CN102603875A - 鲍曼复合醋酸钙不动杆菌oxa-23 突变基因的荧光定量pcr 试剂盒 - Google Patents
鲍曼复合醋酸钙不动杆菌oxa-23 突变基因的荧光定量pcr 试剂盒 Download PDFInfo
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Abstract
本发明公开了鲍曼复合醋酸钙不动杆菌OXA-23突变基因的荧光定量PCR试剂盒及其方法。本发明根据新筛选出的鲍曼复合醋酸钙不动杆菌D类β-内酰胺酶耐药基因OXA-23的突变体的序列设计了特异寡核苷酸引物和探针。本发明所述的荧光定量PCR试剂盒可以用于检测鲍曼复合醋酸钙不动杆菌。
Description
技术领域
本发明涉及耐药基因的检测试剂盒及方法。具体而言，本发明根据新筛选的OXA-23基因突变的鲍曼复合醋酸钙不动杆菌菌株，设计了检测OXA-23突变的鲍曼复合醋酸钙不动杆菌的荧光定量PCR试剂盒，本发明也涉及其检测方法。
背景技术
不动杆菌是一类非发酵、严格需氧的革兰阴性杆菌，普遍存在于自然界中。不动杆菌属至少可分为32个基因种型，其中鲍曼不动杆菌、醋酸钙不动杆菌、不动杆菌基因种型3及13TU通过常规表型鉴定难以区分，合称为鲍曼复合醋酸钙不动杆菌复合体。
鲍曼不动杆菌为不动杆菌属中最常见的一种革兰阴性杆菌，广泛存在于自然界的水及土壤、医院环境及人体皮肤、呼吸道、消化道和泌尿生殖道中，为条件致病菌。该菌在医院环境中分布很广且可以长期存活，极易造成危重患者的感染，因此常从被感染患者的血、尿、脓液及呼吸道分泌物等标本中分离出，在非发酵菌中感染仅次于假单胞菌。
鲍曼不动杆菌在医院分布以ICU病区最多，其次为呼吸内科患者。感染的病人多是老年患者、危重疾病及机体抵抗力弱的患者，以及使用各种侵入性操作和长期使用广谱抗生素治疗的患者。又因为该菌对湿热紫外线及化学消毒剂有较强抵抗力，常规消毒只能抑制其生长而不能杀灭，而抵抗力弱或有创伤的 患者可能被从医务人员的手或消毒不彻底的医疗器械所带有的细菌感染的机会较多。
近年来，不动杆菌属在临床样本中的分离率逐年提高，而鲍曼不动杆菌约占其中的80％-90％。鲍曼不动杆菌可引起呼吸机相关肺炎、***、败血症、创口感染及中枢神经***感染等。由于鲍曼不动杆菌具有极强的环境适应能力和获得外源性耐药基因的能力，因而极易造成医院内的播散流行。鲍曼不动杆菌可以通过质粒、转座子和整合子等可移动基因元件，获得对多种抗菌药物的耐药性。对1株欧洲克隆Ⅰ多药耐药菌株的全基因组序列分析发现，在一段长约86kb的染色体片段上聚集了45个耐药基因，包括VEB-1、AmpC、多种氨基糖苷类抗菌药物修饰酶，形成了被称为AbaR1的耐药基因岛。该耐药基因岛与假单胞菌属、沙门菌属和大肠埃希菌的基因片段具有同源性，证实为获得性外来序列。在另1株欧洲克隆Ⅱ的MDRAB(ACICU)的染色体中亦发耐药基因岛AbaR2。鲍曼不动杆菌整合外源性基因的能力，使其具有对临床所用抗菌药物产生耐药性的巨大潜力。在众多的耐药基因中β-内酰胺酶基因是鲍曼不动杆菌最主要的耐药基因，包括超广谱β-内酰胺酶类(属A类，包括TEM-型、SHV-型、SIM-型、PER-型等基因)、金属酶类(属B类，包括IMP和VIM基因)、OXA型酶(属D类)等。OXA-23型酶，水解青霉素和头孢菌素、碳青霉烯，使菌株产生对亚胺培南类抗生素的耐药性[Livermore DM，Woodford N.Carbapenemases：a problem in waiting.Curr Opin Microbiol，2000，3(5)：489-495.]。
多药耐药鲍曼复合醋酸钙不动杆菌的出现，给医院感染控制及临床治疗带来了极大的困难，由于感染的病人多是老年患者、危重疾病及机体抵抗力弱的患者，如不及时选择鲍曼复合醋酸钙不动杆菌敏感的抗生素进行治疗，将会危 及患者的生命安全。
发明内容
为了解决上述问题，本发明公开了鲍曼复合醋酸钙不动杆菌OXA-23突变基因的实时荧光定量PCR试剂盒及其方法。
本发明首先公开了鲍曼复合醋酸钙不动杆菌OXA-23突变基因的实时荧光定量PCR试剂盒。本发明的试剂盒包括特异性引物、特异性探针、标准DNA模板以及其他常规荧光定量PCR试剂。本发明的试剂盒可采用以下方法实施：
根据新筛选出的D类β-内酰胺酶耐药基因OXA-23的突变体的序列，所述突变体核苷酸序列为Sequence NO.3，其所编码的蛋白质氨基酸序列为Sequence NO.7。根据Sequence NO.3设计探针和上、下游引物。所述的上游和下游引物的序列分别见Sequence NO.4和Sequence NO.5，所述的探针序列见Sequence NO.6。
