CN112481401B - 同时检测肺炎链球菌、嗜肺军团菌和卡他莫拉菌的试剂盒 - Google Patents
同时检测肺炎链球菌、嗜肺军团菌和卡他莫拉菌的试剂盒 Download PDFInfo
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Abstract
本发明涉及荧光PCR检测技术领域,尤其涉及同时检测肺炎链球菌、嗜肺军团菌和卡他莫拉菌的试剂盒。其中,荧光PCR检测体系包括肺炎链球菌、嗜肺军团菌和卡他莫拉菌的特异性保守序列的引物及TaqMan荧光探针,其DNA序列SEQ ID NO:1~9。本发明的优点在于利用多种荧光定量PCR检测技术,实现了同时检测肺炎链球菌、嗜肺军团菌和卡他莫拉菌三种呼吸道病原体的目的,该检测方法快速、准确,且保证了检测的时效性、特异性和灵敏度,在疾病监测、临床诊断等领域具有重要的应用价值。
Description
技术领域
本发明涉及荧光PCR检测技术领域,尤其涉及同时检测肺炎链球菌、嗜肺军团菌和卡他莫拉菌的试剂盒。
背景技术
呼吸道感染是世界范围内最常见的传染病之一,是影响人类健康的重要问题。呼吸道感染包括上呼吸道感染和下呼吸道感染,能够引起呼吸道感染的病原体很多,包括细菌、病毒、支原体、衣原体等,这些病原体引起的症状接近,依靠临床症状进行区分较为困难。下呼吸道感染是指终末气道,肺泡和肺间质的炎症,可由疾病微生物、理化因素,免疫损伤、过敏及药物所致。在多种多样的呼吸道病原体中,肺炎链球菌、嗜肺军团菌和卡他莫拉菌是三种重要的病原体:
肺炎链球菌(Streptococcus pneumoniae,SP)属链球菌科链球菌属细菌,革兰氏阳性球菌,镜下观察一般呈双球状排列,有荚膜结构,荚膜是肺炎链球菌的主要毒力因子可引起多种感染的重要病原体。肺炎链球菌是引起细菌性肺炎的最主要病原体,约占其2/3左右,全年均可发病,冬春季高发。该菌主要通过飞沫和呼吸道分泌物传播进而引起细菌性肺炎,也可侵入机体其它部位,引起继发性胸膜炎、中耳炎、乳突炎以及心内膜炎等。目前在世界范围内,肺炎链球菌感染所导致的疾病依然是一个严重的公共卫生问题。因此对肺炎链球菌感染要尽快针对不同病情给以恰当的治疗。
嗜肺军团菌(Legionella pneumophila,LP)是一种有鞭毛上的革兰氏阴性菌,军团菌属多形态性的短小球杆菌,兼性胞内寄生菌,嗜肺军团菌为胞内寄生菌,是引起人类军团菌病的重要病原体。该菌主要通过吸入带菌飞沫而感染,常见的感染来源为污染的空调和供水系统,其感染力在抗体和血清补体存在下大大提高,可引起军团菌肺炎、肺外综合征和庞蒂亚克热。军团菌肺炎在非典型肺炎中是病情最严重的一种,未经有效治疗者的病死率高达45%。
卡他莫拉菌(MoraxellaCatarrhalis,MC)为革兰氏阴性非发酵菌。卡他莫拉菌可以引起肺炎,大多数肺部感染是通过气道途径扩散,其临床特点主要有高热、呼吸困难、脉搏加速、呼吸加快、咳痰等症状,并是儿童迁延性细菌性支气管炎的主要病原菌,对儿童健康构成威胁。有研究表明卡他莫拉菌是儿童社区获得性肺炎、上额窦炎、中耳炎以及成人慢性下呼吸道感染的第3位最常见致病菌,与肺炎链球菌,流感嗜血杆菌共同被认为是儿童呼吸道感染的最重要的三大致病菌。
现有技术针对呼吸道肺炎链球菌、嗜肺军团菌和卡他莫拉菌的检测方法主要有分离培养法、免疫检测法、血清学诊断和荧光PCR法。传统的分离不培养法需要培养分离出菌株之后才能鉴定,需要时间较长,且嗜肺军团菌生长条件苛刻,营养要求很高,费时费力,不利于快速检测,并对于操作人员来说具备一定的危险性,因而限制了其在临床的应用。免疫法检测试剂针对的是样本中的抗原或者病原体引起机体产生的特异性抗体,且抗体检测有窗口期,在发病初期容易漏诊。另外,虽然血清学方法技术成熟,有商品试剂盒供应,但是敏感性和特异性有待提高,同时,需要检测急性期和恢复期双份血清才有诊断意义且容易漏诊。而现有技术中的荧光PCR检测,因引物特异性等方面的原因,很难实现对SP、LP、MC的多重检测。
