CN110411807A - 一种幽门螺杆菌荧光染色试剂 - Google Patents
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Abstract
本发明公开了一种幽门螺杆菌荧光染色试剂，属于医药检测试剂领域，按质量计包括有吖啶橙0.5‑2份，醋酸0.5‑1.5份，醋酸钠1‑5份，氯化钙1‑5份，甘油1‑3份，复染剂1‑5份。本发明通过吖啶橙荧光标记，可以将幽门螺杆菌染出橙红色，在荧光显微镜下清晰可见，菌体和背景对比度高，敏感度显著高于传统染色，一步染色，1分钟左右出结果，快速高效；且醋酸和醋酸钠的添加能够使吖啶橙与细胞DNA和RNA结合能力强，染色效果佳；复染剂，具有压制背景的作用，减少背景杂光。本发明染色试剂更加稳定，显色对照鲜明，更利于观察。
Description
技术领域
本发明涉及医药领域，具体涉及一种应用于胃黏膜石蜡切片幽门螺杆菌荧光染色试剂。
背景技术
幽门螺杆菌病是一种螺旋形、微厌氧、对生长条件要求十分苛刻的细菌。1983年首次从慢性活动性胃炎患者的胃黏膜活检组织中分离成功，是目前所知能够在人胃中生存的唯一微生物种类。2017年10月27日，世界卫生组织国际癌症研究机构公布的致癌物清单初步整理参考，幽门螺杆菌(感染)在一类致癌物清单中。
研究指出，幽门螺杆菌生存于人体胃幽门部位，是最常见的细菌病原体之一。世界有多半人口受到过幽门螺杆菌的感染，而在有些国家几乎90％的人都感染过这种细菌。人们通常是在幼年时就受到感染，5岁以下达到50％。这种细菌感染首先引起慢性胃炎，并导致胃溃疡和胃萎缩，严重者则发展为胃癌。据统计，初次感染幽门螺杆菌年龄较早的人群萎缩性胃炎及胃癌发生率高，幽门螺杆菌感染与胃癌死亡率的高低呈现平行关系。幽门螺杆菌寄生在胃黏膜组织中，67％～80％的胃溃疡和95％的十二指肠溃疡是由幽门螺杆菌引起的。慢性胃炎和消化道溃疡患者的普遍症状为：食后上腹部饱胀、不适或疼痛，常伴有其他不良症状，如嗳气、腹胀、反酸和食欲减退等。有些患者还可出现反复发作性剧烈腹痛、上消化道少量出血等。及早发现幽门螺杆菌感染者，及时而有效地用抗菌素杀灭幽门螺杆菌，对预防和控制胃癌有重大意义。
根据是否需进行胃镜检查，幽门螺杆菌(Helicobacter pylori)的诊断性检测可分为侵入性和非侵入性两类。非侵入性检测包括尿素呼气试验(urea breath testing,UBT)、大便抗原检测及血清学检测。而侵入性检测包括活检尿素酶检测、组织学检测、细菌培养。而在这些方法里，以病理科组织学检测为金标准方法。组织学检测通常为胃黏膜活检组织通过石蜡包埋处理，切片后进行染色观察，染色方法包括常规HE染色以及吉姆萨染色，银染等特殊染色。但这些染色方法缺点明显，主要为操作繁琐，都为多步操作，检测时间长；非特异性染色，对幽门螺杆菌染色敏感度差，背景着色后反差小，菌体不易发现，当菌量很少时，易造成漏诊。
发明内容
为了解决上述问题，本发明提出了一种一步染色，染色时间快，且分辨对比明显，显像效果佳的幽门螺杆菌荧光染色试剂。
为了实现上述目的，本发明是通过以下技术方案来实现的：
一种幽门螺杆菌荧光染色试剂，包括以下组分，按质量分为：吖啶橙0.5-2份，醋酸0.5-1.5份，醋酸钠1-5份，氯化钙1-5份，甘油1-3份，复染剂1-5份。
优选的，所述幽门螺杆菌荧光染色试剂包括以下组分，按质量分为：包括以下组分，按质量分为：吖啶橙0.8-1.2份，醋酸0.8-1.2份，醋酸钠2-3份，氯化钙3-5份，甘油1-1.5份，复染剂1-3份。
优选的，所述幽门螺杆菌荧光染色试剂包括以下组分，按质量分为：包括以下组分，按质量分为：吖啶橙1份，醋酸1.05份，醋酸钠2份，氯化钙3.3份，甘油1.26份，复染剂2份。
上述所述复染剂为伊文思兰，甲基蓝，孔雀绿，番红中的任意一种。
吖啶橙：3，6－(二甲胺基)吖啶盐酸盐，分子式C17H19N3·HCl·ZnCl2,分子量438.12g/mol,是一种荧光色素，其检测激发滤光片波长488nm,阻断滤光片波长515nm。它与细胞中DNA和RNA结合量存在差别，可发出不同颜色的荧光，与DNA结合量少发绿色荧光，与RNA结合量多发桔黄色或桔红色荧光。该染料具有膜通透性，能透过细胞膜，使核DNA和RNA染色。在荧光显微镜下吖啶橙能将幽门螺杆菌染成橙红色，而组织细胞为黄绿色，对比显著。
醋酸和醋酸钠：能维持溶液的酸性环境，使得pH在3.5-3.8之间，吖啶橙在这种酸性环境下与细胞DNA和RNA结合能力强，染色效果佳。
氯化钙：主要起分色作用，使染色后菌体和背景组织颜色区分更加明显。
甘油：具有保湿和浸润组织的作用，增加溶液整体保湿度以及折光度，增加浸润效果，减少染色时间。
复染剂：包括伊文思兰、甲基蓝、孔雀绿、番红中的任意一种，其作用为压制背景荧光，减少杂光，增加反差效果。
本发明幽门螺杆菌荧光染色试剂，其有益效果在于：
