CN116254351A - 同时检测草绿色链球菌、β溶血性链球菌和肺炎链球菌的试剂盒 - Google Patents
同时检测草绿色链球菌、β溶血性链球菌和肺炎链球菌的试剂盒 Download PDFInfo
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Abstract
本发明涉及生物检测技术领域,尤其涉及同时检测草绿色链球菌、β溶血性链球菌和肺炎链球菌的试剂盒。本发明引物探针可同时检测草绿色链球菌、β溶血性链球菌和肺炎链球菌,提高链球菌检测的覆盖度,满足临床上对链球菌的快速鉴定需求,同时结合数字PCR技术,进一步提高了检测的灵敏度,并对检测结果进行绝对定量,为监控病情的发展提供重要信息。
Description
技术领域
本发明涉及生物检测技术领域,尤其涉及同时检测草绿色链球菌、β溶血性链球菌和肺炎链球菌的试剂盒。
背景技术
链球菌是革兰氏阳性需氧菌,可引起多种疾病,包括喉炎、肺炎、创面和皮肤感染、菌血症和感染性心内膜炎。症状随感染部位而异。多种链球菌合成毒力因子,包括链球菌毒素、DNA酶和透明质酸酶,有助于破坏组织使感染扩散。少数菌株释放的外毒素能激活特定的T细胞,触发释放细胞因子,包括肿瘤坏死因子-α,白介素和其他免疫调节因子。这些细胞因子能激活补体、凝血和纤溶系统,继而导致休克、器官衰竭和死亡。
链球菌根据在实验室中生长时的外观和其不同的化学组分可分为:α溶血性链球菌,因不完全溶血菌落周围环绕绿色,俗称草绿色链球菌。β溶血性链球菌,在每个菌落周围形成一个清晰的溶血环。
γ链球菌,为非溶血性的链球菌。
草绿色链球菌是感染性心内膜炎主要病原菌,可能造成心内膜炎、脑膜炎。也可能造成心包炎、肺炎、腹膜炎、中耳炎、鼻窦炎、坏死性筋膜炎等疾病。它包括5个主要菌种:变异链球菌,血链球菌,唾液链球菌,缓症链球菌和咽峡炎链球菌群。
咽峡炎链球菌(Streptococcus anginosus)群(也被称为米勒链球菌(S.milleri)群)包括3个不同的链球菌种:咽峡炎链球菌(S.anginosus)、中间型链球菌(S.intermedius)以及星座链球菌(S.constellatus)。咽峡炎链球菌有2个亚种:咽峡炎链球菌咽峡炎亚种(S.anginosus subsp anginosus)和咽峡炎链球菌whileyi亚种(S.anginosus subsp whileyi)。星座链球菌有3个亚种:星座链球菌星群亚种(S.constellatus subsp constellatus)、星座链球菌咽炎亚种(S.constellatus subsppharyngis),以及星座链球菌viborgensis亚种(S.constellatus subsp viborgensis)。
β溶血性链球菌根据特异性抗原可分为18个菌群,与人类感染密切相关的主要是A族和B族溶血性链球菌。A族溶血性链球菌(group A streptococcus,GAS)也称化脓性链球菌,是对人毒力最强的菌种,主要定植于咽喉部、皮肤,及软组织中,与多种化脓性感染及非化脓性疾病相关,可引起咽峡炎、扁桃体炎、伤口和皮肤感染、菌血症、猩红热、风湿热和肾小球肾炎。B族溶血性链球菌(group B streptococcus,GBS)也称无乳链球菌,主要寄居于下消化道及泌尿生殖道,也可定植在婴幼儿上呼吸道中,可引起严重感染,特别是新生儿脓毒症、产后脓毒症,心内膜炎和细菌性关节炎。根据细胞壁上特异性荚膜多糖抗原的不同,GBS分为Ⅰa、Ⅰb、Ⅱ~Ⅷ等9个血清型,以Ⅲ型毒力最强。
γ链球菌又称非溶血性链球菌,一般无致病性,常存在于乳品及粪便中。
目前,链球菌的检测主要有以下方法:
(1)传统的细菌培养法:该方法的检测结果准确,但检测时间较长,一般需要24-96小时才能出结果,效率较低,不能满足于临床快速鉴定的需求;
(2)血清学方法,该方法利用胶体金技术,操作简单,提供定性检测结果,但是灵敏度低,特别是感染量少的话,可能会出现漏检、误检的情况;并且血清学方法存在滞后性,不能准确反映当前是否感染,无法满足早期诊断的需求;
(3)细菌核酸检测:通过检测临床样本中细菌的核酸,能够对细菌感染作出早期诊断,给病情的快速分析和控制提供有力的技术支撑。该类方法具有快速、准确和高灵敏性等优点,其中的荧光定量PCR平台已成为广泛使用的细菌检测方法。
由于链球菌种类众多,目前针对链球菌的荧光定量PCR检测主要针对β溶血性链球菌中的A族和B族链球菌,通过一组A族或B族链球菌的引物及对应的荧光探针检测相应的链接菌是否存在。荧光定量PCR方法虽然有较高的灵敏度,但是,目前已有的检测方法主要针对某些特定类型的链球菌,由于链球菌属包括多个种,对链球菌整体覆盖度较低。