或者包含有上述序列的向5’端延长的序列；
或者与上述序列同源性大于85％的序列。
所述探针5’端标记荧光发色基团FAM，3’端连接荧光淬灭基团BHQ。采用25μl反应体积，每个反应中含12.5μl通用PCR反应混合物[TaqMan 
Universal PCR Master Mix(2×)]；上、下游引物各10nM(上下引物序列分别为Sequence NO.4和Sequence NO.5)；400nM探针(探针序列为Sequence NO.6)；2μl DNA模板；补无菌水至25μl。反应条件为95℃10min后，以95℃40s和60℃1min循环45次。
本发明采用VITEK32细菌鉴定仪及GNS448药敏板进行药敏试验，检测D类β-内酰胺酶耐药基因OXA-23的突变体菌株对抗生素的敏感性，结果显示， 所述突变体菌株对头孢噻肟、丁胺卡拉、氨苄西林、头孢唑林、头孢吡肟、头孢西丁、头孢他啶、头孢呋辛钠、头孢呋辛酯、庆大霉素、亚胺培南、哌拉西林/他唑巴坦耐药，对头孢哌酮/舒巴坦和左氧氟沙星较为敏感。
本发明提供了检测鲍曼复合醋酸钙不动杆菌OXA-23突变基因的实时荧光定量PCR试剂盒的应用。本发明所述的荧光定量PCR试剂盒可以用于OXA-23基因突变的鲍曼复合醋酸钙不动杆菌菌株的检测。
附图说明
图1.标准质粒各浓度对应循环参数(Ct值)。图中曲线从左到又依次代表标准质粒浓度为108copies/μl、107copies/μl、106copies/μl、105copies/μl、104copies/μl、103copies/μl、102copies/μl时的荧光定量PCR曲线。
图2.OXA-23突变基因与OXA-23基因序列比对图。三角形所指的位点为突变位点。
图3.OXA-23突变基因编码的蛋白质氨基酸序列与OXA-23基因编码的蛋白质氨基酸序列比对图。三角形所指的位点为突变位点。
具体实施方式
实施例1：鲍曼复合醋酸钙不动杆菌耐药基因OXA-23新序列的确定
1.菌株来源：从2009年10月至2011年5月我们收集了对抗生素高度耐药(特别对亚胺培南耐药)的鲍曼复合醋酸钙不动杆菌32株。所述32株标本来源于病人的痰液。
2.OXA-23引物设计：本发明设计了一对D类β-内酰胺酶耐药基因OXA-23的特异性引物，所述引物的序列见SEQ ID NO：1和SEQ ID NO：2。
3.OXA-23基因的扩增：以上述32株鲍曼复合醋酸钙不动杆菌裂解液为模板，进行PCR扩增，扩增体系为(25ul体系)：模板DNA(细菌裂解液)2ul，引物1和2各2ul，TaqE：0.25ul(0.5U)，10XBuffer 2.5ul，dNTP 2ul，用ddH2O补足至25ul。PCR仪反应条件如下：
预变性：95℃5min
循环(35个)：变性：94℃30秒
退火：55℃30秒
延伸：72℃30秒
延伸：72℃5min
4.OXA-23基因扩增产物回收：将PCR产物做琼脂糖电泳，在紫外灯下切下目的条带，利用QIANGEN快速胶回收试剂盒回收PCR产物。将回收的产物送上海生工测定。
5.测序结果与BLASTn比对，只发现其中1株编号为AB37的菌株OXA-23群阳性，确定与OXA-23基因相似，其核苷酸序列见SEQ ID NO：3，蛋白质序列见SEQ ID NO：7，所述核苷酸突变位点见图2，所述核苷酸序列所编码的蛋白质突变位点见图3。
实施例2：检测OXA-23新序列的实时荧光定量PCR试剂盒制备
1.引物设计与探针合成
根据上述实施例1得到的SEQ ID NO：3序列。采用ABI primer Express 2.0软件设计探针和引物(上海基康公司合成)。上游引物序列见SEQ ID NO：4和下游引物序列见SEQ ID NO：5，探针序列见SEQ ID NO：6。所述探针5’端标记荧光发色基团FAM，3’端连接荧光淬灭基团BHQ。
2.标准DNA模板制备
采用上一步所述的上下游引物对以上述的AB37的菌株为模板扩增出200bp片段，PCR产物纯化(QIAgen，German)并连接到PGM-T克隆载体后转化到DH5α感受态细胞。特异引物筛选阳性克隆后提取质粒DNA，此质粒DNA即为标准DNA模板，为本领域研究人员根据设计的引物和所述模板通过分子学实验可直接得到其序列信息，此处不再赘述。采用NanoDrop ND-1000核酸定量仪定量质粒DNA，使标准质粒浓度范围在108～102copies/μl，作为所述荧光定量试剂盒的标准品。
3.荧光定量PCR反应液：10mmol/L的dNTP、25mmol/L的MgCl2。
4.荧光定量PCR 10xBuffer：500mM KCl、100mM Tris-HCl、1％Triton X-100。
