CN110823785A - 一种真菌检测方法 - Google Patents
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Abstract
本申请公开一种真菌检测方法,所述真菌检测方法包括:样品预处理和流式细胞术检测,其中,所述样品预处理包括:将样品在固定剂中振荡,离心,弃去上清液,然后向沉淀中加入干扰细胞清除剂、活细胞染色剂和荧光增白剂。本申请的方法可在临床中实现在单细胞水平形态学上对真菌进行自动化快速检测。
Description
技术领域
本申请涉及真菌感染检测领域,具体地涉及一种真菌检测方法。
背景技术
真菌感染的微生物检查对真菌病的诊断具有重要价值,其传统的方法是用显微镜来观察真菌菌丝和孢子形态来进行诊断,显微镜直接镜检可采用不染色的KOH湿片法或革兰氏染色、墨汁染色、乳酸酚棉蓝染色观察,也可采用化学荧光染色法检查。但漏诊率较高,需要采用多次取材并做多张涂片,以提高检测阳性率。真菌培养法可以直接观察到病原菌生长,验证直接镜检的结果,但是培养一般所需时间较长,不能快速诊断,且有一定的假阴性率。目前已将生化反应及血清学分析方法运用到真菌检查,并逐步向分子水平深入,比如真菌抗原检测、核酸杂交技术已被用于深部感染真菌的特异性鉴定,也存在其局限性,最主要的是干扰因素较多,故临床上依然把真菌的形态学检查作为金标准。
目前临床上诊断真菌感染大多依靠直接涂片镜检,在普通显微镜下寻找孢子和菌丝的形态作为真菌感染的证据。但常规镜检的缺陷主要在于:全程需要人工操作,效率低,人力投入大;由于是直接镜检,没有浓缩,涂片取样量较少,漏检率高;临床样本中孢子常与人体细胞黏连,在不染色的情况下直接镜检对检验人员的专业要求较高;容易造成实验室污染;干扰因素较多,容易出现假阴性或假阳性结果;普通流式分析只能从系数上进行辨别分群,但无法观察到形态,而临床样本内容物复杂,无法使用此类方法。
要解决这些问题,需要一种快速,直观以及准确率较高的方法来观察真菌的菌体或菌丝;能够使得样本中的人体细胞与真菌有一定的区分。
发明内容
以下是对本文详细描述的主题的概述。本概述并非是为了限制权利要求的保护范围。
本申请通过对真菌细胞壁和细胞核进行双染,在单细胞流式成像仪中可以观察到单个真菌细胞呈现蓝色细胞壁和绿色细胞核,从而进行鉴别诊断临床样本中真菌细胞,并进行亮度排序。该方法可操作性强,可在临床中实现在单细胞水平形态学上对真菌进行自动化快速检测。
具体地,本申请提供一种真菌检测方法,所述真菌检测方法包括:样品预处理和流式细胞术检测,其中,
所述样品预处理包括:将样品在固定剂中振荡,离心,弃去上清液,然后向沉淀中加入干扰细胞清除剂、活细胞染色剂和荧光增白剂。
在本申请中,所述流式细胞术检测包括:在355nm-440nm波长激发荧光增白剂,电压为0.50mV;在355nm-440nm波长激发活细胞染色剂,电压为50.00mV。
在本申请中,所述固定剂可以为丙酮、或多聚甲醛和Triton X-100。4%多聚甲醛和0.1%Triton X-100以1:1的体积比混合。固定剂适当破坏孢子细胞壁与细胞膜,利于核染色剂的进入,同时可以起到固定细胞形态的作用。
在本申请中,所述干扰细胞清除剂可以选自NaOH、KOH、十二烷基硫酸钠(SDS)、TritonX-100和NP40中的任一种或更多种。干扰细胞清除剂可以使人体细胞破碎,起到了减少人体细胞对结果观察的干扰。
在本申请中,所述活细胞染色剂可以为SYTOX green、4',6-二脒基-2-苯基吲哚或Hoechst 33342,优选地为SYTOX Green。SYTOX green是一种不可以透过活细胞的DNA荧光染剂,488蓝光激发后呈绿色荧光。
在本申请中,所述荧光增白剂可以为荧光染色剂28或钙荧光白,优选地为钙荧光白。钙荧光白(Calcofluor white,CFW)染色剂主要与真菌细胞壁成分几丁质的β-1,4糖苷键特异性结合的荧光染色剂,紫外线激发后呈蓝色荧光。
在本申请中,所述样品与所述固定剂的体积比可以为0.5-0.8:1;
所述样品与所述干扰细胞清除剂的体积比可以为10-30:1;