发明内容
有鉴于此,本发明提供了同时检测草绿色链球菌、β溶血性链球菌和肺炎链球菌的试剂盒,提高链球菌检测的覆盖度,满足临床上对链球菌的快速鉴定需求,同时结合数字PCR技术,进一步提高检测的灵敏度,并对检测结果进行绝对定量,为监控病情的发展提供重要信息。
为了实现上述发明目的,本发明提供以下技术方案:
引物探针组合,包括:
核苷酸序列如SEQ ID NO:1~2所示的上、下游引物;
和核苷酸序列如SEQ ID NO:3所示的探针。
本发明中,所述探针的5’端标记荧光报告基团,3’端标记淬灭基团;
所述荧光该报告基团为FAM、VIC、ROX、CY5、CY5.5或A425中的任何一种;
所述淬灭基团为MGB-NFQ。
一些具体实施例中,SEQ ID NO:3所示核苷酸序列的探针的5’端标记有荧光报告基团FAM,3’端标记有荧光淬灭基团MGB-NFQ。
本发明所述的引物探针组合在制备链球菌检测的试剂盒中的应用。
本发明所述引物探针可检测出草绿色链球菌、β溶血性链球菌和肺炎链球菌中的至少一种。
本发明还提供了同时检测草绿色链球菌、β溶血性链球菌和肺炎链球菌的试剂盒,包括权利要求1~3任一项所述的引物探针组合。
本发明所述的试剂盒中,还包括数字PCR的检测试剂;所述检测试剂包括无菌水、DNA扩增酶、dNTP mix、10×AceTaq PCR Buffer中的至少一种。
本发明所述的试剂盒,还包括提取待测样本DNA的提取试剂。
本发明对提取待测样本DNA的提取试剂没有特殊限制,根据不同的样本类型,可采用合适的提取试剂及方案进行微生物核酸的提取,操作步骤按照核酸提取试剂说明书进行。
本发明所述的试剂盒适用于多种样本的检测,可用于诊断目的的检测,如对检测样本肺泡灌洗液、血液、脑脊液、痰液和分泌物等进行检测;也可用于非诊断目的的检测,如对环境或水体等样本的检测。
本发明还提供了非诊断目的的检测链球菌的方法,利用本发明所述的引物探针组合或所述的试剂盒对样本进行检测。
本发明提供的检测链球菌的方法基于数字PCR平台进行检测,有效地提高了检测的灵敏度,并对检测结果进行绝对定量,为监控病情的发展提供重要信息。
本发明检测方法中,PCR扩增的程序包括:95℃5min,[95℃15s,60℃30s]×40cycles,25℃1min。
本发明引物探针可同时检测草绿色链球菌、β溶血性链球菌和肺炎链球菌的试剂盒,提高链球菌检测的覆盖度,满足临床上对链球菌的快速鉴定需求,同时结合数字PCR技术,进一步提高了检测的灵敏度,并对检测结果进行绝对定量,为监控病情的发展提供重要信息。
附图说明
图1链球菌引物探针扩增性能测试的原始液滴图(1-a)及一维散点图(1-b);
图2通过Probit回归分析所确定的咽峡炎链球菌(2-a)、无乳链球菌(2-b)和肺炎链球菌的LoD(2-c)。
具体实施方式
本发明提供了同时检测草绿色链球菌、β溶血性链球菌和肺炎链球菌的试剂盒。本领域技术人员可以借鉴本文内容,适当改进工艺参数实现。特别需要指出的是,所有类似的替换和改动对本领域技术人员来说是显而易见的,它们都被视为包括在本发明。本发明的方法及应用已经通过较佳实施例进行了描述,相关人员明显能在不脱离本发明内容、精神和范围内对本文的方法和应用进行改动或适当变更与组合,来实现和应用本发明技术。
本发明采用的试材皆为普通市售品,皆可于市场购得。
下面结合实施例,进一步阐述本发明:
实施例1
1、引物探针
1.1以往关于链球菌的检测主要是针对具体的链球菌菌种设计特定的引物探针,检测多种链球菌时需要针对每种链球菌分别设计引物探针;由于序列上的相似性,针对链球菌菌属的引物探针往往与其它菌种有交叉,容易产生误检,难以做到对链球菌属广泛覆盖同时又能保证特异性。本发明通过大量序列分析,筛选到一段在链球菌、肠球菌、乳球菌、芽孢杆菌上保守的序列,将上游引物和探针设计在该保守序列,下游引物设计在链球菌特异片段上,实现了对链球菌属的广泛覆盖,并能避免与其它菌种的交叉。具体序列如表1所示。
表1引物探针组合序列
1.2扩增性能测试
1.2.1反应体系:
表2
组分 | 用量(μL) |
10x AceTaq PCR Buffer(Mg2+plus) | 1.5 |
dNTP mix(10mM) | 0.3 |
AceTaq(5U/μL) | 0.3 |
Forward primer(10μM) | 1.5 |
