CN111534618B - 一种23SrRNA基因A380C突变位点的应用 - Google Patents
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Abstract
一种23SrRNA基因A380C突变位点的应用，用于对Hp的耐药性检测，研究Hp菌株耐药突变的规律与机制，有望提高分子生物学检测耐药变异的准确率，从而针对性地选择敏感抗生素对患者进行个体化治疗。
Description
技术领域
本发明属于生物技术领域，具体涉及一种23SrRNA基因A380C突变位点的应用。
背景技术
幽门螺杆菌（Helicobacter pylori，Hp）是慢性胃炎、消化性溃疡、消化不良和胃粘膜相关淋巴组织（MALT）淋巴瘤的主要致病因子，并与胃癌的发生关系密切，目前已被WHO癌症研究委员会列为Ⅰ类致癌因子，而Hp的根除可有效防治上述疾病。全球有超过50%的人感染Hp，在我国，成人中感染率高达40~90%。由于临床抗生素的不合理使用，Hp的耐药性呈现逐年上升趋势，含克拉霉素的标准三联疗法的根除率已经低于或远低于80%。近年来国内外新推荐了左氧氟沙星三联疗法等根除方案，但在治疗中也逐渐产生了耐药性。因此，专家意见及最新共识均认为，已有耐药迹象的患者在治疗前，应先做耐药性试验，以便指导合适药物的选择。
近年来研究表明，Hp相关基因的单核苷酸多态性（SNP）可导致克拉霉素耐药，Hp23SrRNA基因V区的A2142G和A2143G突变是克拉霉素耐药的主要原因，另外还存在一些常见的低水平耐药突变位点，如A2142C 等；Hp gyrA的Asn87和Asp91突变是喹诺酮耐药的主要原因。
经本发明人研究发现Hp突变位点具有特异性的特点，因此，研究Hp菌株耐药突变的规律与机制，对于提高分子生物学检测耐药变异的准确率，从而针对性地选择敏感抗生素对患者进行个体化治疗是十分有必要的。
发明内容
针对上述存在的问题，本发明的目的是提供一种23SrRNA基因A380C突变位点的应用，目前还没有该突变位点与Hp耐药性有关的相关报道。
具体地，一种23SrRNA基因A380C突变位点的应用，用于对Hp的耐药性检测。
本发明的目的在于提供A380C突变位点在制备Hp耐药性检测产品中的应用。
进一步地，检测产品中至少包含一对特异性扩增23SrRNA基因A380C突变位点的引物，包括：正向引物：F：GTTGAGGACTGCAACATCCACGAGAACGCTTTAGCAGAGT；
反向引物：R：TGGATGTTGCAGTCCTCAACCCCGAATGCAAGCACTCGGT。
所述Hp耐药性为Hp对克拉霉素的耐药性。
具体实施方式
一、材料与方法
（1）收集地域性标本：收集湖州市胃炎、胃溃疡患者共492例，每例距幽门附近2公分处夹取 2份胃黏膜样本：1份样本A置于含5%甘油的牛脑心浸液中；另1份样本B置于装有Tris-EDTA溶液的无菌EP管中，于-80℃保存待用，得到A组样本和B组样本。
（2）Hp的分离培养与鉴定：将A组各个样本的组织研磨混匀液接种于含5%新鲜去纤维蛋白绵羊血的哥伦比亚琼脂平板上，于37 ℃微需氧环境中(5%O2, 10%CO2, 85%N2)培养3~11 d，观察结果；挑选菌落形态典型、经涂片镜检菌体形态符合且氧化酶、过氧化氢酶和尿素酶试验阳性的Hp，在增菌培养后收取菌苔，采用生理盐水稀释浓度至2个麦氏单位，保存备用。
（3）抗生素敏感性测试并分组：采用平板掺入法，用琼脂将克拉霉素和左氧氟沙星稀释到临界点耐药浓度(1μg/mL和2μg/mL)，倾注平板；加2μL菌悬液于平板上，待其干燥后，置于微需氧环境(5%O2, 10%CO2, 85%N2和湿度>80%)中培养72 h后判读药敏结果；实验均重复2次，以标准菌株26695作为质控菌株，根据表型药敏试验结果将获得的临床分离株分为克拉霉素敏感组、克拉霉素耐药组、左氧氟沙星敏感组和左氧氟沙星耐药组。
（4）提取幽门螺杆菌DNA：将B组样本按照TIANamp Genomic DNA Kit试剂盒说明书抽提菌株的DNA，-20℃保存备用。
（5）PCR扩增目的基因：用上述提取的Hp基因组DNA作为模板，根据 Hp 基因(GenBank:U26695)序列设计 23S rRNA、gyrA和gyrB 基因片段扩增及测序基因引物，如下表1所示：
反应体系25μL: 2×Taq PCR MasterMix 12.5μL、上下游引物（10μmol/L）各0.5 μL、DNA 1μL、ddH2O 11.5μL；PCR产物用琼脂糖凝胶电泳检测，最后用TIANgel MidiPurification Kit进行DNA的回收和纯化。
（6）筛查耐药位点：通过Vector NTI suite 软件将扩增片段序列和Hp标准株26695序列进行比对，筛选出湖州地区人群感染Hp后克拉霉素/左氧氟沙星耐药的突变位点。
（7）应用 SPSS22.0 统计软件进行分析，因总样本量n＜40，应用 Fisher 确切概率法统计，P＜0.05 为差异有统计学意义。
二、结果
