CN114414798A - 沙眼衣原体/淋球菌/生殖支原体抗原联合检测试剂盒及其制备方法 - Google Patents
沙眼衣原体/淋球菌/生殖支原体抗原联合检测试剂盒及其制备方法 Download PDFInfo
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Abstract
沙眼衣原体/淋球菌/生殖支原体抗原联合检测试剂盒及其制备方法,涉及检测试剂盒领域;包括检测卡,所述检测卡包括底板、加样板、乳胶结合垫、硝酸纤维素膜和吸水纸,所述加样板、乳胶结合垫、硝酸纤维素膜和吸水纸依次首尾连接并固定于所述底板上,所述乳胶结合垫固定有第一乳胶微球标记的沙眼衣原体/淋球菌/生殖支原体特异性抗体和第二乳胶微球标记的链霉亲和素,所述硝酸纤维素膜设有包被沙眼衣原体/淋球菌/生殖支原体抗体的检测线和包被生物素‑BSA的质控线。本发明的检测试剂盒,仅需一次取样及样本处理过程的条件下,同时完成沙眼衣原体/淋球菌/生殖支原体的抗原检测,效率高,且特异性强,灵敏度高。
Description
技术领域
本发明属于检测试剂盒领域,具体涉及沙眼衣原体/淋球菌/生殖支原体抗原联合检测试剂盒及其制备方法。
背景技术
沙眼衣原体是一种细胞内寄生的微生物,可侵犯眼、生殖道和其它脏器,是一种常见的性传播疾病,除引起泌尿生殖道感染外,还是增加艾滋病病毒(HIV)感染的危险因素之一。
淋病奈瑟菌(Nei sseria gonorrhoeae.NG)俗称淋球菌,是人体寄生微生物,形态与脑膜炎球菌类似,主要寄居在急性尿道炎和阴道炎的脓性分泌物的白细胞中,是淋病的病原菌,主要通过性传播,只感染人类,男性常引起急性尿道炎,女性则一般表现为无症状感染,但病菌可向深部组织播散导致宫颈炎或不孕等严重后果。
生殖支原体是所有支原体中基因组最小的一种,最简单的原核生物,缺乏细胞壁,仅有一层细胞膜。生殖支原体是近年来确认的性传播疾病(STD)病原体之一,在男性引起非淋球菌性尿道炎(NGU),在女姓导致宫颈炎、子宫内膜炎及盆腔炎等,如不治疗可致异位妊娠、不孕等不良结局。
沙眼衣原体感染的实验室诊断方法主要有三类:(1)以细胞培养为代表的细胞生物学方法;(2)以直接荧光抗体测定(DFA)法、酶免疫法(EIA)和胶体金法为代表的免疫学方法;(3)以核酸探针检测法和核酸扩增技术(PCR,LCR)为代表的分子生物学检测方法。细胞培养法自上世纪70年代以来一直被作为诊断CT感染的“金标准”,是唯一能检测衣原体活体的方法,具有高度特异性,但敏感性较低,且受实验条件、费用等影响,目前只局限于实验室研究,还未被用作常规的诊断方法。直接荧光抗体测定(DFA)法可以检测衣原体各种类型的标本。这种技术敏感、特异、快速,是一种操作性和实用性都很强的诊断技木平。但此项技术操作要求高。且长时间观察荧光显微镜,容易引起视觉疲劳,不适于大规模的临床筛查。较传统的检测方法敏感性显著提高,是迄今为止诊断和筛查衣原体感染最敏感的方法,但其对技术条件和实验室条件要求高,受到的影响因素很多,如临床标本容易受非衣原体、非淋球菌及污染等影响,容易出现假阳性和假阴性结果,特异性降低。
淋球菌诊断主要依赖于实验室对淋球菌的检测:(1)涂片法;(2)培养基培养法;(3)聚合酶链反应技术。涂片检查法:取泌尿生殖道脓性分泌物涂片革兰染色,镜下可见大量多形核白细胞,白细胞内、外可见数量不等的革兰阴性双球菌。本法对有大量脓性分泌物的单纯性急性淋菌性前尿道炎,尤其是男性淋病性尿道炎,其敏感性可达95%,对慢性淋病患者敏感性较低,女性患者容易出现假阳性。培养检查法:培养法被认为是诊断淋病奈瑟菌的“金标准”,但其缺点是费时、操作繁琐。淋病奈瑟菌培养对症状很轻或无症状的女性和男性患者敏感性均高,因此培养法是目前世界卫生组织推荐的筛查和诊断淋病患者的主要实验室方法。聚合酶链反应技术:PCR(聚合酶链反应)是1985年建立起来的一种体外DNA扩增试验。其基本原理是酶促DNA合成反应,即在模板DNA、引物和脱氧核糖核酸存在下,在DNA聚合酶的作用下,使DNA链扩增延伸。由于试验的敏感性和特异性高,对标本采集的要求远不如培养那么严格,使它在医学领域得到广泛应用。其缺点是对技术条件和实验室条件要求高。
