寄生蟲種類之分子鑑定

臺灣的衛生條件進步之後，寄生蟲病例已較少見，不過還是有零星病例發生，外勞的飲食習慣、進口食材及國際旅遊頻繁等都是可能產生這些零星病例的原因之一。近幾年來本實驗室收到魚肉絛蟲（Diphyllobothrium）節片五例、貓糞便中Spirometra之蟲卵及Taenia節片各一例、石臘包埋之鉤蟲（hookworm）及口腔滴蟲（Trichomonas）培養液各兩例。由於種間形態特徵太接近而容易誤判、僅蟲卵無成蟲期或檢體重要部位殘缺無法鑑定等因素，我們決定利用分子生物學技術來鑑定所得檢體明確的蟲種。鑑定方法主要有蟲種專一性聚合酶連鎖反應（species-specific PCR）、multiplex PCR、序列排比（sequence alignment）及演化樹分析（phylogenetic analysis），利用的基因有核糖體核糖核酸（rRNA）、傳送核糖核酸（tRNA）、cytochrome c oxidase（cox）及NADH dehydrogenase（ND）這些在細胞內重複數多的基因作為標的或參考基因。五例魚肉絛蟲中確認一例為廣節裂頭絛蟲（Diphyllobothrium latum）、兩例為日本海裂頭絛蟲（D. nihokaiense）；貓糞中Spirometra蟲卵為猬迭宮絛蟲（Spirometra erinacei），又名曼氏裂頭絛蟲（S. mansoni）；一例Taenia為亞洲絛蟲（Taenia asiatica）；兩例鉤蟲均為錫蘭鉤蟲（Ancylostoma ceylanicum）；口腔癌病人口腔之primary cell culture裡之Trichomonas為口腔毛滴蟲（Trichomonas tenax）。自兩件經福馬林固定甚至已用卡紅染色封片之魚肉絛蟲樣本萃取之DNA，經PCR反應無法偵測到產物，可能原因是樣本受福馬林影響導致DNA萃取效率降低以及DNA已片段化（fragmentation）。故欲利用分生技術鑑定蟲種仍以文獻建議之冷凍或酒精保存者為佳。

關鍵字

寄生蟲；分生鑑定；聚合酶連鎖反應；絛蟲；鉤蟲；毛滴蟲

並列摘要

With the advancement in public hygiene, reports on cases of parasitic disease become less frequently seen in Taiwan. Nonetheless, sporadic cases still emerge from time to time. Immigrants’ dietary habits, imported food items as well as frequent international traveling may be the reasons why parasitic diseases still occur. We came across with five specimens of the fish tapeworm, one specimen each of Spirometra eggs in stool and Taenia segment, and two specimens each of hookworm embedded in paraffin block and primary tissue culture of oral trichomonad in recent years. Due to factors such as interspecies overlaping in morphologic features, only parasite eggs without adult stage is available and only portion of the parasite without morphologic characteristics for species identification, we decided to utilize molecular techniques for species identification. Methods employed included species-specific PCR, multiplex PCR, sequence alignment and phylogenetic analysis. Ribosomal RNA (rRNA), transfer RNA (tRNA), cytochrome c oxidase (cox) and NADH dehydrogenase (ND) genes that are highly redundant in copy numbers in a single cell were used as target or reference gene. Among the five fish tapeworm specimens, we identified one as Diphyllobothrium latum and two as D. nihonkaiense. We also identified the Spirometra eggs as S. erinacei, the Taenia segment as T. asiatica, the two paraffin embedded hookworms as Ancylostoma ceylanicum and the oral trichomonads as Trichomonas tenax. The remaining two fish tapeworm specimens that came fixed in formalin or even processed to become carmine stained whole mount specimen failed to yield detectable PCR product after DNA extraction. Formalin fixation may have lowered the efficiency of DNA extraction or caused DNA fragmentation. Thus it is better to use frozen or ethanol fixed specimens for molecular identification as suggested in the literatures.

並列關鍵字

parasites ； molecular identification ； PCR ； tapeworms ； hookworms ； trichomonads

參考文獻

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國際替代計量

寄生蟲種類之分子鑑定

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