應用單一步驟免疫層析法開發牛乳房炎快速檢測試劑

Abstract

牛乳房炎肇因於乳牛的乳腺組織受到細菌如大腸桿菌或金黃色葡萄球菌等的感染而造成的發炎反應。乳房炎很容易因為共用榨乳機器而傳染給牧場中其他的牛隻，進而影響牛乳的品質及產量，對於酪農業有很大的影響。由於在發炎前期其症狀輕微因此很容易被酪農戶所忽略。根據 International Dairy Federation (IDF) 所頒布的標準檢測法，乳房炎可藉由細菌培養判定病原菌或測量牛奶中體細胞數的高低來判定感染與否，但是這兩種方法皆相當耗時且需要精密儀器及經專業訓練的人員才能進行，不適用於例行檢測。 文獻發現在乳牛受到感染或發炎時牛奶中的體細胞數會大量增加且九成以上都屬嗜中性球 (neutrophil)。近年來本實驗室發現 Haptoglobin (Hp) 是由嗜中性球 (neutrophil) 所分泌至牛奶中，在乳房炎牛奶中含量是健康牛奶的十倍以上，因此適合做為監測指標。 本研究開發出一種簡單、快速且適用於田間試驗的牛乳房炎快速檢測試劑，可以在2至4分鐘內半定量 (semi-quantitatively) 牛奶中的 Hp ，相同原理並可以應用至其他臨床檢測。 在48個臨床牛奶樣品中，快速檢測的呈色強度與 Hp (r = 0.79; p < 0.001) 及 SCC (r = 0.74; p < 0.001) 的都有顯著的相關性。本方法在檢測牛乳房炎的敏感性及特異性分別為 87.5% 及 96.2%。本檢測試劑提供了一個簡單快速的檢測方法檢測牛乳房炎，即便沒有實驗儀器或未受過專業訓練的酪農戶也可以在牧場操作。

Mastitis is an infectious disease that may spread over a dairy herd and severely affect the production and quality of milk. Because the symptom in cows with subclinical mastitis is subtle, it is often ignored leaving cows without treatment. Bacteriological sampling and milk somatic cell count (SCC) has been recommended by the International Dairy Federation (IDF) to be a standard procedure to diagnose mastitis but not practical as a routine test. Recently, we have found that bovine haptoglobin (Hp) is drastically expressed in the milk of cows with clinical mastitis due to a sharp increase in neutrophils over the somatic cells. The major purpose of this report was to seek an alternative that may be used in local farms without sophisticated equipment. Here, we developed a novel “mini” colorimetric strip test that not only can semi-quantitatively determine milk Hp within 2 to 4 min, but also can apply it for other clinical diagnosis. The chromogeneity of the test correlated significantly with Hp levels (r = 0.79; p < 0.001) and SCC (r = 0.74; p < 0.001) from a total of 48 cows. It visually detected the mastitic samples with a specificity and sensitivity of 96.2% and 87.5%, respectively. This novel strip test may provide a reference value for future rapid diagnosis over the field and offer an aid when SCC is not readily available. We also give a detailed description of the methods involved in this technical platform in this report.