| 摘要: | 金黃色葡萄球菌(Staphylococcus aureus)與許多人類疾病相關，並且也是造成各國食物中毒的五大病原菌之一，導致人類各式各樣的毒性及化膿性感染，它會導致皮膚表面損傷如化膿、癤病等；更嚴重的情況會造成細菌性肺炎、乳腺炎、靜脈炎、腦膜炎、尿路感染等，還有慢性病如骨髓炎和心內膜炎等。S. aureus 是造成院內感染例如外科創傷和傳染、及存在醫療設備中的主因。S. aureus 會透過釋放產腸毒素入食物造成食物中毒，並且將超級抗原釋入血液中引起毒性休克症狀群。
金黃色葡萄球菌（Staphylococcus aureus）為葡萄球菌科(Staphylococcaceae) 葡萄球菌屬(Staphylococcus)的一員；為Gram（＋）球菌，直徑1μm，無鞭毛及運動性， S. aureus 菌型具莢膜，有助於抵抗白血球之呑噬。人和動物皮膚及與外界相通腔道也是本菌寄居之處，有些人皮膚和鼻咽部可帶致病性葡萄菌，是重要傳染源。
本研究的目的主要係利用檢測S. aureus 細胞壁上所特有的Protein A 作為此可拋棄式免疫層析試片(immunochromatographic strip ) 開發的主軸並於環境中之實際應用。Protein A 是一種存在於S. aureus 的高穩定性細胞表面受器，由一條分子量為42 kDa 的單一多肽鏈所組成，Protein A 能夠與鍵結在大多數種類的免疫球蛋白之Fc portion 結合，特別是IgGs。
本研究首先進行膠體金粒子( colloidal gold particles ) 之合成工作，因其為奈米級，故又稱為奈米金。並將Protein A 多株抗體與25 nm 膠體金結合，成功地開發出免疫層析試片。實驗結果顯示此自製免疫層析試片能分別於緩衝溶液、大腸桿菌(Escherichia coli）、沙門氏菌（Salmonella typhi)檢測，且無交叉反應出現，檢測所需時間約10-20 分鐘。本試片可有效應用於環境檢測包括：提款機、紅茶店飲料、電腦鍵盤、醫院病床、醫院置物櫃、用過的粉撲、量販店的手扶梯及飲水機，所採集樣品共計36 件；其中，所有檢測樣品中陽性反應以提款機比例較高。本研究所開發之試片預期可應用於食品衛生檢驗、居家照護( homecare )、護理站( point-of-care )及醫院院內感染之檢測，故極具應用價值。
本研究所製備之可拋棄式免疫層析試片，具有下列優點：1.樣品不需前處理；2.所需樣品量少；3.操作方便；4.檢測快速；5.低成本；6.有效檢測；7.不需要任何儀器之使用；8.沒有交叉反應；9.靈敏度高；10.可長期保存。
Staphylococcus aureus is Gram-positive spherical bacteria. It causes a variety of suppurative (pus-forming)infections and toxicity in human. It causes superficial skin lesions such as boils, styes and furunculosis; more seriousinfections such as pneumonia, mastitis, phlebitis, meningitis, and urin ary tractinfections; and deep- seated infections, such as osteomyelitis and endocarditis. S.aureus is a major cause of hospital acquired (nosocomial) infection of surgicalwounds and infections associated with indwelling medical devices. S.aureus causes food poisoning by releasing endotoxins into food, and toxic shoch syndrome by releasing superantigens into the blood stream.
Conventional methods for determining S. aureus were colony hybridization,preculture and selective medium and biotype test etc. However, these methods needed long procedure, specific machine for determination. The objective of this study was to develop a self-assembled immunochromatographic strip for rapid determination of S. aureus using rabbit anti-Protein A polyclonal antibodies(pAbs). Protein A is a surface protein of S. aureus. This self-assembled immunochromatographic strip was convenient, rapid, low price and no machine needed in clinical diagnosis. The colloidal gold nanoparticles were made from the chloroauric acid (HAuCl4) and anti-Protein A were labeled with 25 nm of colloidal gold.
This immunochromatographic strip prepared could determine for 0.01-100 μg/mL Protein A and no cross-reaction. Moreover, the detection limit IVof this strip for S. aureus samples was about 196cells/mL, and the detection time was 10-20 min.
This study was to develop a self-assembled immunochromatographic strip for determination of S. aureus in environments. The samples were collected from automatic teller machines (ATMs), tea drinks, computer keyboards,hospital beds, cabinets of hospital, used powder puffs, hand staircases with a handrail and water fountains. The total numbers of samples were 36. In which,the majority of positive reactions in all samples were obtained from the determination of ATMs.
The advantages of the self-assembled immunochromatographic strip were high specificity, high sensitivity, no pretreatment, low sample requirement, easy operation, rapid determination, low price, no cross-reaction, long-term preservation, no machine needed etc. |
