CN104630328B - 肺炎支原体23S rRNA 2064位点A:G突变检测特异性引物和探针 - Google Patents

肺炎支原体23S rRNA 2064位点A:G突变检测特异性引物和探针 Download PDF

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CN104630328B
CN104630328B CN201310550193.3A CN201310550193A CN104630328B CN 104630328 B CN104630328 B CN 104630328B CN 201310550193 A CN201310550193 A CN 201310550193A CN 104630328 B CN104630328 B CN 104630328B
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杜君卿
李杨霞
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Abstract

本发明公开一种用于肺炎支原体23S rRNA 2064位点A:G突变的特异性引物和探针，属于生物技术领域，核苷酸序列包括上游引物MPF序列为5’ GGTGTAACCATCTCTTGA 3’，下游引物MPR序列为5’ CCTGATCAATATTAAGCTACAG 3’和探针2064P序列为5’FAM CGGGGTCTCTCCGTCC BHQ1 3’。

Description

肺炎支原体23S rRNA 2064位点A:G突变检测特异性引物和 探针
技术领域
本发明是一种用于检测肺炎支原体23S rRNA 2064位点A:G突变的特异性引物和探针。
背景技术
肺炎支原体（Mycoplasma pneumoniae，MP）是一种常见的呼吸道病原体，通常导致轻微的上呼吸道感染，如喉咙痛、咽喉炎和气管炎，其引起的肺炎占非细菌性肺炎的50%，还可引起肺外并发症，累及一个或多个系统、器官，如皮肤、胃肠道、心血管、骨骼肌肉和肾脏，严重时可致中枢和外周神经系统病变，甚至死亡。通过飞沫以气溶胶形式传播，存留在鼻、喉、气管和痰液中，密切接触可能引起肺炎支原体的暴发。肺炎支原体无细胞壁，对影响细胞壁合成的抗生素如青霉素等不敏感，但是对影响细菌蛋白合成的抗生素如大环内酯类、喹诺酮类等敏感。大环内酯类是治疗肺炎支原体感染的首选药物，可结合于肺炎支原体核糖体50S亚基的转肽酶中心与肽输出通道之间的部分，通过机械阻塞通道而抑制肽链的延伸，从而达到抑制蛋白合成的目的，其结合位点由23S rRNA结构域Ⅴ的核苷酸构成，其中2064位点是主要的组成部分。近年来，随着大环内酯类抗生素的广泛使用，临床分离出了耐药菌株，提示肺炎支原体耐药现象的存在。国内外的研究表明，产生耐药的肺炎支原体在23S rRNA序列上发生点突变，其中最常见的是2064位点A到G的突变。
目前，临床上对肺炎支原体耐药性的检查，主要是依靠肺炎支原体的分离及药敏实验，由于肺炎支原体分离较为困难，所以灵敏度较低，而且耗时耗力，不利于及时指导用药。本发明采用的荧光PCR技术，操作简便、检测迅速、检出率高，通过一次试验即可明确是否有耐药肺炎支原体的感染，有利于对疾病进行及时诊断及合理选择治疗方案，与传统分离培养检测技术相比，提高了检出效率、降低了漏检率。可对多种临床样本进行高效可靠的筛查，以及对临床病情和用药效果进行监测，指导合理用药。
发明内容
本发明的目的在于提供一种用于检测肺炎支原体23S rRNA 2064位点A:G突变的引物和探针。
基于上述目的，本发明采用以下技术方案：
用于检测肺炎支原体23S rRNA 2064位点A:G突变的特异性引物和探针序列包括：上游引物MPF序列为5’ GGTGTAACCATCTCTTGA 3’，下游引物MPR序列为5’CCTGATCAATATTAAGCTACAG 3’和探针2064P序列为 5’FAM CGGGGTCTCTCCGTCC BHQ1 3’。
