台灣地區反芻獸Q熱回溯性調查及螢光即時環形核酸增幅檢測法之建立與應用

Abstract

Q熱是由病原Coxiella burnetii所引起的重要人畜共通傳染病。台灣自1993年首次有人感染Q熱的病例報告，且對南台灣居民的血清學調查結果顯示盛行率約是4.2%，顯示台灣地區確實有Q熱病原存在。動物的感染情形於2006年初首次病例報告之後，實際發生情況是否因症狀不明顯而被低估有待較完整的流行病學調查與探討。 本研究即利用巢式聚合酶鏈反應及酵素結合免疫吸附反應進行台灣地區反芻獸Q熱回溯性之調查，方法為收集1989到2000年之羊隻與牛隻血清樣本共1279個進行檢測中，結果羊隻與牛隻之抗體陽性率分別為19% (173/900)及12% (44/379)；此外以巢式聚合酶鏈反應檢測2002到2005年間草食動物流產病例檢體，結果羊流產病例Q熱陽性率為63％ (15/24)，牛流產病例Q熱陽性率為0％ (0/26)。結果顯示至少自1989年起，Q熱就發生於台灣畜牧產業中，且穩定地存在於環境之中，特別是在羊隻牧場， Q熱已經成為羊隻流產的主要原因之一以及造成經濟上的損失。而檢測2008年採集之全臺灣各縣市的3588個牛羊血清樣本中，羊隻抗體陽性率為18 % (384/2142)，顯著高於牛隻的9 % (125/1446) (P<0.0001)；分析各縣市羊隻抗體陽性率與人病例發生率，均與羊隻牧場平均飼養數量相關，並與2008年1月到5月份累計降雨量與降雨日數成負相關。顯示出在台灣的Q熱受到羊隻的飼養情形與氣候的影響較大。 Q 熱為重要的人畜共通傳染病，因此建立快速的篩檢方式以提高檢測效益，將有助於Q 熱的診斷與疫情調查。近年來大量應用於疾病檢測的環形核酸增幅法，有特異性高、反應效率佳、反應時間短以及恆溫進行等優點，更可以直接觀察濁度的變化，得知是否有核酸增幅反應。反應之前添加鈣黃綠素(calcein)的環形核酸增幅法，應用範圍更廣，不僅容易肉眼辨識，目前應用於即時螢光基因定量儀敏感度可達5個板模/反應，更可以達到即時監測的功能，亦能提升昂貴儀器之使用率。

Q fever is an important zoonotic disease caused by Coxiella burnetii. In Taiwan, the first human case of Q fever was reported in 1993. A seroprevalence investigation in inhabitants in southern Taiwan showed 4.2% seropositive rate in 2000. These data showed that the pathogen of Q fever existed in Taiwan indeed. Although the first animal case was reported in early 2006, epidemic situation may be underestimated in Taiwan owing to inapparent syndrome. By using ELISA, total of 1279 ruminant serum samples that were collected from 1989 to 2000 in Taiwan were tested for the antibodies against Q fever. The seropositive rate of goats and bovine were 19% (173/900) and 12% (44/379), respectively. Samples from abortive cases of goats and bovine were collected during 2002 to 2005 and tested by nested polymerase chain reaction (nested-PCR). The results showed 63% (15/24) of the cases were positive. The results indicate that Q fever was already in Taiwan at least since 1989, and steadily existed till now. Q fever becomes one of the major causes of the abortion in ruminant and causes economic loss in Taiwan, especially in goat farms. Additional 3588 serum collected in 2008 were also tested by ELISA, the seropositive rate in goats was 18 % (384/2142) and was significant higher than the rate of bovine which was 9 % (125/1446) (P<0.0001). Both the seroprevalence of goat and the incidence of human case are highly correlative to the average number of goat per farm and inversely proportional to the accumulative rainfall and raining days during January to May in 2008. The relationship shows that Q fever in Taiwan is strongly affected by the raising of goats and condition of weather. Q fever is an important zoonosis in the world. Developing fast screening method would be helpful for diagnosis of Q fever and investigation of epidemiology. The loop-mediated isothermal amplification method (LAMP) is extensively applied for disease diagnosis. It uses 4 primers to recognize 6 positions on the target sequence and has the ability of amplifying a few copies of DNA to large amount of copies within an hour under isothermal condition. The byproduct of reaction reacts with magnesium ion causing the change of turbidity for visual identifying. By combining the simple, rapid and specific LAMP method and fluorescent metal indicator (calcein) detection technique enable presentation of remarkably clear results. When the modified LAMP method was applied to the detection of C. burnetii on real-time quantitative machine, the limitation is 5 copies per reaction with the function of real-time detection. Indirectly, it also creates new usage of expensive instrument.