CN111417729B - 基于Quantamatrix多重分析平台的幽门螺杆菌及可确认有关其菌的抗生素耐性的基因的检测的诊断法及其应用 - Google Patents
基于Quantamatrix多重分析平台的幽门螺杆菌及可确认有关其菌的抗生素耐性的基因的检测的诊断法及其应用 Download PDFInfo
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Abstract
本发明涉及一种能够鉴定幽门螺杆菌的同时确认幽门螺杆菌的耐药剂基因的方法、使用于其的组合物及其试剂盒,上述方法包括:步骤a),从样本试样分离脱氧核糖核酸(DNA);步骤b),使用序列1至序列8的引物,从上述脱氧核糖核酸进行聚合酶链式反应;以及步骤c),杂交形成偶联有序列9至序列23的低聚物探针的盘和从上述步骤b)中获得的聚合酶链式反应扩增产物。本发明基于Quantamatrix多重分析平台(QMAP)可以确认鉴定幽门螺杆菌时一起使用的干预第一药剂的克拉霉素(clarithromycin)、四环素(Tetracycle)及氟喹诺酮类(fluoroquinolones)的23S核糖体脱氧核糖核酸(rDNA)、16S核糖体脱氧核糖核酸、旋转酶(gyrase)基因是否具有耐性。
Description
技术领域
本发明涉及基于Quantamatrix多重分析平台(QMAP)的幽门螺杆菌及可确认有关其菌的抗生素耐性的基因的检测的诊断法及其应用。
背景技术
螺杆菌(Helicobacter pylori,H.pylori)是一种寄生于胃粘膜和粘液之间的螺旋形革兰氏阴性细菌(Owen R.J.Helicobacter-species classification andidentification.British Medical Bulletin.1998;54(1):17-30.doi:10.1093/oxfordjournals.bmb.a011667.),自1983年首次在胃粘膜中培养以来,已知它是引起胃炎、胃溃疡、慢性宫颈胃炎、胃癌和与粘膜相关的淋巴样组织(MALT,mucosa-as-sociatedlymphoid tissue)淋巴瘤的细菌。
幽门螺杆菌是一种带有数个鞭毛的螺旋形细菌,而螺杆菌是已知唯一能够在强酸性环境的人胃中生存的细菌,主要感染胃肠道粘膜、胃炎、胃溃疡,十二指肠溃疡、胃腺癌和胃淋巴瘤等。发现于胃肠道粘膜的表面活胃肠的黏液,通过穿透胃肠道粘膜细胞本身来感染是非常罕见的。在强酸性胃肠道环境中具有脲酶(urease),这在通过创造碱性环境来帮助细菌在胃肠道粘膜中存活起着必要作用。
据报道,在被螺杆菌感染的情况下,胃癌的风险要高达3.8倍,1994年国际卫生组织(WHO)旗下的国际癌症研究所(IARC)判定它是胃癌的起因,并规定为人类的一流致癌因素。已知螺杆菌的感染率为全世界成年人的50%左右,据报道,发展中国家的螺杆菌感染率达到80~90%(Ndip R.N.,Malange Takang A.E.,Ojongokpoko J.E.A.,etal.Helicobacter pylori isolates recovered from gastric biopsies of patientswith gastro-duodenal pathologies in Cameroon:current status ofantibiogram.Tropical Medicine and International Health.2008;13(6):848-854;Tanih N.F.,Ndip L.M.,Clarke A.M.,Ndip R.N.An overview of pathogenesis andepidemiology of helicobacter pylori infection.African Journal of MicrobiologyResearch.2010;4(6):426-436.),尤其,与西方国家相比,韩国人中螺杆菌感染率很高,韩国的胃癌发生率是世界上最高的,已知螺杆菌感染者中65%患有胃炎,10~20%患有胃溃疡,并且,在60~80%的胃溃疡患者以及90~95%的十二指肠溃疡患者中发现螺杆菌,感染螺杆菌的人患胃癌的概率是未感染螺杆菌的人的两三倍。
