgA antibodies in cat sera using an indirect immunofluorescence antibody test, Vet. Parasitol., 60 (1-2), 37-43.
Neumann N. F., Gyürek L. L., Gammie L., Finch G. R. and Belosevic M., 2000, Comparison of animal infectivity and nucleic acid staining for assessment of Cryptosporidium parvum viability in water, Appl. Environ. Microbiol., 66 (1), 406-412.
Pieniazek N. J., Bornay-Llinares F. J., Slemenda S. B., da Silva A. J., Moura I. N. S., Arrowood M. J., Ditrich O. and Addiss D. G., 1999, New Cryptosporidium genotypes in HIV-infected persons, Emerg. Infect. Dis., 5 (3), 444-449.
Puech M. C., McAnulty J. M., Lesjak M., Shaw N., Heron L. and Watson J. M., 2001, A statewide outbreak of cryptosporidiosis in New South Wales associated with swimming in public pools, Epidemiol. Inf., 126 (3), 389-396.
Quintero-Betancourt W., Peele E. R. and Rose J. B., 2002, Cryptosporidium parvum and Cyclospora cayetanensis: a review of laboratory methods for detection of these waterborne parasites, J. Microbiol. Methods, 49 (3), 209-224.
Quintero-Betancourt W., Gennaccaro A. L., Scott T. M. and Rose J. B., 2003, Assessment of methods for detection of infectious Cryptosporidium oocysts and Giardia cysts in reclaimed effluents, Appl. Environ. Microbiol., 69 (9), 5380-5388.
Richardson A. J., Frankenberg R. A., Buck A. C., Selkon J. B., Colbourne J. S., Parsons J. W. and Mayon-White R. T., 1991, An outbreak of waterborne cryptosporidiosis in Swindon and Oxfordshire, Epidemiol. Inf., 107 (3), 485-495.
Rimhanen-Finne R., Ronkainen P. and Hänninen M. -L., 2001, Simultaneous detection of Cryptosporidium parvum and Giardia in sewage sludge by IC-PCR, J. Appl. Microbiol., 91 (6), 1030-1035.
Rimhanen-Finne R., Hörman A., Ronkainen P. and Hänninen M. -L., 2002, An IC-PCR method for detection of Cryptosporidium and Giardia in natural surface waters in Finland, J. Microbiol. Methods, 50 (3), 299-303.
Rochelle P. A., De Leon R., Stewart M. H. and Wolfe R. L., 1997, Comparison of primers and optimization of PCR conditions for detection of Cryptosporidium parvum and Giardia lamblia in water, Appl. Environ. Microbiol., 63 (1), 106-114.
Rose J. B., Gerba C. P. and Jakubowski W., 1991, Survey of potable water supplies for Cryptosporidium and Giardia, Environ. Sci. Technol., 25 (8), 1393-1400.
Rose J. B., Huffman D. E. and Gennaccaro A., 2002, Risk and control of waterborne cryptosporidiosis, FEMS Microbiol. Rev., 26 (2), 113-123.
Sambrook J. and Rusell D. W., 2001, Molecular Cloning: A Laboratory Manual, 3rd edn. Cold Spring Harbor Laboratory, Cold Spring Harbor, N. Y.
Simmons Ш O. D., Sobsey M. D., Heaney C. D., Schaefer Ш F. W. and Francy D. S., 2001, Concentration and detection of Cryptosporidium oocysts in surface water samples by method 1622 using ultrafiltration and capsule filtration, Appl. Environ. Microbiol., 67 (3), 1123-1127.
Sluter S. D., Tzipori S. and Widmer G., 1997, Parameters affecting polymerase chain reaction detection of waterborne Cryptosporidium parvum oocysts, Appl. Microbiol. Biotechnol., 48 (3), 325-330.
Smith H. V. and Rose J. B., 1998, Waterborne cryptosporidiosis: current status, Parasitol. Today, 14 (1), 14-22.
Smith J. J., Gunasekera T. S., Barardi C. R. M., Veal D. and Vesey G., 2004, Determination of Cryptosporidium parvum oocyst viability by fluorescence in situ hybridization using a ribosomal RNA-directed probe, J. Appl. Microbiol., 96 (2), 409-417.
Straub T. M., Daly D. S., Wunshel S., Rochelle P. A., DeLeon R. and Chandler D. P., 2002, Genotyping Cryptosporidium parvum with an hsp 70 single-nucleotide polymorphism microarray, Appl. Environ. Microbiol., 68 (4), 1817-1826.
USEPA, 2001a, Method 1622: Cryptosporidium in Water by Filtration/IMS/FA, EPA-821-R-01-026, Office of Water, Washington, D. C.
USEPA, 2001b, Method 1623: Cryptosporidium and Giardia in Water by Filtration/IMS/FA, EPA-821-R-01-025, Office of Water, Washington, D. C.