所述样品与所述活细胞染色剂的体积比可以为100-400:1;
所述样品与所述荧光增白剂的体积比可以为10-40:1。
在本申请中,荧光增白剂和活细胞染色剂可以同时进行染色,或可以先用活细胞染色剂进行染色,再用荧光增白剂进行染色。
染色结果:真菌孢子或菌丝为细胞壁CFW+(蓝色),同时细胞核SYTOX green+(绿色),人类细胞为CFW-,SYTOX green+。
在本申请中,使用生理盐水作为溶剂均,尽量维持细胞稳定的形态,利于样本的储存。
本申请还提供一种真菌检测组合物,所述真菌检测组合物包括0.01%-0.02%质量份的荧光增白剂,0.001%-0.01%质量份的活细胞染色剂,55%-60%质量份的固定剂和5%-10%质量份的干扰细胞清除剂。
本申请还提供所述真菌检测组合物应用于检测体液中的真菌。
本申请还提供所述真菌检测组合物应用于检测尿液、肺泡灌洗液或胸腹腔积液中的真菌。
本申请还提供所述真菌检测组合物应用于检测念珠菌或烟曲霉。
本申请还提供所述真菌检测组合物应用于白带检测,或应用于肺脏、泌尿系统或胸腹腔中的感染性疾病的辅助检测。
在本申请中,术语“真菌”指的是能感染人体,引起相应临床症状和体征的真核细胞型微生物,包括酵母菌和丝状菌,例如白念珠菌,隐球菌和烟曲霉。
相比于现有技术,本申请可以先进行SYTOX green染色,再进行CFW染色,也可以CFW和SYTOX green同时进行染色。本申请的方法:检测全程自动化,节省大量人力时间;结果可按任意一种荧光强度排序,增加了检出率。形态学检测是临床诊断真菌感染的金标准,但往往检测期间耗时长,效率低,本方法通过利用流式成像技术,改变了现有的完全依靠人力的检测方法,节省了大量人力,可以进行批量全自动的检测。
植物细胞壁钙荧光白和细胞核酸染料SYTOX green(SYTOX green Nucleic AcidStain)两种荧光染料可对真菌细胞壁和细胞核进行双染。CFW与真菌细胞壁几丁质结合时显示增强的荧光;SYTOX green可以穿透细胞膜对其细胞核进行荧光染色;KOH使人体细胞破碎,减少人体细胞对结果的干扰。最终在单细胞流式成像仪中可以观察到单个真菌细胞呈现蓝色细胞壁和绿色细胞核,这样就对临床样本中真菌细胞进行鉴别诊断。在单细胞流式成像仪上,最后结果可以按CFW亮度排序,真菌排在较前部分。此种方法可操作性强,故可在临床中实现在单细胞水平形态学上对真菌进行自动化快速检测。
本申请的其它特征和优点将在随后的说明书中阐述,并且,部分地从说明书中变得显而易见,或者通过实施本申请而了解。本申请的目的和其他优点可通过在说明书、权利要求书以及附图中所特别指出的结构来实现和获得。
附图说明
附图用来提供对本申请技术方案的进一步理解,并且构成说明书的一部分,与本申请的实施例一起用于解释本申请的技术方案,并不构成对本申请技术方案的限制。
图1是念珠菌和曲霉在流式成像下的形态图片。
图2是荧光图片按照CFW的荧光强度由强到弱排序后的结果。
图3是对比例中的样本进行荧光染色后,在荧光显微镜下的观察结果。
具体实施方式
为使本申请的目的、技术方案和优点更加清楚明白,下文中将结合附图对本申请的实施例进行详细说明。需要说明的是,在不冲突的情况下,本申请中的实施例及实施例中的特征可以相互任意组合。
仪器:Amnis ImageStreamX MarkII量化成像分析流式细胞仪(美国)实施例
实施例1
1)取500微升含有念珠菌的尿液,加入750μL丙酮中震荡混匀。
2)离心10000rpm/s,10分钟。
3)吸出上清后,加入50μL 10%KOH悬浮,依次加入2μL 0.5mM的SYTOX green染色剂和5μL的0.1%CFW染色剂,混匀后使用德国密理博Amnis高速成像流式细胞仪进行检测。所有试剂的溶剂均为生理盐水(0.9%NaCl)。CFW荧光使用405nm波长激光激发,设置电压为0.50mV,SYTOX green的荧光使用405nm波长激光激发,设置电压为50.00mV,设置明场通道为Ch01。
4)分析数据,可在IDEAs软件(Amnis高速成像流式细胞仪的分析软件)里打开最后的结果图,并按CFW的亮度进行排序。