因此,预期螺杆菌除菌治疗可能在预防胃癌中起重要作用。幽门螺杆菌感染的治疗利用铋剂和抗生素的组合疗法。
目前,最有代表性的组合疗法是将基于质子泵抑制剂(proton pump inhibitor,PPI)的克拉霉素(clarithromycin)(500mg每日两次(bid))和(amoxicillin)阿莫西林(1g每日两次)的联合疗法用作第一治疗剂,或者将氟喹诺酮类(fluoroquinolones)和四环素(tetracycline)等抗生素用作第二治疗剂(Lee J.W.,Kim N.,Kim J.M.,etal.Prevalence of primary and secondary antimicrobial resistance ofHelicobacter pylori in Korea from 2003 through 2012.Helicobacter.2013;18(3):206-214.)。在用第一治疗剂进行除菌治疗的情况下,已知除菌率为55~90%,根据韩国的一项研究,从1991年至1998年,标准第一治疗法的除菌率为90%以上,非常有效,相反,据报道,2000年之后的除菌率小于80%,因此正在进行用于提高除菌率的研究[The KoreanJournal of Helicobacter and Upper Gastrointestinal Research,Vol.11,No.1,26-36,June 2011]。
然而,螺杆菌对抗生素的耐性增加已成为全球性问题,导致治疗失败(MahmoudiS,Antibiotic susceptibility of Helicobacter pylori strains isolated fromIranian children:High frequency of A2143G point mutation associated withclarithromycin resistance.J Glob Antimicrob Resist.2017 Jul 15;10:131-135.)。
在抗生素治疗法中,除菌率下降的原因在于,螺杆菌存在于胃肠道粘膜表面活黏液中,因此抗生素有效成分无法到达存在螺杆菌的地方的情况较多,在多次暴露于抗生素的情况下,还有一个原因是对药物产生耐性而不易治疗。并且,由于所使用的药物有剧毒,在服用抗生素过程中任意停止服用的情况下,存在因抗生素耐性而可能难以进行下一步治疗的问题点。幽门螺杆菌是一种增殖慢且需要较长的培养时间的细菌,因此难以培养,进而存在难以确定对抗生素的耐性的问题点。在某些情况下,有一种可以确认是否对克拉霉素具有耐性的诊断产品,但是,由于难以确认对第一药剂的总体耐性,因此需要一种可以确认对耐抗生素性的耐性诊断产品。
Quantamatrix多重分析平台系统(Quantamatrix Multiplexed Assay Platformsystem,QMAP)移向Quantamatrix公司的原始技术(专利授权号1011013100000(2011年12月26日)/1015823840000(2015年12月28日),基于悬浮点阵技术(suspension arraytechnology),将探针与50μm大小的带磁性的盘(Microdisk)结合,并使其与聚合酶链式反应(PCR)产物反应后,可用荧光确认是否突变的检查法,Quantamatrix多重分析平台的最大特征是可以通过在盘上刻上唯一的代码来区分盘而不会产生干扰,从技术上来看,可以使用1024个代码,因此可使用1024种类型的盘进行多次检查,每个盘都有自己的代码,并且由于所有过程均在96孔板上进行,因此是一种具有高流通量(high throughput)的系统。
现有专利文献:
专利公开号1020030024129
发明内容
技术问题
本发明鉴于上述需要提出,本发明的目的在于,提供一种可鉴定新型幽门螺杆菌的同时确认幽门螺杆菌的耐药剂基因的方法。
本发明的另一目的在于,提供可鉴定新型幽门螺杆菌的同时确认幽门螺杆菌的耐药剂基因的引物、探针、它们的组合物及试剂盒。