(1)传统HE染色以及特殊染色，如吉姆萨染色以及银染，操作繁琐，步骤繁多，且染色时间长，不能达到立刻出结果。本发明为单剂型荧光试剂，一步染色，1分钟左右出结果，快速高效，可用于快速筛查病理科石蜡组织幽门螺杆菌感染。与现有技术相比，具有意想不到的效果。
(2)本发明通过吖啶橙荧光标记，可以将幽门螺杆菌染出橙红色，在荧光显微镜下清晰可见，菌体和背景对比度高，敏感度显著高于传统染色，且醋酸和醋酸钠的添加能够使吖啶橙与细胞DNA和RNA结合能力强，染色效果佳；复染剂，具有压制背景的作用，减少背景杂光。本发明染色试剂更加稳定，显色对照鲜明，更利于观察。
附图说明
为了更清楚地说明本发明实施例或现有技术中的技术方案，下面将对实施例或现有技术描述中所需要使用的附图作简单地介绍，显而易见地，下面描述中的附图仅仅是本发明的一些实施例，对于本领域普通技术人员来讲，在不付出创造性劳动性的前提下，还可以根据这些附图获得其他的附图。
图1是采用传统HE染色的胃黏膜组织荧光染色图；
图2是采用本发明染色的胃黏膜组织荧光染色图。
具体实施方式
下面结合具体实施例及附图来进一步详细说明本发明。
实施例1
一种幽门螺杆菌荧光染色试剂，包括以下组分，吖啶橙0.5g，醋酸10ml，醋酸钠1g，氯化钙1g，甘油1ml，复染剂1g。
实施例2
一种幽门螺杆菌荧光染色试剂，包括以下组分，吖啶橙2g，醋酸10ml，醋酸钠1.5g，氯化钙5g，甘油10ml，复染剂5g。
实施例3
一种幽门螺杆菌荧光染色试剂，包括以下组分，吖啶橙1.2g，醋酸10ml，醋酸钠3g，氯化钙3g，甘油10ml，复染剂3g。
实施例4
一种幽门螺杆菌荧光染色试剂，包括以下组分，吖啶橙0.8g，醋酸10ml，醋酸钠2g，氯化钙3.5g，甘油10ml，复染剂2.5g。
实施例5
一种幽门螺杆菌荧光染色试剂，包括以下组分，吖啶橙0.1g，醋酸1ml，醋酸钠0.2g，氯化钙0.33g，甘油1ml，复染剂0.2g。
对照组：
采用感染幽门螺杆菌的胃粘膜组织，分别利用传统HE染色和本发明实施例5的荧光染色剂进行染色，得到的的显微图片分布分别如图1和图2。即图1为传统荧光染色剂染色，图2为本发明实施例5的荧光染色剂。从两组对照可以看出，本发明采用的荧光剂标注的幽门螺杆菌更加明显，菌体和背景对比度高，更观察显现。
以上对本发明实施例所提供的技术方案进行了详细介绍，本文中应用了具体个例对本发明实施例的原理以及实施方式进行了阐述，以上实施例的说明只适用于帮助理解本发明实施例的原理；同时，对于本领域的一般技术人员，依据本发明实施例，在具体实施方式以及应用范围上均会有改变之处，综上所述，本说明书内容不应理解为对本发明的限制。
Claims (4)
1.一种幽门螺杆菌荧光染色试剂，其特征在于：包括以下组分，按质量分为：吖啶橙0.5-2份，醋酸0.5-1.5份，醋酸钠1-5份，氯化钙1-5份，甘油1-3份，复染剂1-5份。
2.根据权利要求1所述幽门螺杆菌荧光染色试剂，其特征在于：包括以下组分，按质量分为：吖啶橙0.8-1.2份，醋酸0.8-1.2份，醋酸钠2-3份，氯化钙3-5份，甘油1-1.5份，复染剂1-3份。
3.根据权利要求1所述幽门螺杆菌荧光染色试剂，其特征在于：包括以下组分，按质量分为：吖啶橙1份，醋酸1.05份，醋酸钠2份，氯化钙3.3份，甘油1.26份，复染剂2份。
4.根据权利要求1-3任意项所述幽门螺杆菌荧光染色试剂，其特征在于：所述复染剂为伊文思兰，甲基蓝，孔雀绿，番红中的任意一种。
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| CN116067742A (zh) * | 2023-03-29 | 2023-05-05 | 北京汉氏联合生物技术股份有限公司 | CTCs染色试剂盒和染色方法 |
| CN116626302A (zh) * | 2023-03-13 | 2023-08-22 | 宁波国际旅行卫生保健中心(宁波海关口岸门诊部) | 一种用于骨肽干预治疗骨质疏松中的生物标志物 |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN116626302A (zh) * | 2023-03-13 | 2023-08-22 | 宁波国际旅行卫生保健中心(宁波海关口岸门诊部) | 一种用于骨肽干预治疗骨质疏松中的生物标志物 |
| CN116626302B (zh) * | 2023-03-13 | 2023-11-10 | 宁波国际旅行卫生保健中心(宁波海关口岸门诊部) | 一种用于骨肽干预治疗骨质疏松中的生物标志物 |
| CN116067742A (zh) * | 2023-03-29 | 2023-05-05 | 北京汉氏联合生物技术股份有限公司 | CTCs染色试剂盒和染色方法 |
| CN116067742B (zh) * | 2023-03-29 | 2023-09-01 | 北京汉氏联合生物技术股份有限公司 | CTCs染色试剂盒和染色方法 |
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