发明内容
有鉴于此,本发明要解决的技术问题在于提供同时检测肺炎链球菌、嗜肺军团菌和卡他莫拉菌的试剂盒,该试剂盒具有良好的灵敏性、特异性。
本发明提供了肺炎链球菌的检测引物和探针,
肺炎链球菌的正向引物,其核酸序列如SEQ ID NO:1所示;
肺炎链球菌的反向引物,其核酸序列如SEQ ID NO:2所示;
肺炎链球菌的探针,其核酸序列如SEQ ID NO:3所示。
本发明还提供了嗜肺军团菌的检测引物和探针,
嗜肺军团菌的正向引物,其核酸序列如SEQ ID NO:4所示;
嗜肺军团菌的反向引物,其核酸序列如SEQ ID NO:5所示;
嗜肺军团菌的探针,其核酸序列如SEQ ID NO:6所示。
本发明还提供了卡他莫拉菌的检测引物和探针,
卡他莫拉菌的正向引物,其核酸序列如SEQ ID NO:7所示;
卡他莫拉菌的反向引物,其核酸序列如SEQ ID NO:8所示;
卡他莫拉菌的探针,其核酸序列如SEQ ID NO:9所示。
本发明提供的针对肺炎链球菌、嗜肺军团菌和卡他莫拉菌的引物和探针具有良好的特异性,且彼此之间互补感染,因此,可以在一个体系中对样品进行多重检测。
本发明提供了同时检测肺炎链球菌、嗜肺军团菌和卡他莫拉菌的试剂盒,其包括肺炎链球菌、嗜肺军团菌、卡他莫拉菌的检测引物和探针。
本发明以SEQ IDNO:10所示核酸序列的核酸作为内标,该片段及其相应的引物和探针不会干扰其他引物和探针对于致病菌的检测。
所述试剂盒中还包括内标的引物和探针;所述内标的序列为SEQ ID NO:10;
内标的正向引物,其核酸序列如SEQ ID NO:11所示;
内标的反向引物,其核酸序列如SEQ ID NO:12所示;
内标的探针,其核酸序列如SEQ ID NO:13所示。
本发明所述试剂盒的探针中,
所述肺炎链球菌的探针5’端标记FAM荧光基团,3’端标记BHQ 1荧光淬灭基团;
所述嗜肺军团菌的探针5’端标记ROX荧光基团,3’端标记BHQ 1荧光淬灭基团;
所述卡他莫拉菌的探针5’端标记CY5荧光基团,3’端标记BHQ 1荧光淬灭基团;
所述内标的探针5’端标记HEX荧光基团,3’端标记BHQ 1荧光淬灭基团。
本发明所述的试剂盒中,还包括反应液,其中包括:反应缓冲液、dNTPs、MLV酶、Taq酶、MgCl2和水。
本发明所述的试剂盒中,还包括样品处理试剂;所述样品处理试剂包括NaOH溶液、生理盐水和细菌裂解液。所述NaOH溶液中,NaOH的质量分数为4%。本发明所述的试剂盒中,还包括阴性质控品和阳性质控品;
所述阳性质控品为肺炎链球菌、嗜肺军团菌和卡他莫拉菌培养物1:1:1混合;所述阴性质控品为β-珠蛋白假病毒用病毒。
本发明所述的试剂盒适用于多种样品的检测,其可以用于诊断目的的检测,例如,对于痰液等样品的检测,也可用于非诊断目的的检测,例如对于环境中样品的检测,例如,对于取自空调的样品的检测,对于水体的检测等。
本发明还提供了非诊断目的的同时检测肺炎链球菌、嗜肺军团菌和卡他莫拉菌的方法,以本发明所述的试剂盒对样品进行扩增,根据荧光信号判断样品是否存在肺炎链球菌、嗜肺军团菌或卡他莫拉菌。
本发明所述检测的方法的反应体系包括:5μL样品提取物和25μL反应液。
本发明所述检测的方法的反应程序包括:50℃2min;94℃3min;94℃变性10sec,60℃退火30sec,40个循环,每个循环在退火阶段采集荧光信号。