实施例2
采用含有烟曲霉的肺泡灌洗液,进行与实施例1相同的检测。
从图1A和图1B可以看出,使用丙酮对样本中的白念珠菌的细胞膜打孔和固定不同时间,短时间和长时间CFW和SYTOX green均能成功染色,且在高速成像流式细胞仪中能够成功被检测到;从图1C和图1D可以看出,使用丙酮对样本中的烟曲酶菌丝和孢子的细胞膜打孔和固定不同时间,短时间仅CFW能成功染色,在高速成像流式细胞仪中能够成功被检测到,长时间CFW和SYTOX green均能成功染色,且在高速成像流式细胞仪中能够成功被检测到。图2结果显示,将荧光图片按照CFW染色后的荧光强度由高到底进行排序,排在最上面的为CFW荧光强度最高的图片,这时这些图片中SYTOX Green成功染色的即为真菌孢子,这样易于对阳性结果进行挑选和观察,提高了检测效率。
对比例
手工使用上述相同的染色方法对样本进行荧光染色后,在荧光显微镜下肉眼进行观察检测,人工检测的速度慢,效率低,而且误诊和漏诊率高。如图3所示,虽然使用双染色可以在镜下观察到真菌孢子,但是容易受到标本中杂质的影响,而且标本中孢子的数量也会影响镜检的结果:在白光下,圆形或椭圆形的干扰物与孢子形态相似,因此在白光下,这些干扰物很有可能被认为是真菌孢子;当标本中的孢子数量很少时,直接取10μL-20μL的样本去涂片镜检,涂片的这10μL-20μL的样本中没有真菌孢子的几率很大,其中并没有真菌孢子的存在,因此镜检可能被认为是阴性结果。
虽然本申请所揭露的实施方式如上,但所述的内容仅为便于理解本申请而采用的实施方式,并非用以限定本申请。任何本申请所属领域内的技术人员,在不脱离本申请所揭露的精神和范围的前提下,可以在实施的形式及细节上进行任何的修改与变化,但本申请的专利保护范围,仍须以所附的权利要求书所界定的范围为准。
Claims (13)
1.一种真菌检测方法,包括:样品预处理和流式细胞术检测,其中,
所述样品预处理包括:将样品在固定剂中振荡,离心,弃去上清液,然后向沉淀中加入干扰细胞清除剂、活细胞染色剂和荧光增白剂。
2.根据权利要求1所述的真菌检测方法,其中,所述流式细胞术检测包括:在355nm-440nm波长激发荧光增白剂,电压为0.50mV;在355nm-440nm波长激发活细胞染色剂,电压为50.00mV。
3.根据权利要求1所述的真菌检测方法,其中,所述固定剂为丙酮、或多聚甲醛和Triton X-100。
4.根据权利要求1所述的真菌检测方法,其中,所述干扰细胞清除剂选自NaOH、KOH、十二烷基硫酸钠、TritonX-100和乙基苯基聚乙二醇中的任一种或更多种。
5.根据权利要求1所述的真菌检测方法,其中,所述活细胞染色剂为SYTOX green、4',6-二脒基-2-苯基吲哚或Hoechst 33342,优选地为SYTOX green。
6.根据权利要求1所述的真菌检测方法,其中,所述荧光增白剂为荧光染色剂28或钙荧光白,优选地为钙荧光白。
7.根据权利要求1至6中任一项所述的真菌检测方法,其中,
所述样品与所述固定剂的体积比为0.5-0.8:1;
所述样品与所述干扰细胞清除剂的体积比为10-30:1;
所述样品与所述活细胞染色剂的体积比为100-400:1;
所述样品与所述荧光增白剂的体积比为10-40:1。
8.根据权利要求1至6中任一项所述的真菌检测方法,其中,荧光增白剂和活细胞染色剂同时进行染色,或先用活细胞染色剂进行染色,再用荧光增白剂进行染色。
9.一种真菌检测组合物,包括0.01%-0.02%质量份的荧光增白剂,0.001%-0.01%质量份的活细胞染色剂,55%-60%质量份的固定剂和5%-10%质量份的干扰细胞清除剂。
10.权利要求9所述的真菌检测组合物应用于检测体液中的真菌。
11.权利要求9所述的真菌检测组合物应用于检测尿液、肺泡灌洗液或胸腹腔积液中的真菌。
12.权利要求9所述的真菌检测组合物应用于检测念珠菌或烟曲霉。
13.权利要求9所述的真菌检测组合物应用于白带检测,或应用于肺脏、泌尿系统或胸腹腔中的感染性疾病的辅助检测。
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