本发明的具体原理是利用一对靶核酸序列的特异性引物和探针，采用实时荧光定量PCR技术，通过PCR实现靶核酸序列片断的扩增。所使用的探针为两端分别标记荧光报告基团（R）和荧光淬灭基团（Q）的寡核苷酸。在探针完整时，报告基团发出的荧光被淬灭基团吸收，在PCR扩增过程中，DNA聚合酶的5’端外切酶活性将特异结合在靶核苷酸片断上的荧光探针酶切降解，使报告基团的荧光信号可以被检测，荧光信号量的变化与扩增产物量成正比，从而可以通过荧光强弱来判断待测样本中靶核苷酸序列的存在。
附图说明
图1是利用引物对MPF/MPR和探针2064P检测肺炎支原体23S rRNA 2064位点A:G突变阳性样本的荧光PCR扩增图。
具体实施方式
1.引物和探针的设计：通过分别对所有已知的肺炎支原体23S rDNA序列进行比较分析，选择2064位点所在区段，设计多对引物和探针，引物长度一般为20碱基左右。最优引物探针序列组合如下：
上游引物MPF： 5’ GGTGTAACCATCTCTTGA 3’
下游引物MPR： 5’ CCTGATCAATATTAAGCTACAG 3’
探针2064P： 5’ FAM CGGGGTCTCTCCGTCC BHQ1 3’。
2. 反应体系的优化：利用临床分离的耐药肺炎支原体灭活液作为待检样品，用磁珠裂解法抽提肺炎支原体核酸，分装后贮存于－80℃。
2.1 引物浓度的优化 在反应体系中其它条件相同的情况下，将肺炎支原体引物浓度分别从0.1μmol/L至1.6μmol/L作倍比连续稀释，通过对试验结果的分析比较，确定最佳引物浓度是0.2μmol/L。
2.2 探针浓度的优化 在反应体系中其它条件相同的情况下，将肺炎支原体突变检测用探针浓度分别从0.1μmol/L至0.5μmol/L作倍比连续稀释，通过对试验结果的分析比较，确定最佳探针浓度是0.2μmol/L。
利用上述引物和探针进行反应体系的建立，最后确定采用的肺炎支原体23S rRNA2064位点A:G突变的实时荧光PCR反应体系为25μL。按照荧光定量PCR试剂盒说明进行反应液的配置。
3. 仪器检测通道的选择
选择的荧光检测通道应与探针所标记的报告荧光基团一致，具体按照仪器使用说明书进行设置。
4. PCR反应条件如下
50℃ 2min进行UNG酶反应；95℃ 1min，1 个循环；91℃ 15s，64℃ 1min，40 个循环，在64℃ 1min阶段收集荧光。
5. 检测结果分析
如果待检样本中含有发生2064位点A:G突变的肺炎支原体，则显示阳性扩增曲线，其检测灵敏度可以达到1000 copies/mL，如果待检样本中没有2064位点A:G突变的肺炎支原体，则显示阴性扩增曲线，即无扩增信号，提示上述引物和探针具有良好的特异性和灵敏度。
本发明的优点在于：
（1） 本发明提供的引物和探针的检测灵敏度可以达到1000 copies/mL，说明其灵敏度良好。
（2） 本发明提供的引物和探针对不含2064位点A:G突变的肺炎支原体的样本没有检测信号，说明其特异性强。
（3） 由于本发明是针对2064A:G突变位点，进行了程序和体系优化，避免了假阴性和假阳性结果的出现。

Claims (2)

1.一种用于检测和鉴定肺炎支原体23S rRNA 2064位点A:G突变的特异性引物序列，其特征在于所述的引物序列包括上游引物MPF序列为5’ GGTGTAACCATCTCTTGA 3’，下游引物MPR序列为5’ CCTGATCAATATTAAGCTACAG 3’。
2.一种用于检测和鉴定肺炎支原体23S rRNA 2064位点A:G突变的特异性探针序列，其特征在于所述的探针序列为 5’FAM CGGGGTCTCTCCGTCC BHQ1 3’。
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