環保專案成果報告查詢系統

專案計畫編號	EPA-99-E3S5-02-01
經費年度	099
計畫經費	1,344 千元
專案開始日期	2010/02/03
專案結束日期	2010/12/31
專案主持人	張長泉
主辦單位	環檢所
承辦人	許令宜
執行單位	國立成功大學醫學檢驗技術研究中心
專案分類	環境監測及檢測
中文關鍵字	巨噬細胞感染增強蛋白基因;寡核苷酸晶片;退伍軍人菌
英文關鍵字	Macrophage infectivity potentiator;oligonucleotide array;Legionella spp.
協同主持人
共同主持人
計畫聯絡人	蘇淑琍
計畫聯絡電話	06-2353535#5774
計畫聯絡信箱	shuli0827@yahoo.com.tw

中文摘要	退伍軍人菌(Legionella spp.)在自然界中分布甚廣，調查人員曾在河川、湖泊、沼澤、井水、蓮蓬頭及冷卻塔之冷卻水中檢出。人體主要透過吸入該細菌而引發急性呼吸道疾病，依據症狀不同分為退伍軍人病與龐提亞克熱 (Pontiac fever)。退伍軍人菌所引起肺炎感染病狀，無法僅靠症狀與其他肺炎鑑別，其病程發展迅速，病菌培養不易且耗時，醫療機關常因診斷延誤，無法給予病患適當處置，可能造成死亡或病危。因此，有需要發展出一套快速之檢測方法。本研究利用生物晶片，以退伍軍人菌macrophage infectivity potentiator (mip)為目標基因，發展晶片以檢測退伍軍人菌。本研究之晶片可鑑定18種退伍軍人菌，經測試51株目標菌株(target strain)，晶片之敏感性為100% (51/51)。另外測試了71株(62種)非目標菌株(nontarget strain)，晶片之特異性為100% (71/71)。若以PCR放大mip基因後再進行晶片雜合反應，檢測極限(detection limit)為10 pg (L. micdadei ATCC 33218T, L. pneumophila ATCC 33152T)。進一步利用此晶片進行環境水樣品中退伍軍人菌之檢測，在50個水樣品中2個水樣品可檢測到L. micdadei，2個水樣品可檢測到L. pneumophila，2個水樣品可檢測到L. micdadei及L. pneumophila，其檢出率為12.0% (6/50)。部分水樣品也以傳統方法分離出退伍軍人菌，經mip定序結果與晶片鑑定結果一致。顯示此晶片為一種可鑑定水樣品中退伍軍人菌之良好工具。
中文關鍵字	巨噬細胞感染增強蛋白基因;寡核苷酸晶片;退伍軍人菌

中文摘要

退伍軍人菌(Legionella spp.)在自然界中分布甚廣，調查人員曾在河川、湖泊、沼澤、井水、蓮蓬頭及冷卻塔之冷卻水中檢出。人體主要透過吸入該細菌而引發急性呼吸道疾病，依據症狀不同分為退伍軍人病與龐提亞克熱 (Pontiac fever)。退伍軍人菌所引起肺炎感染病狀，無法僅靠症狀與其他肺炎鑑別，其病程發展迅速，病菌培養不易且耗時，醫療機關常因診斷延誤，無法給予病患適當處置，可能造成死亡或病危。因此，有需要發展出一套快速之檢測方法。本研究利用生物晶片，以退伍軍人菌macrophage infectivity potentiator (mip)為目標基因，發展晶片以檢測退伍軍人菌。本研究之晶片可鑑定18種退伍軍人菌，經測試51株目標菌株(target strain)，晶片之敏感性為100% (51/51)。另外測試了71株(62種)非目標菌株(nontarget strain)，晶片之特異性為100% (71/71)。若以PCR放大mip基因後再進行晶片雜合反應，檢測極限(detection limit)為10 pg (L. micdadei ATCC 33218T, L. pneumophila ATCC 33152T)。進一步利用此晶片進行環境水樣品中退伍軍人菌之檢測，在50個水樣品中2個水樣品可檢測到L. micdadei，2個水樣品可檢測到L. pneumophila，2個水樣品可檢測到L. micdadei及L. pneumophila，其檢出率為12.0% (6/50)。部分水樣品也以傳統方法分離出退伍軍人菌，經mip定序結果與晶片鑑定結果一致。顯示此晶片為一種可鑑定水樣品中退伍軍人菌之良好工具。

