CN111378770A - 一种检测肺炎支原体的试剂盒 - Google Patents
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Abstract
本发明公开了肺炎支原体的基于LAMP技术的检测试剂盒。LAMP引物根据肺炎支原体的特异保守序列设计,每组引物包含4条寡核苷酸,通过LAMP反应体系利用恒温扩增仪对肺炎支原体进行荧光鉴定检测。本发明为肺炎支原体测提供了一个新的技术平台,适宜在基层单位、现场监测和床旁检测中推广应用。
Description
技术领域
本发明属于以等温扩增为代表的分子生物学检测方法在肺炎支原体检测方面的应用,即肺炎支原体的环介导等温扩增检测方法和试剂盒。
背景技术
肺炎支原体(M.Pneumonia,Mpn)是兼性厌氧、能独立生活的最小微生物,大小为200 nm。无菌胞壁,可在无细胞的培养基上生长与***繁殖,含有RNA和DNA,经代谢产生能量,对抗生素敏感。支原体为动物多种疾病的致病体,目前已发现8种类型,其中只有肺炎支原体肯定对人致病,主要是呼吸***疾病。在20%马血清和酵母的琼脂培养基上生长良好,初次培养于显微镜下可见典型的呈圆屋顶形桑葚状菌落,多次传代后转呈煎蛋形状。支原体肺炎的病理改变以间质性肺炎为主,有时并发支气管肺炎,称为原发性非典型性肺炎。肺炎支原体主要经飞沫传染,潜伏期2~3周,发病率以青少年最高。临床症状较轻,甚至根本无症状,若有也只是头痛、咽痛、发热、咳嗽等一般的呼吸道症状,但也有个别死亡报道。一年四季均可发生,但多在秋冬时节。
近年来,分子生物学方法不断被应用到呼吸道病原体的快速检测中,呼吸道病原体的实验室诊断方法取得了很大的进展,核酸检测已经成为了呼吸道病原体实验室诊断的发展方向。相对于传统的实验室检测法,分子诊断技术具有无可比拟的检测速度、特异性和灵敏度,已成为实验室诊断的新标准。
其中,环介导等温扩增法具有检测灵敏度高、结果判断直观,且不需要荧光定量PCR仪等价格昂贵的设备等优点,具有广泛应用前景。
环介导等温扩增技术(Loop-mediated isothermal amplification, LAMP)是一种新型等温核酸扩增技术,具有快速、简便、经济、灵敏等优点,目前已被广泛应用于核酸快速检测领域。LAMP的原理是针对靶基因上6个区域设计2对引物(FIP[F1c+F2]、BIP[B2+B1c]、F3、B3),在链置换型DNA聚合酶的作用下,于等温条件在短时间内(15~90min)进行核酸扩增。
在反应液中添加荧光物质SYBR GREEN,利用恒温扩增仪检测荧光曲线,有荧光曲线表示待测样品中存在肺炎支原体(阳性),无扩增曲线表示待测样品中无肺炎支原体(阴性)。
与传统PCR相比,LAMP具有操作简便、灵敏度高、特异性强、结果判定简单、成本低廉等特点。LAMP检测灵敏度至少比普通PCR 高出2个数量级。此外,等温扩增仅需一个水浴锅或恒温装置,对设备的要求简单,其操作过程耗时较短,可在1小时内完成。通过SYBRGREEN等荧光染料对PCR扩增产物的显色,LAMP反应结果的检测可通过等温扩增仪进行可视化呈现,是一种高效、简便、快捷、高通量的检测方法。
因此,开发一种针对肺炎支原体的环介导等温扩增试剂盒具有重要意义。
发明内容
本发明的目的在于:提供一种用于肺炎支原体核酸检测的方法;另一目的在于提供一种用于该方法的试剂盒。
1. 肺炎支原体的LAMP检测专用引物
根据肺炎支原体基因组特异性保守序列,应用在线引物设计软件Primer ExplorerV5,上传靶序列后,即可初步得到多组引物序列。
根据LAMP引物设计的关键因素,主要包括引物末端的稳定性、GC含量、引物间的距离和二级结构对其进行筛选,最终得到的LAMP引物。具体来说,为了在反应中能使F1c和B1c更加容易弯转,可以形成双茎环结构,引物F1c和B1c要高于其他几条引物的Tm值5℃左右。为提高核苷酸越与模板退火结合效率,每条引物最末端的六个碱基自由能⊿G值≤-4Kcal/mol,F3/B3,F2/B2的3’末端⊿G值≤-4Kcal/mol,F1c和B1c的5’端的⊿G值≤-4Kcal/mol。对于引物的GC含量设定范围在40%~60%之间。对于引物之间的距离,从F2的5’端到B2的5’端应在120~180bp之间,从F2的5’端到F1c的3’端也就是茎环段在40~60bp之间,从F2的5’端到F3的3’端在0~20bp之间。最后要特别注意引物之间不能形成二级结构。通过以上设计原则筛选即得到最终引物如下。
F3: 5’-TGCTGCTATTCTCAATCCGG-3’;
B3: 5’-GACCCCACAAGGTTGAACC-3’;
FIP:
5’-CGCTCGTACTCGTTAGCAGCAATTAGCAGCTCTTCCCGACA-3’;
BIP:
5’-AACGGTAGCTCCTACCCAAGGAGGTGGAGAAACGGGAAAGC-3’。
2. 肺炎支原体的LAMP检测方法
本发明所提供的肺炎支原体的LAMP检测方法,主要包括以下步骤:
[1]核酸提取:使用上海复星长征医学科学有限公司生产的磁珠法核酸提取试剂(核酸提取及纯化试剂)对待测样本进行核酸提取。
[2]LAMP扩增