Reverse prime(10μM) | 1.5 |
Probe(25μM) | 0.18 |
ROX染料(50X) | 0.1 |
模板 | 5 |
ddH2O | 补齐至15μL |
其中,模板为肺炎链球菌DNA(15copies/μL)
1.2.2微滴生成
(1)取加样后的反应液14μL,分别加入到数字PCR微滴式芯片的各通道进样杯中。
(2)使用液滴生成仪DG32进行液滴生成,操作步骤按照液滴生成仪DG32说明书进行。
1.2.3PCR扩增
将液滴生成后的芯片放入PCR扩增仪TC1,按照以下PCR参数进行反应。
95℃5min,[95℃15s,60℃30s]×40cycles,25℃1min
1.2.4芯片扫描
PCR结束后,将芯片放入生物芯片阅读仪CS7的芯片托架内,选择FAM/ROX通道,设置芯片孔位,进行芯片扫描和分析,结果见图1。
1.2.5结果分析
原始图片(图1-a)和一维散点图(图1-b)均表明,引物探针的扩增性能良好。
实施例2引物探针对链球菌的覆盖度及特异性测试
使用实施例1中的检测体系及操作步骤,将肺炎链球菌替换成要测试的菌株,对链球菌菌株及临床上常见的其它菌株的DNA进行检测,结果见表3和表4。
表3引物探针对链球菌的覆盖度
表4引物探针的特异性
结果显示,对以上多种病原菌,本发明试剂盒只检测出草绿色链球菌、β溶血性链球菌和肺炎链球菌,不会检出其他的病原菌。说明,本发明试剂盒特异性高,能准确地、特异地将三类链球菌检测出来。
实施例3最低检出限(Limit of Detection,LoD)分析
选取咽峡炎链球菌作为草绿色链球菌的代表菌株、无乳链球菌作为β溶血性链球菌的代表菌株,将咽峡炎链球菌、无乳链球菌和肺炎链球菌基因组DNA各自梯度稀释后,分别作为模板,按实施例1中的反应体系和操作步骤进行检测,检测结果进行Probit回归分析,计算试剂盒的LoD。分析结果如图2所示,结果表明,咽峡炎链球菌、无乳链球菌和肺炎链球菌的LoD分别为:2.1、1.9、2.4拷贝/反应。
实施例4临床样本测试
2.1核酸提取
对发生血流感染的110例儿童抽取1mL血液,使用血液cfDNA试剂盒提取cfDNA,,操作步骤按照核酸提取试剂说明书进行;同时抽血进行血培养。
2.2样本检测
参照实施例1中的反应体系和操作步骤,使用5μL提取后的cfDNA进行检测。
2.3检测结果
110例血液样本,共检测到19例阳性,其与血培养的结果见表5,对3例血培养阴性而数字PCR阳性的样本,使用一代测序进一步复核,测序结果均显示为肺炎链球菌,证明了数字PCR检测的准确性。
表5临床样本的数字PCR检测结果
以上仅是本发明的优选实施方式,应当指出,对于本技术领域的普通技术人员来说,在不脱离本发明原理的前提下,还可以做出若干改进和润饰,这些改进和润饰也应视为本发明的保护范围。
Claims (10)
1.引物探针组合,其特征在于,包括:
核苷酸序列如SEQ ID NO:1~2所示的上、下游引物;
和核苷酸序列如SEQ ID NO:3所示的探针。
2.根据权利要求1所述的引物探针组合,其特征在于,所述探针的5’端标记荧光报告基团,3’端标记淬灭基团;
所述荧光报告基团为FAM、VIC、ROX、CY5、CY5.5或A425中的任何一种;
所述淬灭基团为MGB-NFQ。
3.根据权利要求2所述的引物探针组合,其特征在于,
SEQ ID NO:3所示核苷酸序列的探针的5’端标记有荧光报告基团FAM,3’端标记有荧光淬灭基团MGB-NFQ。
4.权利要求1~3任一项所述的引物探针组合在制备链球菌检测的试剂盒中的应用。
5.根据权利要求4所述的应用,其特征在于,所述链球菌包括草绿色链球菌、β溶血性链球菌和肺炎链球菌中的至少一种。
6.同时检测草绿色链球菌、β溶血性链球菌和肺炎链球菌的试剂盒,其特征在于,包括权利要求1~3任一项所述的引物探针组合。
7.根据权利要求6所述的试剂盒,其特征在于,还包括数字PCR的检测试剂;所述检测试剂包括无菌水、DNA扩增酶、dNTPmix、10×AceTaqPCR Buffer中的至少一种。
8.根据权利要求6所述的试剂盒,其特征在于,还包括提取待测样本DNA的提取试剂。
9.根据权利要求8所述的试剂盒,其特征在于,所述样本包括肺泡灌洗液、血液、脑脊液、痰液和分泌物。
10.非诊断目的的检测链球菌的方法,其特征在于,利用权利要求1~3任一项所述的引物探针组合或权利要求6~9任一项所述的试剂盒对样本进行检测。
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