建立分子生物診斷法檢測實驗動物囓齒類毛蟎並與傳統診斷法比較

Development of a Molecular Assay for Laboratory Rodent Fur Mite Diagnosis and Comparison with the Traditional Diagnostic Methods

周知林(Chih-Lin Chou)

指導教授 ： 萬灼華

國立臺灣大學/獸醫專業學院/分子暨比較病理生物學研究所/碩士(2019年)

https://doi.org/10.6342/NTU201903951

摘要

實驗鼠感染外寄生蟲毛蟎(fur mite)是實驗動物中心常見且難徹底解決的問題之一，乃主要受限於外寄生蟲診斷方法敏感度較差、檢測樣本不易具有代表性及常用的衛兵鼠健康監測系統並不適用於監控實驗鼠的外寄生蟲感染情形。為了提升毛蟎檢測的敏感性與效率，本研究建立了一個多重引子聚合酶鏈鎖反應檢測法(multiplex PCR)，此檢測法能同時偵測並區別不同種類毛蟎，包括Myocoptes musculinus (COP)、Myobia musculi (MOB)/Radfordia spp. (RAD)以及於臺灣發現的新種毛蟎(species A；SPA)，更可藉由同時檢測囓齒動物管家基因(housekeeping gene)以確定檢體的品質。當多種毛蟎等量感染時，此檢測法能同時偵測出僅10 copies的各種毛蟎。而當同時有多種不等量的毛蟎感染時，此檢測法仍能偵測到感染量相差10至100倍的不同毛蟎感染。為了比較此多重引子聚合酶鏈鎖反應與多種傳統診斷法(拔毛測試、膠帶測試、毛皮檢查)，本研究分別以不同診斷法來檢測48隻囓齒動物與25個鼠籠的毛蟎汙染情形。於診斷個體動物毛蟎感染情形，此多重引子聚合酶鏈鎖反應檢測法的敏感度與準確度(86 %與95.1 %)明顯高於所有傳統診斷方法(敏感度：6 % - 46 %，準確度： 67.4 % - 81.3 %)。更值得一提的是，當應用於檢測鼠籠擦拭樣本(環境樣本)時，此毛蟎多重引子聚合酶鏈鎖反應法可完全正確地區別檢測出每個鼠籠的不同毛蟎的汙染情形，其敏感度與準確度均達100 %。本研究所建立的毛蟎多重引子聚合酶鏈鎖反應檢測法(COP/MOB-RAD/SPA/Actin multiplex PCR assay)是一可靠的替代診斷方法，能應用於實驗動物中心的例行性病原監測(不論動物或環境樣本)，更可用於實驗動物中心疑似毛蟎感染的追蹤調查。

關鍵字

囓齒類毛蟎 ； Myocoptes musculinus ； Myobia musculi ； Radfordia spp. ； 臺灣新種囓齒類毛蟎 ； 多重引子聚合酶鏈鎖反應 ； 環境監測

並列摘要

Rodent fur mite infestation is a persistent and intractable problem in laboratory rodent colonies, due to insensitive diagnostics, unrepresentative samples for testing, and improper sentinel system. To improve the sensitivity and efficiency of fur mite detection, a multiplex PCR assay was developed to simultaneously detect and differentiate different species of fur mites, including Myocoptes musculinus (COP), Myobia musculi (MOB) and/or Radfordia spp. (RAD), and species A (SPA; a novel rodent fur mite identified in Taiwan), with the existence of a rodent housekeeping gene. This multiplex PCR could specifically detect as low as 10 copies of each species in equal-amount triple infestation. Super-infestation with 10 to 100-fold differences in mite burdens could be also detected. In comparison of the multiple PCR and traditional methods (pluck test, tape test, and pelt exam) for fur mite diagnosis, 48 rodents and 25 cage environment samples were evaluated for the fur mite infestation. In screening the status of various fur mites on individual animals, the multiplex PCR assay showed distinctly higher in sensitivity and accuracy (86 % and 95.1 %) than that of traditional methods (sensitivity: 6 % - 46 %, accuracy: 67.4 % - 81.3 %). Interestingly, by using cage wipe environmental samples, the multiplex PCR assay exhibited 100 % in both sensitivity and accuracy on the fur mite detection and differentiation. The COP/MOB-RAD/SPA/Actin multiplex PCR assay developed in this study could be a reliable alternative method for routine pathogen monitoring (animal or environment) or for tracing the suspect fur mite outbreak in rodent colonies.

並列關鍵字

rodent fur mite ； Myocoptes musculinus ； Myobia musculi ； Radfordia spp. ； novel rodent fur mite in Taiwan ； multiplex PCR ； environmental monitoring

參考文獻

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