生殖支原体主要检测方法:(1)分离培养法;(2)分子生物学方法;(3)DNA探针技术。分离培养法:由于Mg对营养要求苛刻,生长极其缓慢,从临床标本中分离出在无细胞培养基上生长的Mg是非常困难的。分离培养法仍因其所需时间过长、培养过程中菌株容易死亡等原因不适用于临床快速检测。血清学方法:由于Mg和肺炎支原体(Mp)具有许多相似的结构特征和广泛的抗原交叉反应性,因此血清学方法应用于Mg临床诊断缺乏足够的特异性。近年来,人们建立了许多血清学检测方法并在流行病学研究中进行了应用。但是,至今没有一种血清学方法能够有效应用于临床患者的诊断。Jurstrand等中应用脂结合膜蛋白-酶免疫测定法(LAMP-EIA)检测了血清Mg抗体,与其他支原体无交叉反应发生。基于脂结合膜蛋白的酶联免疫吸附试验检测法(LAMPELISA)虽然具有较高的种属特异性,但其检测结果随时间波动较大,影响因素较多,亦很难应用于临床诊断。DNA探针技术:DNA探针技术对Mg有一些探索研究。斑点杂交检测Mg灵敏度可达到0.1ng.DNA探针技术较培养法快速、阴性率高,同时克服了Mg血清学与Mp的交叉反应性。其缺点:费用较高,操作复杂,放射性同位素存在污染等。
综上所述,沙眼衣原体/淋球菌/生殖支原体的现有检测方法大致相同。涂片法虽简便快捷,但敏感性差;培养法具有高敏感性但耗时多;分子生物学技术虽然敏感度和特异性都俱佳,并且耗时短,但是需要昂贵的仪器配套以及专业的技术人员,对其在临床上推行有较大的限制作用。鉴于沙眼衣原体、淋球菌和生殖支原体感染泌尿生殖道的部位相同,症状相似,且常合并感染,有必要研究一种可完成三种病原体的检测,以方便患者及临床检验人员,提高检测效率。
发明内容
为了克服现有技术的不足,本发明的目的之一在于提供沙眼衣原体/淋球菌/生殖支原体抗原联合检测试剂盒,仅需一次取样及样本处理过程的条件下,同时完成沙眼衣原体/淋球菌/生殖支原体的抗原检测,效率高,且特异性强,灵敏度高。
本发明的目的之二在于提供上述沙眼衣原体/淋球菌/生殖支原体抗原联合检测试剂盒的制备方法。
本发明的目的之一采用如下技术方案实现:
沙眼衣原体/淋球菌/生殖支原体抗原联合检测试剂盒,包括分别测定沙眼衣原体/淋球菌/生殖支原体抗原的检测卡,所述检测卡包括底板、加样板、乳胶结合垫、硝酸纤维素膜和吸水纸,所述加样板、乳胶结合垫、硝酸纤维素膜和吸水纸依次首尾连接并固定于所述底板上,所述乳胶结合垫固定有第一乳胶微球标记的沙眼衣原体/淋球菌/生殖支原体特异性抗体和第二乳胶微球标记的链霉亲和素,所述硝酸纤维素膜设有包被沙眼衣原体/淋球菌/生殖支原体抗体的检测线和包被生物素-BSA的质控线。
进一步地,所述第一乳胶微球为呈红色的聚苯乙烯微球,粒径为200-400nm;所述第二乳胶微球为呈蓝色的聚苯乙烯微球,粒径为200-400nm。
进一步地,所述加样板设有三个平行的加样孔,每个所述加样孔分别对应用于沙眼衣原体、淋球菌和生殖支原体的加样处理。
进一步地,还包括样本处理液A、样本处理液B,所述样本处理液A为0.1-0.3M NaOH溶液,所述样本处理液B为0.1-0.3M HCl溶液。
沙眼衣原体/淋球菌/生殖支原体抗原联合检测试剂盒的使用方法,包括以下步骤:
S1,将取样拭子置于样本处理液A中,搅拌后静置,再添加样本处理液B,搅拌后静置,移除取样拭子,得到样本液;
S2,将样本液滴加到检测卡的加样板中,15-25min内观察并判断结果。
进一步地,使用方法的步骤S1中,加入适量的样本处理液A和样本处理液B,静置时间为1-5min。
进一步地,使用方法的步骤S1中,所述取样拭子为从女性宫颈或男性尿道中取样。
进一步地,使用方法的步骤S2中,判断结果的具体步骤为:若样本液中含有沙眼衣原体/淋球菌/生殖支原体抗原,则在检测线的对应位置上呈现相应的色条带;若待测样本中不含沙眼衣原体/淋球菌/生殖支原体抗原,则在检测线上不显色;同时无论样本液中有无沙眼衣原体/淋球菌/生殖支原体抗原,质控线都应呈现色条带,否则,检测无效。
本发明的目的之二采用如下技术方案实现:
沙眼衣原体/淋球菌/生殖支原体抗原联合检测试剂盒的制备方法,包括以下步骤:
1)采用第一乳胶微球标记沙眼衣原体/淋球菌/生殖支原体特异性抗体,得到抗体-乳胶体复合物;采用第二乳胶微球标记链霉亲和素,得到亲和素-乳胶体复合物;将所述抗体-乳胶体复合物和亲和素-乳胶体复合物混合后喷涂到吸附膜上,形成乳胶结合垫;
2)将沙眼衣原体/淋球菌/生殖支原体抗体喷涂在硝酸纤维素膜上,形成检测线;将生物素-BSA喷涂在位于检测线另一侧的硝酸纤维素膜上,形成质控线,干燥,制得设有检测线和质控线的硝酸纤维素膜;
3)在底板上顺次搭接的加样板、乳胶结合垫、硝酸纤维素膜和吸水板,即得检测卡。
相比现有技术,本发明的有益效果在于:
本申请的沙眼衣原体/淋球菌/生殖支原体抗原联合检测试剂盒,采用乳胶层析法对沙眼衣原体/淋球菌/生殖支原体的抗原进行联合检测,其中,采用彩色乳胶微球标记沙眼衣原体/淋球菌/生殖支原体特异性抗体和链霉亲和素制备乳胶结合垫,分别在硝酸纤维素膜包被沙眼衣原体/淋球菌/生殖支原体抗体和生物素-BSA,形成检测线和质控线,得到的检测试剂盒仅需一次取样及样本处理过程的条件下,同时完成沙眼衣原体/淋球菌/生殖支原体的抗原检测,且效率高,特异性强,灵敏度高,可重复性强。本发明可快速对待测样本进行沙眼衣原体/淋球菌/生殖支原体的定性检测判断,效率高,特异性强,灵敏度高。
本发明的沙眼衣原体/淋球菌/生殖支原体抗原联合检测试剂盒的制备方法,可快速制备上述检测试剂盒,工艺简单,制备成本低。
附图说明
图1是本发明实施例1中检测卡的结构示意图。
图2是本发明实施例1中检测卡的检测示意图。
图中:1、加样板;2、乳胶结合垫;3、硝酸纤维素膜;4、吸水纸;5、底板。
具体实施方式
下面,结合附图与具体实施方式,对本发明做进一步描述,需要说明的是,在不相冲突的前提下,以下描述的各实施例之间或各技术特征之间可以任意组合形成新的实施例。
实施例1
沙眼衣原体/淋球菌/生殖支原体抗原联合检测试剂盒,参照图1-2,包括三种分别测定沙眼衣原体/淋球菌/生殖支原体抗原的检测卡,所述检测卡包括底板5、加样板1、乳胶结合垫2、硝酸纤维素膜3和吸水纸4,所述加样板1、乳胶结合垫2、硝酸纤维素膜3和吸水纸4依次首尾连接并固定于所述底板5上。
所述乳胶结合垫2固定有第一乳胶微球标记的沙眼衣原体/淋球菌/生殖支原体特异性抗体和第二乳胶微球标记的链霉亲和素,所述第一乳胶微球呈红色的聚苯乙烯微球,粒径为300nm;所述第二乳胶微球呈蓝色的聚苯乙烯微球,粒径为300nm。
所述硝酸纤维素膜3设有包被沙眼衣原体/淋球菌/生殖支原体抗体的检测线(T线)和包被生物素-BSA的质控线(C线)。
所述加样板1设有三个平行的加样孔,每个所述加样孔分别对应用于沙眼衣原体、淋球菌和生殖支原体的加样处理。
上述沙眼衣原体/淋球菌/生殖支原体抗原联合检测试剂盒的制备方法,包括以下步骤:
1)采用第一乳胶微球标记沙眼衣原体/淋球菌/生殖支原体特异性抗体,得到抗体-乳胶体复合物;采用第二乳胶微球标记链霉亲和素,得到亲和素-乳胶体复合物;将所述抗体-乳胶体复合物和亲和素-乳胶体复合物喷涂到吸附膜上,烘干,形成乳胶结合垫2;烘干的温度为50℃、相对湿度≤20%干燥1h;
2)将沙眼衣原体/淋球菌/生殖支原体抗体喷涂在硝酸纤维素膜上,干燥,形成检测线;将生物素-BSA喷涂在位于检测线另一侧的硝酸纤维素膜上,形成质控线,烘干,制得设有检测线(T线)和质控线(C线)的硝酸纤维素膜3;烘干的温度为57℃、相对湿度≤20%干燥14-16h;
3)在底板5上顺次搭接的加样板1、乳胶结合垫2、硝酸纤维素膜3和吸水纸4,即得检测卡。
性能分析
本实施例通过对沙眼衣原体/淋球菌/生殖支原体抗原联合检测试剂盒的检出限进行测试分析;结果表明,本实施例的检测试剂盒中,沙眼衣原体阳性判断值为≥104IFU/mL,淋球菌阳性判断值为≥103CFU/mL,生殖支原体阳性判断值为≥103CCU/mL。即本实施例的检测试剂盒的检出限较低,且检测效率高,操作简单,特异性强,灵敏度高,检测结果可供临床参考。
实施例2
沙眼衣原体/淋球菌/生殖支原体抗原联合检测试剂盒的使用方法,应用实施例1所述的沙眼衣原体/淋球菌/生殖支原体抗原联合检测试剂盒,包括以下步骤:
S1,对女性宫颈进行拭子样本处理,然后将取样拭子滴加适量样本处理液A,转动拭子15次后静置2min,再滴加适量样本处理液B,转动拭子15次后静置1min,沿管壁挤压拭子后取出拭子弃之,得到样本液;所述样本处理液A为0.2M NaOH溶液,所述样本处理液B为0.2M HCl溶液;
S2,将样本液滴加到检测卡的加样板中,15-25min内观察并判断结果;
判断结果:在检测线上呈现红色条带,质控线都应呈现蓝色条带,结果为:阳性。阳性结果表明:检测线表示样本中含有沙眼衣原体抗原/淋球菌抗原/生殖支原体抗原。
检验结果的解释:
阳性(+):
同时出现两条条带;其中,一条红色条带位于检测线(T线)内,另一条蓝色条带位于质控线(C线)。阳性结果表明:检测的样本中含有沙眼衣原体抗原/淋球菌抗原/生殖支原体抗原。
阴性(-):
仅质控线(C线)出现一条蓝色条带。在检测线(T线)内无红色条带出现。阴性结果表明:样本中不含有沙眼衣原体/淋球菌/生殖支原体抗原,或是含量低于可检测范围。
无效:
质控线(C线)未出现蓝色条带,表明操作过程不正确或检测卡已变质损坏。
实施例3
沙眼衣原体/淋球菌/生殖支原体抗原联合检测试剂盒的使用方法,应用实施例1所述的沙眼衣原体/淋球菌/生殖支原体抗原联合检测试剂盒,包括以下步骤:
S1,对女性宫颈进行拭子样本处理,然后将取样拭子滴加适量样本处理液A,转动拭子15次后静置2min,再滴加适量样本处理液B,转动拭子15次后静置1min,沿管壁挤压拭子后取出拭子弃之,得到样本液;所述样本处理液A为0.2M NaOH溶液,所述样本处理液B为0.2M HCl溶液;
S2,将样本液滴加到检测卡的加样板中,15-25min内观察并判断结果;
判断结果:仅质控线(C线)出现一条蓝色条带,在检测线(T线)内无红色条带出现,结果为:阴性。阴性结果表明:样本中不含有沙眼衣原体/淋球菌/生殖支原体抗原,或是含量低于可检测范围。
实施例4
沙眼衣原体/淋球菌/生殖支原体抗原联合检测试剂盒的使用方法,应用实施例1所述的沙眼衣原体/淋球菌/生殖支原体抗原联合检测试剂盒,包括以下步骤:
S1,对男性尿道进行拭子样本处理,然后将取样拭子滴加适量样本处理液A,转动拭子15次后静置2min,再滴加适量样本处理液B,转动拭子15次后静置1min,沿管壁挤压拭子后取出拭子弃之,得到样本液;所述样本处理液A为0.3M NaOH溶液,所述样本处理液B为0.3M HCl溶液;