以上述提取的待测物核酸为模板,在专用引物的介导下进行扩增。其中,LAMP反应体系(20 μl)包括:待测物的基因组DNA 3μL,20mM Tris·HCl (pH8.8),10mM(NH4)2SO4,50mMKCl,2mM MgSO4,0.1% Tween20,1M Betaine,0.4mM dNTP each,8U Bst DNA polymerase,SYBR GREEN(1×DMSO PCR级),0.2 μM F3,0.2 μM B3,0.8 μM FIP和0.8 μM BIP。LAMP扩增条件可设定为58~68℃恒温反应15~60min,优选为63℃恒温反应30min。
[3]结果判定
利用恒温扩增仪检测荧光曲线,有荧光曲线表示待测样品中存在肺炎支原体(阳性),无扩增曲线表示待测样品中无肺炎支原体(阴性)。
3. 肺炎支原体的LAMP检测试剂盒
本发明所提供的肺炎支原体的LAMP检测试剂盒,包含有上述LAMP检测的专用引物、主要试剂和反应参数,本发明具有以下优点:
[1]高特异性
4条引物对靶序列的6个特异区域的识别保证了LAMP扩增的高度特异性,即LAMP能够从相差仅一个核苷酸的基因标本中寻找出相应的靶序列进行扩增;
[2]高效扩增
灵敏度比普通PCR高约100倍;
[3]操作简便
只需将待检样品(靶核酸)和检测试剂置于约63℃恒温扩增仪中反应30min左右即可判断结果;
[4]结果直观
利用恒温扩增仪检测荧光曲线观察反应结果。本发明可以在等温条件下简便快速(63℃恒温反应30min)、高效特异地检测到肺炎支原体。该法不需要复杂仪器,为肺炎支原体的检测提供了一个新的技术平台,尤其适用于人群筛查,具有广阔的市场前景和较大的经济、社会效益,适于大范围推广应用。
具体实施方式
现以本发明技术方案为前提,列举出详细的实施方式和具体的操作过程,但本发明的保护范围不限于下述的实施例子。下述实施例中所用方法如无特别说明均为常规方法。
实施例1、用于对肺炎支原体进行LAMP检测的引物设计
利用NCBI数据库检索获得肺炎支原体基因组特异性保守序列,应用在线引物设计软件PrimerExplorer V5,上传靶序列后,即可初步得到多组引物序列。根据LAMP引物设计的关键因素,主要包括引物末端的稳定性、GC含量、引物间的距离和二级结构对其进行筛选,最终得到的LAMP引物。
实施例2、用于肺炎支原体进行LAMP检测方法的建立
用实施例1获得的用于对肺炎支原体进行LAMP检测的引物对鼻咽拭子样本核酸提取液进行LAMP检测,具体操作步骤如下:
[1]反应体系
使用上海复星长征医学科学有限公司生产的磁珠法核酸提取试剂(核酸提取及纯化试剂)对待测样本进行核酸提取,将提取产物作为模板,在实施例1获得的LAMP专用引物的引导下进行等温扩增。其中,LAMP反应体系(20 μl)包括:待测物的基因组DNA 3μL,20mMTris·HCl (pH8.8),10mM(NH4)2SO4,50mM KCl,2mM MgSO4,0.1% Tween20,1M Betaine,0.4mM dNTP each,8U Bst DNA polymerase,SYBR GREEN(1×DMSO PCR级),0.2μM F3,0.2μM B3,0.8μM FIP和0.8μM BIP。
[2]结果判定
利用恒温扩增仪检测荧光曲线,有荧光曲线表示待测样品中存在肺炎支原体(阳性),无扩增曲线表示待测样品中无肺炎支原体(阴性)。
实施例3、制备肺炎支原体的LAMP检测试剂盒
将LAMP反应发生混合物即LAMP反应引物(4μM F3和B3;16μM FIP和BIP)、LAMP反应酶(8U/μL Bst DNA polymerase)、反应缓冲液(40mM Tris·HCl, pH8.8;20mM (NH4)2SO4;100mM KCl;4mM MgSO4;0.2% Tween20;2M Betaine;0.8mM dNTP each、SYBR GREEN(2×DMSO PCR级))和阳性对照(含有肺炎支原体基因组特异性保守序列的质粒稀释液)共同包装,得到肺炎支原体的LAMP检测试剂盒。
Claims (4)
1.一种用于肺炎支原体的LAMP检测方法和反应体系。
2.一种肺炎支原体的LAMP检测方法,主要包括以下步骤:以待测物的基因组DNA为模板,在权利要求1所述专用引物的引导下进行LAMP扩增,通过在反应液中添加SYBR GREEN,利用恒温扩增仪检测荧光曲线,有荧光曲线表示待测样品中存在肺炎支原体、结果为阳性,无扩增曲线表示待测样品中无肺炎支原体、结果为阴性。
3.根据权利要求2所述的检测方法,其特征在于:使用磁珠法对待测样本进行核酸提取;LAMP反应体系包括:待测物的基因组DNA 3μL,20mM Tris·HCl (pH8.8),10mM(NH4)2SO4,50mM KCl,2mM MgSO4,0.1% Tween20,1M Betaine,0.4mM dNTP each,8U Bst DNApolymerase,SYBR GREEN(1×DMSO PCR级),0.2μM F3,0.2μM B3,0.8μM FIP和0.8μM BIP。
4.根据权利要求2所述的检测方法,其特征在于LAMP扩增条件为:反应管置于58~68℃恒温反应15~60min,优选为63℃恒温反应30min。
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