（1）Hp分离培养：共492份胃黏膜样本进行Hp培养，经生化反应鉴定显示过氧化氢酶、氧化酶和尿素酶者确定为Hp，共成功分离出Hp菌株247株。
（2）Hp耐药检测：对成功分离的247株Hp菌株进行耐药性检测，发现59株对克拉霉素耐药，占总菌数的23.9% ，58株对左氧氟沙星耐药，占总菌数的23.5%。
（3）23SrRNA基因突变检测：选择对克拉霉素耐药（16株）和敏感 （16株）的Hp菌株进行23S rRNA 基因序列分析，寻找与克拉霉素耐药相关的突变位点，共发现43个突变位点。其中，T465G，A757T，A1821G，G1826A，T1830C，G2864A，G2917A突变在敏感与耐药菌株中均存在，且突变率为 100%；其余36个突变位点在耐药组和敏感组中的分布情况如下表2所示：
表2.32株幽门螺杆菌菌株（包括16株克拉霉素耐药菌株和16株克拉霉素敏感菌株）23S rRNA的突变位点分布
上表结果表明，A380C和A2143G两组间比较差异有统计学意义（P<0.05）。
（4）gyrA基因突变检测：选择对左氧氟沙星耐药（16 株）和敏感（16 株）的 Hp 菌株进行 gyrA 基因序列分析，寻找与左氧氟沙星耐药相关的突变位点。共发现52个突变位点。其中，G468E，P484Q，K527R，S539N，E632G，S637Y，V656I，H669Y，E680D，G734E，G756S，R785K，G788I，A792T，N799D，M805V，V807A/T，T821N突变在敏感与耐药菌株中均存在，且突变率为 100%；其余34个突变位点在耐药组和敏感组中的分布情况如下表3所示：
表3.32株幽门螺杆菌菌株（包括16株左氧氟沙星耐药菌株和16株左氧氟沙星敏感菌株）gyrA的突变位点分布
上表结果表明，N87K/I、 D91G/N/Y两组间比较差异有统计学意义（P<0.05）。
（5）gyrB基因突变检测：选择对左氧氟沙星耐药（16 株）和敏感（15 株）的 Hp 菌株进行 gyrB 基因序列分析，寻找与左氧氟沙星耐药相关的突变位点。共发现24个突变位点。其中，T149I/V，G298D，K304R，L610F，V614I突变在敏感与耐药菌株中均存在，且突变率为 100%；其余19个突变位点在耐药组和敏感组中的分布情况如下表4所示：
表4.31株幽门螺杆菌菌株（包括16株左氧氟沙星耐药菌株和15株左氧氟沙星敏感菌株）gyrB的突变位点分布
上表结果表明，突变位点均不具统计学意义（P>0.05）。
三、讨论
1.本研究结果显示，gyrA基因上N87K/I 和 D91G/N/Y 的氨基酸突变方式是本地区 Hp 耐左氧氟沙星的主要突变方式，且多为单一位点突变，而gyrB基因上不存在明显与左氧氟沙星耐药相关的突变。
2.本研究结果显示，16株克拉霉素耐药菌株中在23SrRNA基因V区外的A380C位点发生突变，且存在统计学差异（P<0.05），突变率为31.3%，说明本地区Hp的23SrRNA基因上除了A2143G的经典突变位点外，还存在A380C的突变位点，上述两个突变位点是本地区 Hp 耐克拉霉素耐药的主要突变位点，因此湖州地区 Hp对克拉霉素耐药的分子机制除了Hp23SrRNA基因 A2143G 突变外，还可继续深入研究A380C突变作为该地区对克拉霉素耐药的分子机制，为临床个体化治疗提供指导，从而控制 Hp 相关性疾病的发生与发展。
综上所述，本发明人针对上述实验数据提出了一种针对湖州地区Hp耐药性检测的检测产品，具体包括对左氧氟沙星和克拉霉素的耐药性检测，除了A2143G、N87K/I 和D91G/N/Y经典位点的扩增引物外，还额外包含一对特异性扩增A380C突变位点的引物：
正向引物：F：GTTGAGGACTGCAACATCCACGAGAACGCTTTAGCAGAGT；
反向引物：R：TGGATGTTGCAGTCCTCAACCCCGAATGCAAGCACTCGGT。
采用本发明对本地区Hp耐药基因进行检测和判定，可以有针对性的鉴定出耐药基因位点突变的具体情况，有助于本地区Hp的耐药机制的研究，能为临床个体化治疗提供指导，从而控制 Hp 相关性疾病的发生与发展。
本文中所描述的具体实施例仅仅是对本发明精神作举例说明。本发明所属技术领域的技术人员可以对所描述的具体实施例做各种各样的修改或补充或采用类似的方式替代，但并不会偏离本发明的精神或者超越所附权利要求书所定义的范围。
Claims (4)
1.一种23SrRNA基因A380C突变位点的应用，用于非疾病诊断目的的Hp耐药性检测。
2.根据权利要求1所述的一种23SrRNA基因A380C突变位点的应用，用于制备Hp耐药性检测的检测产品。
3.根据权利要求2所述的一种23SrRNA基因A380C突变位点的应用，其特征在于，所述检测产品中至少包含一对特异性扩增A380C突变位点的引物。
4.根据权利要求1~3任一所述的一种23SrRNA基因A380C突变位点的应用，其特征在于，所述Hp耐药性为Hp对克拉霉素的耐药性。
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