可攜帶型隔絕式恆溫聚合酶連鎖反應檢測鯨豚弓蟲感染之研發

Development of a field-deployable insulated isothermal PCR (iiPCR) for the detection of Toxoplasma gondii in cetaceans

謝孟容(Meng-Jung Hsieh)

指導教授 ： 楊瑋誠

國立臺灣大學/獸醫專業學院/獸醫學研究所/碩士(2021年)

https://doi.org/10.6342/NTU202104557

摘要

弓蟲病是一個重要的人畜共通傳染病，不管是在獸醫學以及公共衛生上都十分重視。在鯨豚身上發現陸地上的弓蟲病，可以視為是一個從陸地到海洋的病源污染。目前，弓蟲診斷需要在專業的實驗室裡進行，因此弓蟲的病例報告主要來自某些特定的國家。此外，當擱淺事件發生在含有許多島嶼的熱帶國家，並不是每個小島都有後送的實驗室。由於交通運輸不便，運送樣本時間過長，加上天氣炎熱，以至於擱淺動物樣本難以良好保存，使得DNA容易降解讓疾病監測更加困難。為了監測全球的擱淺鯨豚的弓蟲感染情形，我們研發可攜帶性之隔絕式恆溫PCR (insulated isothermal PCR, iiPCR)，搭配小型自動磁珠萃取，達到野外檢測之目的。我們使用B1基因當作檢測弓蟲的目標基因，同時使用鯨豚的β2-microglobulin (B2M)基因當作內部控制。將弓蟲質體加入鯨豚大腦、小腦、心肌、肌肉模擬臨床樣本，進行靈敏度分析。結果顯示B1/B2M (B1 gene/ β2-microglobulin) iiPCR在25 mg的組織下，只要21~86個緩殖體就可以檢測出陽性，B1/B2M iiPCR與qPCR有幾乎完美一致性(92%; 95% CI: 0.78–0.90; κ=0.84)。雙重B1/B2M iiPCR檢測平台不只方便攜帶去野外檢測，且敏感性高也更快速得到檢驗結果，適合運用在擱淺鯨豚之弓蟲檢驗。此平台提供一個有效方法，以鯨豚作為哨兵動物，評估並監測目前海洋環境生態的健康。

關鍵字

可攜帶式 ； 隔絕式恆溫聚合酶連鎖反應 ； 鯨豚 ； 弓蟲 ； 污染

並列摘要

Toxoplasmosis is a zoonotic disease with veterinary and public health importance worldwide. Toxoplasma gondii infection in cetaceans is an indicator of land-to-sea oocyst pollution. However, there is a critical knowledge gap within the distribution of the T. gondii infection in cetaceans. For facilitating the global surveillance of this important zoonotic pathogen, we developed a field-deployable duplex insulated isothermal PCR (iiPCR) with automated magnetic bead-based DNA extraction for the on-site detection of T. gondii in stranded cetaceans. It targets the B1 gene of T. gondii combined with β2-microglobulin (B2M) gene of cetaceans as an internal control. Compared with conventional qPCR assay, B1/B2M duplex iiPCR assay showed comparable sensitivity (21~86 bradyzoites in 25 mg of tissue) to detect spike-in standard of T. gondii DNA in cerebrum, cerebellum, skeletal muscle and myocardium tissues. Moreover, the overall agreement between the duplex iiPCR and qPCR was almost perfect agreement (92%; 95% CI: 0.78–0.90; κ=0.84) in detecting synthetic spike-in standards. The B1/B2M iiPCR assay coupled with a field-deployable system provides a prompt (~1.5 h), feasible, highly sensitive and specific on-site diagnostic tool for T. gondii in stranded cetaceans. This platform provides one approach to evaluating aquatic ecosystem health and early warnings about negative impacts on humans and marine animals.

並列關鍵字

insulated isothermal PCR ； cetaceans ； Toxoplasma ； pollution ； field-deployed

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