技术方案
为了实现上述目的,本发明提供一种可鉴定幽门螺杆菌的同时确认幽门螺杆菌的耐药剂基因的方法,包括:步骤a),从样本试样分离脱氧核糖核酸(DNA);步骤b),使用序列1至序列8的引物从上述脱氧核糖核酸进行聚合酶链式反应扩增;以及步骤c),杂交形成偶联有序列9至序列23的低聚物探针的盘和从上述步骤b)中获得的聚合酶链式反应扩增产物。
在本发明的一实例中,优选地,上述药剂为选自由克拉霉素、四环素及氟喹诺酮类组成的组中的药剂,但并不限定于此。
在本发明的另一实例中,优选地,在上述扩增产物为螺杆菌的情况下大小为130bp,在克拉霉素的情况下大小为121bp,在四环素的情况下大小为143bp,以及在氟喹诺酮类的情况下大小为141bp,但并不限定于此。
并且,本发明提供一种可鉴定菌包含偶联有序列1至序列8的引物及序列9至序列23的低聚物探针的盘的幽门螺杆菌的同时确认幽门螺杆菌的耐药剂基因的试剂盒。
优选地,上述试剂盒为用于进行聚合酶链式扩增反应的试剂,还包含脱氧核糖核酸聚合酶、三磷酸碱基脱氧核苷酸(dNTPs)及缓冲液,但并不限定于此。
并且,本发明提供可鉴定包含偶联有序列1至序列8的引物及序列9至序列23的低聚物探针幽门螺杆菌的同时确认幽门螺杆菌的耐药剂基因的组合物。
本发明通过开发基于Quantamatrix多重分析平台系统的分子诊断检查法,其可确认鉴定幽门螺杆菌的鉴定时一起使用的干预第一药剂的克拉霉素、四环素及氟喹诺酮类的23S核糖体脱氧核糖核酸(rDNA)、16S核糖体脱氧核糖核酸、旋转酶(gyrase)基因是否具有耐性,从而评估其有用性。
发明的效果
通过本发明可以看出,本发明基于Quantamatrix多重分析平台,可以确认与幽门螺杆菌的鉴定一起使用的干预第一药剂的克拉霉素、四环素及氟喹诺酮类的23S核糖体脱氧核糖核酸、16S核糖体脱氧核糖核酸、旋转酶基因是否具有耐性。
附图说明
图1为有关Quantamatrix多重分析平台系统的图。
具体实施方式
下面,通过非限定性例来进一步详细说明本发明。但是,下述实施例仅用于示例本发明,本发明的范围不应解释为限定于下述实施例。
实施例1.材料
为了检验开发的引物和探针,临床分离的检验完成的野生型/突变型(wild/mutant)幽门螺杆菌使用延世大学原州校区临床病理学与微生物学教室提供的,为了检验QMAP HP-DR法的特异度,使用15种26个标准菌株[大肠杆菌(Escherichia)(2)、志贺氏杆菌(Shigella)(4)、沙门氏菌(Salmonella)(2)、克雷伯氏菌(Klebsiella)(1)、肠杆菌(Enterobacter)(1)、柠檬酸杆菌(Citrobacter)(1)、假单胞菌(Pseudomonas)(1)、不动杆菌(Acinetobacter)(1)、嗜血杆菌(Haemophlius)(1)、弯曲杆菌(Campylobacter)(1)、耶尔森氏菌(Yersinia)(2)、葡萄球菌(Staphylococcus)(4)、链球菌(Streptococcus)(2)、肠球菌(Enterococcus)(1)及李斯特菌(Listeria)(2)]进行测试。
实施例2.脱氧核糖核酸的提取
通过如下方法分离核酸,即,取一部分通过培养液分离的分离菌加入100μL的脱氧核糖核酸提取液(extraction solution)后涡流(vortex)1分钟,在100℃的温度下加热10分钟后,在室温下以13000rpm离心3分钟来取得到的上清液。分离的虎算用作用于进行QMAPHP-DR的聚合酶链式反应的模板。
实施例3.QMAP HP-DR的基因的收集及分析
在国家生物技术信息中心(National center for biotechnology information,NCBI,http://www.ncbi.nlm.nih.gov)基因库(Genbank)中,作为靶标的聚合酶链式反应引物和寡核苷酸探针通过搜索用于准确地鉴定幽门螺杆菌的尿素酶(urease)基因,以及可以看出幽门螺杆菌是否对抗生素具有耐性的克拉霉素(23S核糖体脱氧核糖核酸)、四环素(16S核糖体脱氧核糖核酸)和氟喹诺酮类(旋转酶A)基因来收集了碱基序列。进行多重比较(http://multalin.toulouse.inra.fr/multalin)后,在作为靶标的每个基因共有的碱基序列位点设计了两对引物(primer),其中,在反向引物(reverse primer)中,将生物素(biotin)连接到5’末端以适用于Quantamatrix多重分析平台。而且,设计了可在两对引物内部中存在的菌特异性位点检测每个靶标菌种的寡核苷酸探针,每个寡核苷酸探针均的5’端连接有一个胺基,以使肽与薄膜结合。设计的引物和寡核苷酸探针委托百奥尼(Bioneer)(韩国大田)合成。
