CN111289337A - 一种真菌荧光染色液及其制备方法 - Google Patents
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Abstract
本发明涉及一种真菌荧光染色液及其制备方法,由以下原料组成:5‑羟基荧光素、真菌引物ITS4、促溶剂、抗干扰荧光助剂肌醇六磷酸钾、稳定剂、渗透剂、背景染料、去离子水。本发明真菌荧光染色液的制备方法包括,将5‑羟基荧光素和真菌引物ITS4溶于去离子水中,搅拌均匀后,缓慢加入促溶剂,轻轻搅拌充分溶解,然后置于37℃恒温摇床上,摇匀后获得溶液A;将抗干扰荧光助剂、稳定剂、渗透剂以及背景染料溶液溶于剩余去离子水中,搅拌均匀后,获得溶液B;将溶液A缓慢倒入溶液B中,搅拌均匀后即得真菌荧光染色液。本发明制备的真菌荧光染色液不仅简便快速、灵敏性高、辨识度高,且解决了荧光染色液使肺孢子菌着色困难的问题。
Description
技术领域
本发明涉及一种真菌荧光染色液及其制备方法,属于医学临床检验中病原菌生物学检验技术领域。
背景技术
真菌广泛存在于自然界,而致病性真菌约有400余种。随着器官移植的大量开展及免疫抑制剂、肿瘤患者化疗药物、糖皮质类固醇激素的广泛使用,人类免疫缺陷病毒感染的高流行趋势、体内有创检查的大量开展,人口老龄化和糖尿病等慢性疾病患者的增多,真菌发病率和死亡率逐年增加,对人类健康的危害日趋严重。由于临床对很多真菌感染的微生物特性了解较少,加之真菌感染的临床表现多种多样,通常会出现误诊、漏诊,导致病情加重甚至危及生命。这就需要确实有效快速的早期诊断方法。
常见的真菌检测方法包括荧光染色、革兰氏染色、KOH湿片法、血清学方法、分子生物学法以及真菌培养,期中,荧光染色法是一种利用重组几丁质酶来检测人体各种标本中是否存在真菌有形成分的方法。真菌荧光染色液中,经过特殊荧光素标联的重组几丁质酶可以高亲和力与真菌细胞壁上的几丁质结合并发出荧光,在荧光显微镜下可以清楚的观察到真菌形态。荧光染色简便快速、灵敏性高、辨识度高,且适用范围广。
申请号为201710899393.8,发明名称为一种真菌荧光染色液及其制备方法中5-羧基荧光素的羧基与真菌核糖体核糖核酸间隔区引物4(ITS4)的核苷酸链两端的磷酸基团端氨基残基共价结合标记成荧光引物,在游离状态时该荧光引物形成茎环结构避免5-羧基荧光素衰退,在氢氧化钾溶液中快速进入真菌细胞与真菌核糖体核糖核酸基因间隔区(ITS)碱基发生特异性的结合,此时,茎环结构打开,露出5-羧基荧光素将真菌细胞标记荧光,另外,甘油、双磷脂酰甘油、抗坏血酸形成的抗荧光衰退保护层将露出5-羧基荧光素封闭式包裹阻止荧光衰退,使真菌样本能长期保存,用荧光显微镜在480nm波长下镜检观察背景为黑色,真菌发射出520nm波长的绿色荧光。
但实际检测过程中,荧光染色除了真菌外,动脉壁、肺泡和细支气管上的弹性纤维、外源性的植物纤维、脂肪滴、寄生虫等也可以发出亮蓝色的荧光,结合形态还是容易区分。造成假阴性的因素有未使用适合波长段的滤光片、标本图片太厚、染液量过少、染色时间过短、荧光猝灭物质存在等,现有荧光染色液使肺孢子菌着色困难。
发明内容
本发明针对上述问题,提供了一种真菌荧光染色液及其制备方法,本发明制备的荧光染色液不仅简便快速、灵敏性高、辨识度高,且解决了荧光染色液使肺孢子菌着色困难的问题。
本发明的技术方案如下:
一种真菌荧光染色液,由以下原料组成:
5-羟基荧光素、真菌引物ITS4、促溶剂、抗干扰荧光助剂肌醇六磷酸钾、稳定剂、渗透剂、背景染料、去离子水。
