CN110806478A - 艰难梭菌检测试剂盒 - Google Patents

艰难梭菌检测试剂盒 Download PDF

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CN110806478A
CN110806478A CN201911160862.XA CN201911160862A CN110806478A CN 110806478 A CN110806478 A CN 110806478A CN 201911160862 A CN201911160862 A CN 201911160862A CN 110806478 A CN110806478 A CN 110806478A
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exotoxin
clostridium difficile
igm
glutamate dehydrogenase
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李立和
张晨磊
白霞
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Tianjin Baodi Hospital
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Abstract

本发明公开了一种艰难梭菌检测试剂盒，属于免疫学技术领域，本发明的技术方案是：以艰难梭菌菌体谷氨酸脱氢酶、外毒素A和B三种靶抗原制成生物薄片，加入稀释后血清，再加入和人血清一抗IgM靶抗原对应的生物素化二抗IgM，异硫氰酸荧光素标记的链霉亲和素，荧光显微镜观察；异硫氰酸荧光素在490‑495nm激发，在520‑530nm发射荧光，呈黄绿色，检测有谷氨酸脱氢酶、外毒素A和外毒素B三种靶抗原对应抗体存在，说明检测有艰难梭菌感染。

Description

艰难梭菌检测试剂盒
技术领域
本发明涉及一种艰难梭菌检测试剂盒，或是利用可见光，通过测试反应的结果产生颜色变化来测试材料的方法，特别是涉及一种应用免疫技术检测艰难梭菌的试剂盒。
背景技术
艰难梭菌(Clostridium difficile)是梭菌属的一种专性厌氧菌，对氧十分敏感，很难分离培养，故得名。粗长杆菌，大小(1.3～1.6)μm*(3.6～6.4)μm，能运动或不能运动，运动性菌株为周毛菌，芽胞为卵圆形，位于菌体次极端，部分菌株有周鞭毛，近年来证实该菌有荚膜，革兰染色阳性，但培养2天后有转为革兰阴性的趋向，本菌为严格的专性厌氧菌，用常规的厌氧培养法不易生长，生长温度为25℃～45℃，而最适温度为30℃～37℃，在血琼脂、牛心脑浸液琼脂及CCFA等平板，经48小时培养后，菌落的直径3～5mm，圆形，略凸起，白色或淡黄色、不透明、边缘不整齐、表面粗糙。在血平板上不溶血，在卵黄琼脂平板上不形成乳浊环，CCFA平板上生长的菌落在紫外线照射下可见黄绿色荧光，本菌经肉汤培养2天以上，菌体有溶融现象，艰难梭菌是由于服用抗生素后，打破了肠内菌群的平衡，从而导致艰难梭菌累计了大量的数量，产生大量的外毒素A和B，外毒素A由肠道毒素和细胞毒素组成，外毒素可绑定黏膜细胞从而导致出血，而外毒素B是一种细胞毒素，在体内外毒素B不能与黏膜细胞直接绑定，因为外毒素B只能与破坏的细胞绑定，导致更大的破坏，大多数艰难梭菌种属都产生这两种毒素，从而在不同时期产生不同的作用，在初期外毒素A首先与黏膜细胞绑定，造成初级的破坏，然后外毒素B进而发挥更大的破坏。
发明内容：针对艰难梭菌的细胞壁具有谷氨酸脱氢酶（GDH）和产生外毒素A和B的抗原特性，本发明建立了以谷氨酸脱氢酶（GDH）和产生外毒素A和B三种靶抗原的生物素荧光法检测试剂盒。
本发明的技术方案是：以艰难梭菌菌体谷氨酸脱氢酶（GDH）和产生外毒素A和B三种靶抗原制成生物薄片，加入稀释后血清，再加入和人血清一抗IgM
抗原对应的生物素化二抗IgM，异硫氰酸荧光素标记的链霉亲和素（SABC-FITC），荧光显微镜观察；异硫氰酸荧光素（FITC）在490-495nm激发，在520-530nm发射荧光，呈黄绿色，说明检测有谷氨酸脱氢酶（GDH）、外毒素A和外毒素B三种靶抗原对应抗体存在，有艰难梭菌感染。
附图说明:
图1．本发明试剂盒结构示意图
图2. 本发明生物薄片示意图
图3．本发明荧光反应模式示意图
具体实施方式：
下面通过实施例对本发明试剂盒使用方法做进一步详细说明。
实施例1
试剂盒组成：
本发明的试剂盒包括盒体（1）与盒盖（2），其特征在于：盒体（1）一侧长边内形成有插孔（3）的试剂架（4）上，插孔（3）分别容纳生物素化羊抗人谷氨酸脱氢酶抗体IgM（5），生物素化羊抗人外毒素A抗体IgM（6），生物素化羊抗人外毒素B抗体IgM（7），异硫氰酸荧光素标记的链霉亲和素（8），封片剂（9），盒底的中间设有一个将盒底分成放置缓冲液试剂（10）与放置生物薄片（11）两部分的纸质挡片（12），生物载片（11）外面塑料包装（13）。
图中：1.盒体 2.盒盖
3.插孔 4.试剂架
5.生物素化羊抗人谷氨酸脱氢酶抗体IgM
6.生物素化羊抗人外毒素A抗体IgM
7.生物素化羊抗人外毒素B抗体IgM
8.异硫氰酸荧光素标记的链霉亲和素
9.封片剂 10.缓冲液试剂
11.生物薄片
11a.谷氨酸脱氢酶抗体IgM反应区
11b.外毒素A抗体IgM反应区
11c.外毒素B抗体IgM反应区
12.纸质挡片 13.塑料包装
实施例2
生物薄片的制作:艰难梭菌经培养液增菌后加入10mlpH7.4 PBS缓冲液中,800G离心5min,弃去上清液,再洗一次,继续用10ml PBS-T缓冲液0.5%(V/V)TritonX-100PBS混悬,静置10min,800G离心5min,用PBS调整细菌数至100个/μl。点片到含有多聚赖氨酸玻片上,每张生物薄片制成3个区，冷风吹干后,密封,-70℃低温保存。
实施例3
试剂盒的使用方法:生物薄片放置室温30分钟后，在生物薄片上分别加入1:100倍稀释的血清，30分钟后，0.01MPBS洗2分钟3次，分别滴加0.01M PBS 1：10稀释的生物素化抗谷氨酸脱氢酶IgM、生物素化羊抗人外毒素A抗体IgM、生物素化羊抗人外毒素B抗体IgM，37℃1小时后，0.01MPBS洗2分钟3次；滴加0.01MPBS1:100稀释的SABC-FITC，37℃ 60分钟，0.01MPBS洗2分钟4次；封片剂封片，荧光显微镜观察；FITC在490-495nm激发，在520-530nm发射荧光，呈黄绿色。
在本发明中，如果在荧光显微镜下在生物薄片11a、11b、11c区上艰难梭菌周围出现黄绿色荧光，证明有谷氨酸脱氢酶（GDH）、外毒素A和外毒素B三种靶抗原对应抗体存在，结合艰难梭菌革兰氏染色特征，即可检测出艰难梭菌感染，本发明试剂盒具有良好的社会和经济效益。