实施例4.聚合酶链式反应
为了进行基于Quantamatrix多重分析平台的HP-DR,针对于从样本分离的核酸,使用生物素标记的引物进行如下聚合酶链式反应。以提取的各菌株的基因组脱氧核糖核酸(genomic DNA)为模板,利用商用化的引物Taq预混料(Prime Taq Premix)(2X)(韩国论山Genet Bio)进行聚合酶链式反应。引物Taq预混料(2X)的组成如下,即,1单位/10μl的引物Taq聚合酶(primer Taq polymerase)、2X的反应缓冲液(reaction buffer)、4mM的MgCl2、酶稳定剂(enzyme stabilizer)、沉淀物(sedment)、加载染料(loading dye)、pH 9.0、0.5mM的任意三磷酸腺嘌呤脱氧核苷酸(dATP)、三磷酸胞嘧啶脱氧核苷酸(dCTP)、三磷酸鸟嘌呤脱氧核苷酸(dGTP)、三磷酸胸腺嘧啶脱氧核苷酸(dTTP)。用于聚合酶链式反应的组成如下,即10μl的引物Taq预混料(2X)、分别1μl的10pmole的一对引物、3μl的超纯水(ultrapure water),5μl的各菌株的基因组脱氧核糖核酸,并且总量为20μl。在聚合酶链式反应中,为了进行初始变性过程在95℃的温度下反应5分钟,为了扩增在95℃的温度下反应30秒钟,在60℃的温度下反应30秒钟,并重复40次后,为了完整的延伸反应,在72℃的温度下反应10分钟。聚合酶链式反应完成后,将聚合酶链式反应产物在2%的三硼酸-乙二胺四乙酸二钠盐二水合物(Tris-borate-ethylenediaminetetraacetic acid disodium saltdihydrate,TBE)在琼脂糖基因(agarose gene)(W/V%)上用290筛(bolt)电泳20分钟,用溴化乙锭(ethidium bromide)染色10分钟后,用紫外线透射光确认是否扩增。
实施例5.基于Quantamatrix多重分析平台的HP-DR的进行
利用扩增的聚合酶链式反应产物的QMAP HP-DR是在制造商建议的实验条件下进行的,实验方法如下。在扩增的聚合酶链式反应产物中混合等量的变性溶液(Denaturationsolution)(0.2N的NaOH、0.2mM的乙二胺四乙酸(EDTA))并在室温下放置5分钟后,在杂交缓冲液(hybridization buffer)中稀释,将其添加到准备好的偶联盘(disk)(韩国首尔Quantamatrix)后,在40℃的温度下反应30分钟,在25℃的温度下使用洗涤液(washingsolution,WS)1分钟内洗涤3次后,处理以1∶2000(v/v)稀释的链霉亲和素R-藻红蛋白缀合物(streptavidin R-phycoerythrin conjugate)(加利福尼亚州圣莱安德罗Prozyme),并在室温下反应10分钟。将结束反应的微盘(microdisk)在室温下用洗涤缓冲液(washingbuffer)在1分钟内洗涤3次。针对于用于区分阳性和阴性结果的截止(cut-off)值,在盘的荧光信号值为500以上的情况下提供阳性结果,在500以下的情况下提供阴性结果的通过Quantamatrix多重分析平台软件(QMAP software)自动测量盘的图像,以检测幽门螺杆菌(H.pylori),并可确认克拉霉素是否具有耐性(23S核糖体脱氧核糖核酸)、四环素是否具有耐性(16S核糖体脱氧核糖核酸)、氟喹诺酮类是否具有耐性(gyrA)。
上述实施例的结果如下。
基于Quantamatrix多重分析平台的幽门螺杆菌特异性基因-UreC检测确以