S2,将样本液滴加到检测卡的加样板中,15-25min内观察并判断结果;
判断结果:质控线(C线)未出现蓝色条带,结果为:检测无效。表明操作过程不正确或检测卡已变质损坏。
上述实施方式仅为本发明的优选实施方式,不能以此来限定本发明保护的范围,本领域的技术人员在本发明的基础上所做的任何非实质性的变化及替换均属于本发明所要求保护的范围。
Claims (9)
1.沙眼衣原体/淋球菌/生殖支原体抗原联合检测试剂盒,其特征在于:包括分别测定沙眼衣原体/淋球菌/生殖支原体抗原的检测卡,所述检测卡包括底板、加样板、乳胶结合垫、硝酸纤维素膜和吸水纸,所述加样板、乳胶结合垫、硝酸纤维素膜和吸水纸依次首尾连接并固定于所述底板上,所述乳胶结合垫固定有第一乳胶微球标记的沙眼衣原体/淋球菌/生殖支原体特异性抗体和第二乳胶微球标记的链霉亲和素,所述硝酸纤维素膜设有包被沙眼衣原体/淋球菌/生殖支原体抗体的检测线和包被生物素-BSA的质控线。
2.如权利要求1所述的沙眼衣原体/淋球菌/生殖支原体抗原联合检测试剂盒,其特征在于:所述第一乳胶微球为呈红色的聚苯乙烯微球,粒径为200-400nm;所述第二乳胶微球为呈蓝色的聚苯乙烯微球,粒径为200-400nm。
3.如权利要求1所述的沙眼衣原体/淋球菌/生殖支原体抗原联合检测试剂盒,其特征在于:所述加样板设有三个平行的加样孔,每个所述加样孔分别对应用于沙眼衣原体、淋球菌和生殖支原体的加样处理。
4.如权利要求1所述的沙眼衣原体/淋球菌/生殖支原体抗原联合检测试剂盒,其特征在于:还包括样本处理液A、样本处理液B,所述样本处理液A为0.1-0.3M NaOH溶液,所述样本处理液B为0.1-0.3M HCl溶液。
5.如权利要求1-4任一项所述的沙眼衣原体/淋球菌/生殖支原体抗原联合检测试剂盒,其特征在于:其使用方法包括以下步骤:
S1,将取样拭子置于样本处理液A中,搅拌后静置,再添加样本处理液B,搅拌后静置,移除取样拭子,得到样本液;
S2,将样本液滴加到检测卡的加样板中,15-25min内观察并判断结果。
6.如权利要求5所述的沙眼衣原体/淋球菌/生殖支原体抗原联合检测试剂盒,其特征在于:其使用方法的步骤S1中,加入适量的样本处理液A和样本处理液B,静置时间为1-5min。
7.如权利要求5所述的沙眼衣原体/淋球菌/生殖支原体抗原联合检测试剂盒,其特征在于:其使用方法的步骤S1中,所述取样拭子为从女性宫颈或男性尿道中取样。
8.如权利要求5所述的沙眼衣原体/淋球菌/生殖支原体抗原联合检测试剂盒,其特征在于:其使用方法的步骤S2中,判断结果的具体步骤为:若样本液中含有沙眼衣原体/淋球菌/生殖支原体抗原,则在检测线的对应位置上呈现相应的色条带;若待测样本中不含沙眼衣原体/淋球菌/生殖支原体抗原,则在检测线上不显色;同时无论样本液中有无沙眼衣原体/淋球菌/生殖支原体抗原,质控线都应呈现色条带,否则,检测无效。
9.一种权利要求1-4任一项所述的沙眼衣原体/淋球菌/生殖支原体抗原联合检测试剂盒的制备方法,其特征在于,包括以下步骤:
1)采用第一乳胶微球标记沙眼衣原体/淋球菌/生殖支原体特异性抗体,得到抗体-乳胶体复合物;采用第二乳胶微球标记链霉亲和素,得到亲和素-乳胶体复合物;将所述抗体-乳胶体复合物和亲和素-乳胶体复合物混合后喷涂到吸附膜上,形成乳胶结合垫;
2)将沙眼衣原体/淋球菌/生殖支原体抗体喷涂在硝酸纤维素膜上,形成检测线;将生物素-BSA喷涂在位于检测线另一侧的硝酸纤维素膜上,形成质控线,干燥,制得设有检测线和质控线的硝酸纤维素膜;
3)在底板上顺次搭接的加样板、乳胶结合垫、硝酸纤维素膜和吸水板,即得检测卡。
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