本发明公开了一种同时检测肺炎链球菌、嗜肺军团菌和卡他莫拉菌的荧光PCR检测体系、试剂盒及检测方法。其中,荧光PCR检测体系包括肺炎链球菌、嗜肺军团菌和卡他莫拉菌的特异性保守序列的引物及TaqMan荧光探针,其DNA序列SEQ ID NO:1~9。本发明的优点在于利用多种荧光定量PCR检测技术,实现了同时检测肺炎链球菌、嗜肺军团菌和卡他莫拉菌三种呼吸道病原体的目的,该检测方法快速、准确,且保证了检测的时效性、特异性和灵敏度,在疾病监测、临床诊断等领域具有重要的应用价值。
具体实施方式
本发明提供了同时检测肺炎链球菌、嗜肺军团菌和卡他莫拉菌的试剂盒,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。
表1本发明涉及引物及探针序列
1、引物和TaqMan探针的设计与合成
利用Blast工具对Genbank和国内外文献中的肺炎链球菌、嗜肺军团菌和卡他莫拉菌基因组序列进行分析,分别选择其特异性的保守基因:LytA基因、mip基因和COPB基因作为检测靶序列,并针对这三个检测靶序列设计和合成多套引物和探针(见表1)。引物和探针均由上海英骏生物技术有限公司合成。其中肺炎链球菌的检测探针5’端标记FAM荧光基团,3’端标记BHQ 1荧光淬灭基团,嗜肺军团菌的检测探针5’端标记ROX荧光基团,3’端标记BHQ1荧光淬灭基团,卡他莫拉菌的检测探针5’端标记CY5荧光基团,3’端标记BHQ 1荧光淬灭基团,内标的检测探针5’端标记HEX荧光基团,3’端标记BHQ 1荧光淬灭基团。
2、检测菌种的准备
本实施例中所使用的肺炎链球菌、嗜肺军团菌和卡他莫拉菌均由郑州安图生物工程股份有限公司微生物组提供。
3、质控品的制备
阳性质控品:肺炎链球菌、嗜肺军团菌和卡他莫拉菌培养物1:1:1混合;稀释1000倍,分装0.65mL。阴性质控品:β-珠蛋白假病毒用病毒保存液稀释混匀而成,分装0.65mL。
下面结合实施例,进一步阐述本发明:
实施例1同时检测肺炎链球菌、嗜肺军团菌和卡他莫拉菌的试剂盒的制备
试剂盒包括PCR反应液、阴性质控品、阳性质控品;
其中,PCR反应液的组分如表2:
表2 PCR反应液中各组分以及浓度
组分 | 每个反应体系中的体积 |
反应缓冲液 | 10μL |
dNTPs(10mM) | 2μL |
MLV酶(200U/μL) | 0.1μL |
Taq酶(5U/μL) | 0.3μL |
MgCl2(50mM) | 2μL |
SEQ ID NO:1 | 1μL |
SEQ ID NO:2 | 1μL |
SEQ ID NO:3 | 0.5μL |
SEQ ID NO:4 | 1μL |
SEQ ID NO:5 | 1μL |
SEQ ID NO:6 | 0.5μL |
SEQ ID NO:7 | 1μL |
SEQ ID NO:8 | 1μL |
SEQ ID NO:9 | 0.5μL |
SEQ ID NO:11 | 0.5μL |
SEQ ID NO:12 | 0.5μL |
SEQ ID NO:13 | 0.25μL |
灭菌纯化水 | Up To 25μL |
阳性质控品:肺炎链球菌、嗜肺军团菌和卡他莫拉菌培养物1:1:1混合;稀释1000倍,分装0.65mL。
阴性质控品:β-珠蛋白假病毒用病毒保存液稀释混匀而成,分装0.65mL。
实施例2本发明试剂盒的检测方法