其中,促溶剂为氢氧化钠和氢氧化钾的混合物;稳定剂为山梨醇、渗透剂为二甲基乙酰胺;背景染料为染料溴百里酚蓝;优选的,促溶剂中氢氧化钠和氢氧化钾的重量比为1:1。
进一步的,本发明真菌荧光染色液,由以下重量份数的原料组成:
5-羟基荧光素0.005份、真菌引物ITS4 0.03份、促溶剂氢氧化钠和氢氧化钾按重量比为1:1的比例混合的混合物11份、抗干扰荧光助剂肌醇六磷酸钾1份、稳定剂山梨醇20份、渗透剂二甲基乙酰胺0.5份、背景染料溴百里酚蓝2份、去离子水60份。
本发明真菌荧光染色液的制备方法如下:
(1)将5-羟基荧光素和真菌引物ITS4溶于1/2去离子水中,搅拌均匀后,缓慢加入促溶剂,轻轻搅拌充分溶解,然后置于37℃恒温摇床上,摇床转速为80-110转/min,时间为50-65min,获得溶液A;优选的,摇床转速为100转/min,时间为60min;
(2)将抗干扰荧光助剂、稳定剂、渗透剂以及背景染料溶液溶于剩余去离子水中,搅拌均匀后,获得溶液B;
(3)将溶液A缓慢倒入溶液B中,搅拌均匀后即得真菌荧光染色液。本发明与现有技术相比具有以下优点:
(1)本发明制备的荧光染色液不仅简便快速、灵敏性高、辨识度高,且解决了荧光染色液使肺孢子菌着色困难的问题,如图1所示;
(2)本发明中抗干扰荧光助剂、稳定剂以及渗透剂抗荧光衰退保护层将露出5-羧基荧光素封闭式包裹阻止荧光衰退,使真菌样本能长期保存。
附图说明
图1为本发明实施例1制备的真菌荧光染色液对肺泡切片样品进行染色后,利用荧光显微镜观察结果图;
图2为本发明实施例2制备的真菌荧光染色液对皮屑样品进行染色后,利用荧光显微镜观察结果图;
图3为本发明实施例1制备的真菌荧光染色液对粘液样品进行染色后,利用荧光显微镜观察结果图。
具体实施方式
下面结合具体实施例来进一步描述本发明,本发明的优点和特点将会随着描述而更为清楚。但实施例仅是范例性的,并不对本发明的范围构成任何限制。本领域技术人员应该理解的是,在不偏离本发明的精神和范围下可以对本发明技术方案的细节和形式进行修改或替换,但这些修改和替换均落入本发明的保护范围内。
实例1:一种真菌荧光染色液及其制备方法
本实施例中真菌荧光染色液由以下重量份数的原料组成:
5-羟基荧光素0.005、真菌引物ITS4 0.03、促溶剂氢氧化钠和氢氧化钾按重量比为1:1的比例混合的混合物11、抗干扰荧光助剂肌醇六磷酸钾1、稳定剂山梨醇20、渗透剂二甲基乙酰胺0.5、背景染料溴百里酚蓝2、去离子水60。
制备方法如下:
(1)将5-羟基荧光素和真菌引物ITS4溶于1/2去离子水中,搅拌均匀后,缓慢加入促溶剂,轻轻搅拌充分溶解,然后置于37℃恒温摇床上,摇床转速为100转/min,时间为1h,获得溶液A;
(2)将抗干扰荧光助剂、稳定剂、渗透剂以及背景染料溶液溶于剩余去离子水中,搅拌均匀后,获得溶液B;
(3)将溶液A缓慢倒入溶液B中,搅拌均匀后即得真菌荧光染色液。
实例2:一种真菌荧光染色液及其制备方法
本实施例中真菌荧光染色液由以下重量份数的原料组成:
5-羟基荧光素0.0045份、真菌引物ITS4 0.035份、促溶剂氢氧化钠和氢氧化钾按重量比为1:1的比例混合的混合物12份、抗干扰荧光助剂肌醇六磷酸钾0.75份、稳定剂山梨醇25份、渗透剂二甲基乙酰胺0.45份、背景染料溴百里酚蓝2.5份、去离子水65份。
制备方法如下:
(1)将5-羟基荧光素和真菌引物ITS4溶于1/2去离子水中,搅拌均匀后,缓慢加入促溶剂,轻轻搅拌充分溶解,然后置于37℃恒温摇床上,摇床转速为90转/min,时间为65min,获得溶液A;