中文關鍵字

巨噬細胞感染增強蛋白基因;寡核苷酸晶片;退伍軍人菌

類型	檔名	檔案大小	說明	最新版公開日期
期末報告	99期末報告計畫成果報告（公開版）-EPA-99-E3S5-02-01.pdf	22MB		2011/2/1 上午 12:00:00

摘要	Legionella species, which are fastidious and ubiquitous worldwide in natural water environment such as rivers, lakes and artificial water systems, are the causative agents of Legionnaire’s disease. Legionella pneumophila is the most important human pathogen, followed by L. micdadei, L. longbeachae, L. dumoffii, and other rare species. Accurate identification of Legionella spp. other than L. pneumophila is difficult because of biochemical inertness and phenotypic identity of different species. Clinical manifestations caused by Legionella infection are usually indistinguishable from pneumonia caused by other bacterial pathogens. Since the symptoms are nontypical, it is difficult clinically to identify the actual causative agent. Therefore, the rapid and accurate identification of Legionella species have been of increasingly important. DNA microarray technology displays a high potential in the testing and analysis of environmental samples. The feasibility of using an oligonucleotide array for identification of 18 species of Legionella was evaluated in this study. The method consisted of PCR amplification of the macrophage infectivity potentiator (mip) gene, followed by hybridization of the digoxigenin-labeled PCR products to a panel of 37 oligonucleotide probes (16-27 nucleotides) immobilized on a nylon membrane. A collection of 51 target strains (18 species) and 71 nontarget strains (62 species) were analyzed by the array. Both the sensitivity (51/51 strains) and specificity (71/71 strains) of the array were 100%. The detection limit of the array was 10 pg (L. micdadei ATCC 33218T, L. pneumophila ATCC 33152T) of genomic DNA per assay. The array was also used to directly detect Legionella spp. in water samples, and a detection rate of 12.0% (6/50) was obtained. The array method is reliable and can be used as an accurate alternative to conventional or other molecular methods for identification of Legionella spp..
英文關鍵字	Macrophage infectivity potentiator;oligonucleotide array;Legionella spp.

摘要

Legionella species, which are fastidious and ubiquitous worldwide in natural water environment such as rivers, lakes and artificial water systems, are the causative agents of Legionnaire’s disease. Legionella pneumophila is the most important human pathogen, followed by L. micdadei, L. longbeachae, L. dumoffii, and other rare species. Accurate identification of Legionella spp. other than L. pneumophila is difficult because of biochemical inertness and phenotypic identity of different species. Clinical manifestations caused by Legionella infection are usually indistinguishable from pneumonia caused by other bacterial pathogens. Since the symptoms are nontypical, it is difficult clinically to identify the actual causative agent. Therefore, the rapid and accurate identification of Legionella species have been of increasingly important. DNA microarray technology displays a high potential in the testing and analysis of environmental samples. The feasibility of using an oligonucleotide array for identification of 18 species of Legionella was evaluated in this study. The method consisted of PCR amplification of the macrophage infectivity potentiator (mip) gene, followed by hybridization of the digoxigenin-labeled PCR products to a panel of 37 oligonucleotide probes (16-27 nucleotides) immobilized on a nylon membrane. A collection of 51 target strains (18 species) and 71 nontarget strains (62 species) were analyzed by the array. Both the sensitivity (51/51 strains) and specificity (71/71 strains) of the array were 100%. The detection limit of the array was 10 pg (L. micdadei ATCC 33218T, L. pneumophila ATCC 33152T) of genomic DNA per assay. The array was also used to directly detect Legionella spp. in water samples, and a detection rate of 12.0% (6/50) was obtained. The array method is reliable and can be used as an accurate alternative to conventional or other molecular methods for identification of Legionella spp..

英文關鍵字

Macrophage infectivity potentiator;oligonucleotide array;Legionella spp.