将人痰液样本用移液器混匀,取出200μL痰液加入4倍体积的4%NaOH(自备),摇匀,室温下放置30分钟液化(若痰液仍很粘稠可增加放置时间直至痰液液化),取0.5mL样品至1.5mL离心管种,再加入0.5mL4%NaOH室温放置10分钟后13000rpm离心5分钟。沉淀加入无菌生理盐水1mL打匀,13000rpm离心5分钟;再重复洗涤一次,去尽上清。向沉淀中加入200μL的细菌裂解液(来自北京百奥莱博科技有限公司),充分混匀,100℃水浴10min,13000rpm离心5min备用。
采用实施例1的方法制备的肺炎链球菌、嗜肺军团菌和卡他莫拉菌核酸检测试剂加到PCR反应管中,然后取5μL上述提取好的样品提取物进行检测。
根据扩增片段长短、引物和探针的退火温度以及酶的特性,本发明对反应退火温度和延伸时间进行了优化,最终确定的循环参数为:50℃2min;94℃3min;94℃变性10sec,60℃退火30sec,40个循环,每个循环在退火阶段采集荧光信号。
荧光检测通道选择:(1)选择FAM通道检测肺炎链球菌;(2)选择HEX通道检测内标;(3)选择ROX通道检测嗜肺军团菌;(4)选择CY5通道检测卡他莫拉菌。反应结束后,记录样本Ct值。在扩增结束后按同一条件分析数据,确定各样品的Ct值,具体测试结果分析如下:
(1)当FAM/CY5/ROX通道无值,且HEX通道Ct值≤35(一般为15-30)时,可报告检测结果为阴性。
(2)当FAM/CY5/ROX通道Ct值≤38,且HEX通道Ct值≤35时,可报告检测结果为肺炎链球菌/卡他莫拉菌/嗜肺军团菌阳性。
(3)当FAM/CY5/ROX通道Ct值>38,且HEX通道Ct值≤35时,,可报告样本浓度低于检测下线,结果仅供参考。
(4)当HEX通道Ct值>35时,该检测结果无效,应查找并排除原因,并重复试验。
实施例3本发明试剂盒的可行性试验
1、灵敏度评价
(1)阳性标准品的配制
分别将肺炎链球菌、嗜肺军团菌和卡他莫拉菌的扩增序列克隆于puc57载体中,转化DH5α大肠杆菌,用碱裂解法提取阳性克隆质粒,利用NanoDrop One测定质粒的浓度,然后用超纯水分别稀释成1000copies/μL、750copies/μL、500copies/μL、250copies/μL、100copies/μL的阳性标准品。
(2)实验过程
采用实施例1的方法制备的肺炎链球菌、嗜肺军团菌和卡他莫拉菌核酸检测试剂加到PCR反应管中,然后取5μL上述提取好的样本进行检测,每个浓度做20个复孔,同时分别取5μL阴性质控品和阳性质控品加到检测液中做阴性对照和阳性对照。
(3)实验结果
实验结果表明肺炎链球菌、嗜肺军团菌和卡他莫拉菌检测灵敏度(LOD)分别为5copies/μL、5copies/μL、10copies/μL,本试剂盒3种靶标检测灵敏度较高,操作较为简单,可用于临床对肺炎链球菌、嗜肺军团菌和卡他莫拉菌的辅助诊断。具体结果见表3,表4,表5。
表3:肺炎链球菌最低检测限确认
样本浓度(copies/μL) | 检测重复数 | 阳性检测数 | 阳性检出率 |
0 | 20 | 0 | 0.00% |
2 | 20 | 18 | 90.00% |
5 | 20 | 20 | 100.00% |
10 | 20 | 20 | 100.00% |
20 | 20 | 20 | 100.00% |
表4:嗜肺军团菌最低检测限确认
表5:卡他莫拉菌最低检测限确认
样本浓度(copies/μL) | 检测重复数 | 阳性检测数 | 阳性检出率 |
0 | 20 | 0 | 0.00% |
2 | 20 | 16 | 80.00% |
5 | 20 | 18 | 90.00% |
10 | 20 | 20 | 100.00% |
20 | 20 | 20 | 100.00% |
2、检测特异性的评价
(1)实验样本