(2)将抗干扰荧光助剂、稳定剂、渗透剂以及背景染料溶液溶于剩余去离子水中,搅拌均匀后,获得溶液B;
(3)将溶液A缓慢倒入溶液B中,搅拌均匀后即得真菌荧光染色液。
实例3:一种真菌荧光染色液及其制备方法
本实施例中真菌荧光染色液由以下重量份数的原料组成:
5-羟基荧光素0.005份、真菌引物ITS4 0.032份、促溶剂氢氧化钠和氢氧化钾按重量比为1:1的比例混合的混合物10份、抗干扰荧光助剂肌醇六磷酸钾1、稳定剂山梨醇20份、渗透剂二甲基乙酰胺0.45份、背景染料溴百里酚蓝3份、去离子水60份。
制备方法如下:
(1)将5-羟基荧光素和真菌引物ITS4溶于1/2去离子水中,搅拌均匀后,缓慢加入促溶剂,轻轻搅拌充分溶解,然后置于37℃恒温摇床上,摇床转速为110转/min,时间为50min,获得溶液A;
(2)将抗干扰荧光助剂、稳定剂、渗透剂以及背景染料溶液溶于剩余去离子水中,搅拌均匀后,获得溶液B;
(3)将溶液A缓慢倒入溶液B中,搅拌均匀后即得真菌荧光染色液。
本发明真菌荧光染色液的使用方法:取样品置于载玻片上,滴加本发明真菌荧光染色液,盖上盖玻片,通过荧光显微镜在480nm波长下观察样本,观察时视野背景为黑色,真菌放射出520nm波长的绿色荧光。
试验例
取肺泡切片样品、皮屑样品、粘液样品,置于载玻片上,分别滴加实施例1、实施例2和实施例3制备的真菌荧光染色液,盖上盖玻片,通过荧光显微镜在480nm波长下观察样本,观察时视野背景为黑色,真菌放射出520nm波长的绿色荧光。观察结果如图1、图2和图3所示。
Claims (9)
1.一种真菌荧光染色液,其特征在于,真菌荧光染色液由以下原料组成:
5-羟基荧光素、真菌引物ITS4、促溶剂、抗干扰荧光助剂肌醇六磷酸钾、稳定剂、渗透剂、背景染料、去离子水。
2.根据权利要求1所述的真菌荧光染色液,其特征在于,所述促溶剂为氢氧化钠和氢氧化钾的混合物。
3.根据权利要求1所述的真菌荧光染色液,其特征在于,所述稳定剂为山梨醇。
4.根据权利要求1所述的真菌荧光染色液,其特征在于,所述渗透剂为二甲基乙酰胺。
5.根据权利要求1所述的真菌荧光染色液,其特征在于,所述背景染料为染料溴百里酚蓝。
6.根据权利要求2所述的真菌荧光染色液,其特征在于,所述促溶剂中氢氧化钠和氢氧化钾的重量比为1:1。
7.根据权利要求1所述的真菌荧光染色液,其特征在于,所述真菌荧光染色液由以下重量份数的原料组成:5-羟基荧光素0.005份、真菌引物ITS40.03份、促溶剂氢氧化钠和氢氧化钾按重量比为1:1的比例混合的混合物11份、抗干扰荧光助剂肌醇六磷酸钾1份、稳定剂山梨醇20份、渗透剂二甲基乙酰胺0.5份、背景染料溴百里酚蓝2份、去离子水60份。
8.制备如权利要求1-7任一项所述真菌荧光染色液的方法,具体步骤如下:
(1)将5-羟基荧光素和真菌引物ITS4溶于1/2去离子水中,搅拌均匀后,缓慢加入促溶剂,轻轻搅拌充分溶解,然后置于37℃恒温摇床上,摇床转速为80-110转/min,时间为50-65min,获得溶液A;
(2)将抗干扰荧光助剂、稳定剂、渗透剂以及背景染料溶液溶于剩余去离子水中,搅拌均匀后,获得溶液B;
(3)将溶液A缓慢倒入溶液B中,搅拌均匀后即得真菌荧光染色液。
9.根据权利要求7所述的制备方法,其特征在于,所述步骤(1)中摇床转速为100转/min,时间为60min。
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