Claims (5)

1.一种艰难梭菌检测试剂盒，其特征在于以艰难梭菌菌体谷氨酸脱氢酶、外毒素A和B三种靶抗原制成生物薄片，加入稀释后血清，再加入和人血清一抗IgM对应的生物素化二抗IgM，异硫氰酸荧光素标记的链霉亲和素，荧光显微镜观察；异硫氰酸荧光素在490-495nm激发，在520-530nm发射荧光，呈黄绿色，检测有谷氨酸脱氢酶、外毒素A和外毒素B三种靶抗原对应抗体存在，说明检测有艰难梭菌。
2.根据权利要求1所述的一种艰难梭菌检测试剂盒，其特征在于生物薄片上有3个反应区，按一定间距形成10×10mm的3个方形反应凹槽,凹槽内包被有艰难梭菌，凹槽内容体积为25μl，并分别形成对应的谷氨酸脱氢酶抗体IgM反应区、外毒素A抗体IgM反应区、外毒素B抗体IgM反应区。
3.根据权利要求1所述的一种艰难梭菌检测试剂盒，其特征在于生物素化二抗IgM分别为生物素化羊抗人谷氨酸脱氢酶抗体IgM、生物素化羊抗人外毒素A抗体IgM、生物素化羊抗人外毒素B抗体IgM。
4.根据权利要求1所述的一种艰难梭菌检测试剂盒，其特征在于标记物为异硫氰酸荧光素标记的链霉亲和素。
5.根据权利要求1所述的一种艰难梭菌检测试剂盒，其特征在于检测物质为人体内血液中谷氨酸脱氢酶、外毒素A和外毒素B三种靶抗原对应的抗体。
CN201911160862.XA 2019-11-24 2019-11-24 艰难梭菌检测试剂盒 Pending CN110806478A (zh)

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