Valdez L. M., Dang H., Okhuysen P. C. and Chappell C. L., 1997, Flow cytometric detection of Cryptosporidium oocysts in human stool samples, J. Clin. Microbiol., 35 (8), 2013-2017.
Vesey G., Slade J. S., Byrne M., Shepherd K., Dennis P. J. and Fricker C. R., 1993, Routine monitoring of Cryptosporidium oocysts in water using flow cytometry, J. Appl. Bacteriol., 75 (1), 87-90.
Vesey G., Hutton P., Champion A., Ashbolt N., Williams K. L., Warton A. and Veal D., 1994, Application of flow cytometric methods for the routine detection of Cryptosporidium and Giardia in water, Cytometry, 16 (1), 1-6.
Vesey G., Ashbolt N., Wallner G., Dorsch M., Williams K. and Veal D., 1995, Assessing Cryptosporidium parvum oocyst viability with fluorescent in situ hybridization using ribosomal RNA probes and flow cytometry, In: Betts W. B., Casemore D., Fricker C., Smith H. and Watkins J. (eds.), Protozoan Parasites and Water, Royal Society of Chemistry, Cambridge, 133-138.
Vesey G., Griffiths K. R., Gauci M. R., Deere D., Williams K. L. and Veal D. A., 1997, Simple and rapid measurement of Cryptosporidium excystation using flow cytometry, Int. J. Parasitol., 27 (11), 1353-1359.
Vesey G., Ashbolt N., Fricker E. J., Deere D., Williams K. L., Veal D. A. and Dorsch M., 1998, The use of a ribosomal RNA targeted oligonucleotide probe for fluorescent labelling of viable Cryptosporidium parvum oocysts, J. Appl. Microbiol., 85 (3), 429-440.
Walsh P. S., Metzger D. A. and Higuchi R., 1991, Chelex® 100 as a medium for simple extraction of DNA for PCR-based typing from forensic material, BioTechniques, 10 (4), 506-513.
Ward L. A. and Wang Y., 2001, Rapid methods to isolate Cryptosporidium DNA from frozen feces for PCR, Diagn. Microbiol. Infect. Dis., 41 (1-2), 37-42.
Weber R., Bryan R. T., Bishop H. S., Wahlquist S. P., Sullivan J. J. and Juranek D. D., 1991, Threshold of detection of Cryptosporidium oocysts in human stool specimens: evidence for low sensitivity of current diagnosic methods, J. Clin. Microbiol., 29 (7), 1323-1327.
Webster K. A., Pow J. D. E., Giles M., Catchpole J. and Woodward M. J., 1993, Detection of Cryptosporidium parvum using a specific polymerase chain reaction, Vet. Parasitol., 50 (1-2), 35-44.
Webster K. A., Smith H. V., Giles M., Dawson L. and Robertson L. J., 1996, Detection of Cryptosporidium parvum oocysts in faeces: comparison of conventional coproscopical methods and the polymerase chain reaction, Vet. Parasitol., 61 (1-2), 5-13.
Wiedenmann A., Krüger P. and Botzenhart K., 1998, PCR detection of Cryptosporidium parvum in environmental samples-a review of published protocols and current developments, J. Ind. Microbiol. Biotechnol., 21 (3), 150-166.
Xiao L., Morgan U. M., Limor J., Escalante A., Arrowood M., Shulaw W., Thompson R. C. A., Fayer R. and Lal A. A., 1999, Genetic diversity within Cryptosporidium parvum and related Cryptosporidium species, Appl. Environ. Microbiol., 65 (8), 3386-3391.
Xiao L., Morgan U. M., Fayer R., Thompson R. C. A. and Lal A. A., 2000, Cryptosporidium systematics and implications for public health, Parasitol. Today, 16 (7), 287-292.
Xiao L., Singh A., Limor J., Graczyk T. K., Gradus S. and Lal A., 2001, Molecular characterization of Cryptosporidium oocysts in samples of raw surface water and wastewater, Appl. Environ. Microbiol., 67 (3), 1097-1101.
Zuckerman U., Gold D., Shelef G. and Armon R., 1997, The presence of Giardia and Cryptosporidium in surface waters and effluents in Israel, Wat. Sci. Technol., 35 (11-12), 381-384.
QRCODE
 
 
 
 
 
                                                                                                                                                                                                                                                                                                                                                                                                               
第一頁 上一頁 下一頁 最後一頁 top
無相關期刊