采取10份特异性样本对试剂的特异性进行验证,其中1份为生理盐水、1份为金黄色葡萄球菌样本、1份为肺炎克雷伯氏菌样本、1份为流感嗜血杆菌样本、1份为鲍曼不动杆菌样本、1份为大肠埃希氏菌样本、1份为铜绿单胞菌样本、1份为嗜麦芽窄食单胞菌样本、1份为洋葱伯克霍尔德菌样本、1份为耐甲氧西林葡萄球菌样本。
(2)实验过程
采用实施例1的方法制备的肺炎链球菌、嗜肺军团菌和卡他莫拉菌核酸检测试剂分别检测以上10份特异性样本,分析检测结果,验证试剂的特异性。
(3)实验结果
肺炎链球菌、嗜肺军团菌和卡他莫拉菌多重检测试剂检测10份特异性样本皆为阴性,表明试剂特异性好,无交叉反应情况。具体结果见表6。
表6:试剂特异性验证。
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。
序列表
<110> 郑州安图生物工程股份有限公司
<120> 同时检测肺炎链球菌、嗜肺军团菌和卡他莫拉菌的试剂盒
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Claims (7)
1.同时检测肺炎链球菌、嗜肺军团菌和卡他莫拉菌的试剂盒,其特征在于,包括肺炎链球菌、嗜肺军团菌、卡他莫拉菌的检测引物和探针;
所述肺炎链球菌的检测引物和探针中:
肺炎链球菌的正向引物,其核酸序列如SEQ ID NO:1所示;
肺炎链球菌的反向引物,其核酸序列如SEQ ID NO:2所示;
肺炎链球菌的探针,其核酸序列如SEQ ID NO:3所示;
所述嗜肺军团菌的检测引物和探针中:
嗜肺军团菌的正向引物,其核酸序列如SEQ ID NO:4所示;
嗜肺军团菌的反向引物,其核酸序列如SEQ ID NO:5所示;
嗜肺军团菌的探针,其核酸序列如SEQ ID NO:6所示;
所述卡他莫拉菌的检测引物和探针中:
卡他莫拉菌的正向引物,其核酸序列如SEQ ID NO:7所示;
卡他莫拉菌的反向引物,其核酸序列如SEQ ID NO:8所示;
卡他莫拉菌的探针,其核酸序列如SEQ ID NO:9所示。
2.根据权利要求1所述的试剂盒,其特征在于,还包括内标的引物和探针;所述内标的序列为SEQ ID NO:10;
内标的正向引物,其核酸序列如SEQ ID NO:11所示;
内标的反向引物,其核酸序列如SEQ ID NO:12所示;
内标的探针,其核酸序列如SEQ ID NO:13所示。
3.根据权利要求2所述的试剂盒,其特征在于,
所述肺炎链球菌的探针 5’端标记 FAM 荧光基团,3’端标记BHQ 1荧光淬灭基团;
所述嗜肺军团菌的探针 5’端标记 ROX 荧光基团,3’端标记BHQ 1荧光淬灭基团;
所述卡他莫拉菌的探针 5’端标记 CY5荧光基团,3’端标记BHQ 1 荧光淬灭基团;
所述内标的探针 5’端标记HEX荧光基团,3’端标记BHQ 1荧光淬灭基团。
4.根据权利要求1所述的试剂盒,其特征在于,还包括反应液,其中包括:反应缓冲液、dNTPs、MLV酶、Taq酶、MgCl2和水。
5.根据权利要求1~4任一项所述的试剂盒,其特征在于,还包括样品处理试剂;所述样品处理试剂包括NaOH溶液、生理盐水和细菌裂解液。
6.根据权利要求1~4任一项所述的试剂盒,其特征在于,还包括阴性质控品和阳性质控品;
所述阳性质控品为肺炎链球菌、嗜肺军团菌和卡他莫拉菌培养物1:1:1混合;所述阴性质控品为β-珠蛋白假病毒用病毒。
7.非诊断目的的同时检测肺炎链球菌、嗜肺军团菌和卡他莫拉菌的方法,其特征在于,以权利要求1~4任一项所述的试剂盒对样品进行扩增,根据荧光信号判断样品是否存在肺炎链球菌、嗜肺军团菌或卡他莫拉菌。
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