为了去人对基于Quantamatrix多重分析平台的幽门螺杆菌特异性的基因UreC探针检测的有效性,利用幽门螺杆菌临床菌株进行测试,其结果在所有幽门螺杆菌相关菌株中均检测到ureC基因(表1)。
在下述表1至表5中用红色表示显示阳性的部分。
表1
表1为用于QMAP HP-DR的UreC基因的检测结果
基于Quantamatrix多重分析平台的幽门螺杆菌的克拉霉素耐性基因的检测确认
为了确认基于Quantamatrix多重分析平台的幽门螺杆菌的作为克拉霉素耐性基因的23S rRNA(A2142G、A2142C、A2143G)的检测,利用具有该突变的菌株进行了测试,其结果可以确认,仅在相关耐性基因中分别检测到(表2)。
表2
表2为用于QMAP HP-DR的23S rRNA基因(A2142G、A2142C、A2143G)的检测结果
基于Quantamatrix多重分析平台的幽门螺杆菌的四环素耐性基因的检测确认
为了确认基于Quantamatrix多重分析平台的幽门螺杆菌的作为四环素耐性基因的16s核糖体核糖核酸(rRNA)(AGA965-967TTC)的检测,利用具有该突变的菌株进行了测试,其结果可以确认,仅在相关耐性基因中分别检测到(表3)。
表3
表3为用于QMAP HP-DR的16S rRNA基因(16S AGA-TTC)的检测结果
基于Quantamatrix多重分析平台的幽门螺杆菌的氟喹诺酮类耐性基因的检测确认
为了确认基于Quantamatrix多重分析平台的幽门螺杆菌作为氟喹诺酮类耐性基因的旋转酶A(密码子87-AAT、AAC、AAG、AAA)的检测,利用具有该突变的菌株进行了测试,其结果可以确认,仅在相关耐性基因中分别检测到(表4)。
表4
表4为用于QMAP HP-DR的gyrA基因(密码子87-AAT、AAC、AAG、AAA)的检测结果
并且,为了确认旋转酶A(密码子91-TAT、GGT、AAT)的检测,利用具有该突变的菌株进行了测试,其结果可以确认,仅在相关耐性基因中分别检测到(表5)。
表5
表5为用于QMAP HP-DR的gyrA基因(密码子91-TAT、GGT、AAT)的检测结果
表6
表6为使用于本发明的引物及探针的碱基序列。
Claims (8)
1.一种能够鉴定幽门螺杆菌的同时确认幽门螺杆菌的耐药剂基因的非疾病诊断治疗目的的方法,其特征在于,包括:
步骤a),从样本试样分离脱氧核糖核酸;
步骤b),使用序列1至序列8的引物从上述脱氧核糖核酸进行聚合酶链式反应扩增;以及
步骤c),杂交形成偶联有序列9至序列23的低聚物探针的盘和从上述步骤b)中获得的聚合酶链式反应扩增产物。
2.根据权利要求1所述的能够鉴定幽门螺杆菌的同时确认幽门螺杆菌的耐药剂基因的非疾病诊断治疗目的的方法,其特征在于,上述药剂选自由克拉霉素、四环素及氟喹诺酮类组成的组中。
3.根据权利要求1所述的能够鉴定幽门螺杆菌的同时确认幽门螺杆菌的耐药剂基因的非疾病诊断治疗目的的方法,其特征在于,在上述扩增产物为螺杆菌的情况下大小为130bp,在克拉霉素的情况下大小为121bp,在四环素的情况下大小为143bp,以及在氟喹诺酮类的情况下大小为141bp。
4.一种能够鉴定幽门螺杆菌的同时确认幽门螺杆菌的耐药剂基因的试剂盒,其特征在于,包含序列1至序列8的引物及偶联有序列9至序列23的低聚物探针的盘。
5.根据权利要求4所述的能够鉴定幽门螺杆菌的同时确认幽门螺杆菌的耐药剂基因的试剂盒,其特征在于,上述药剂选自由克拉霉素、四环素及氟喹诺酮类组成的组中。
6.根据权利要求4所述的能够鉴定幽门螺杆菌的同时确认幽门螺杆菌的耐药剂基因的试剂盒,其特征在于,上述试剂盒为用于进行聚合酶链式扩增反应的试剂,还包含脱氧核糖核酸聚合酶、三磷酸碱基脱氧核苷酸及缓冲液。
7.一种能够鉴定幽门螺杆菌的同时确认幽门螺杆菌的耐药剂基因的组合物,其特征在于,包含序列1至序列8的引物及偶联有序列9至序列23的低聚物探针。
8.根据权利要求7所述的能够鉴定幽门螺杆菌的同时确认幽门螺杆菌的耐药剂基因的组合物,其特征在于,上述药剂选自由克拉霉素、四环素及氟喹诺酮类组成的组中。
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