5.阴性质控标准品：无菌双蒸水。
6.荧光定量PCR反应条件
荧光定量PCR仪为Line-Gene K(杭州博日，中国)，25微升反应体系包括：12.5μl 2×TaqMan Universal PCR Master Mix(美国ABI公司)，上、下游引物各10nM(上下引物序列分别为Sequence NO.4和Sequence NO.5)；400nM探针(探针序列为Sequence NO.6)，2μl DNA模板、加ddH2O至25微升。反应程序为95℃10min预变性，接45个循环：95℃40s，60℃1min。无菌水用于阴性对照。
实施例3体外检测实验
1.灵敏性检测
将标准DNA模板进行10倍梯度稀释，使标准质粒浓度范围在108～102copies/μl，作为荧光定量试剂盒的标准品。荧光定量PCR反应条件，25微升反应体系：12.5μl 2×TaqMan Universal PCR Master Mix(美国ABI公司)，上、 下游引物各10nM(上下引物序列分别为Sequence NO.4和Sequence NO.5)，400nM探针(探针序列为Sequence NO.6)，2μl梯度稀释的标准DNA模板、加ddH2O至25微升。反应程序为95℃10min预变性，接45个循环：95℃40s，60℃1min。无菌水用于阴性对照。
检测结果见图1，灵敏分析显示最小能检测到100copies/反应。本实验批内重复3次，批间重复3次以分析其重复性。重复性结果显示批内CV％＜5％内，批间CV％＜10％。说明本试剂盒具有良好的重复性。
2.特异性检测
1)阴性对照菌株：肺炎链球菌、金黄色葡萄球菌、奇异变形杆菌、恶臭假单胞菌、鲍曼复合醋酸钙不动杆菌，以上菌株均来自中国微生物菌株保藏管理委员会。
2)阳性对照株：为本室分离的OXA-23基因突变的鲍曼复合醋酸钙不动杆菌4个克隆。
3)PCR反应体系为：12.5μl 2×TaqMan Universal PCR Master Mix(美国ABI公司)，上、下游引物各10nM(上下引物序列分别为Sequence NO.4和Sequence NO.5)，400nM探针(探针序列为Sequence NO.6)，2μl各菌种DNA提取液和标准DNA质粒、加ddH2O至25微升。反应程序为95℃10min预变性，接45个循环：95℃40s，60℃1min。
特异性结果分析显示，只有本室分离的OXA-23基因突变的鲍曼复合醋酸钙不动杆菌的4个克隆为阳性，其它样品均为阴性，所以本试剂盒检测特异性达到100％。
实施例3鲍曼复合醋酸钙不动杆菌耐药基因OXA-23新突变体菌株的药物敏感实验
本发明采用VITEK32细菌鉴定仪及GNS448药敏板进行药敏试验，检测D类β-内酰胺酶耐药基因OXA-23的突变体菌株对抗生素的敏感性，用铜绿假单胞菌(ATCC 27853)及大肠埃希菌(ATCC25922)作为药敏质控菌株，结果判断按CLSI2000标准进行，结果如下表：
结果显示，所述突变体菌株对头孢噻肟、丁胺卡拉、氨苄西林、头孢唑林、头孢吡肟、头孢西丁、头孢他啶、头孢呋辛钠、头孢呋辛酯、庆大霉素、亚胺培南、哌拉西林/他唑巴坦耐药，对头孢哌酮/舒巴坦和左氧氟沙星较为敏感。
Claims (7)
1.一种OXA-23突变蛋白，其序列如SEQ ID NO：7所示。
2.编码权利要求1所述蛋白的核苷酸。
3.根据权利要求2所述的核苷酸，其特征在于：其序列如Sequence NO.3所示。
4.一种检测权利要求2或3的鲍曼复合醋酸钙不动杆菌OXA-23突变基因的实时荧光定量PCR试剂盒，包括特异性引物和特异性探针，所述特异性引物包括上游引物和下游引物：
上游引物其序列为Sequence NO.4，或者包含Sequence NO.4序列的向5’端延长的序列；
下游引物其序列为Sequence NO.5，或者包含Sequence NO.5序列的向5’端延长的序列；
所述特异性探针其序列为Sequence NO.6，或者包含Sequence NO.6序列的向5’或3’端延长的序列。
5.根据权利要求4所述的试剂盒，其特征在于，所述试剂盒可以用于含有OXA-23突变基因的鲍曼复合醋酸钙不动杆菌引起的疾病早期抗菌药物的选择。
6.根据权利要求5所述的试剂盒，其特征在于，所述的抗菌药物优选左氧氟沙星。
7.权利要求2或3所述的核苷酸在制备检测OXA-23突变的鲍曼复合醋酸钙不动杆菌菌株的试剂盒中的用途。
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| EP2948549A4 (en) * | 2013-05-21 | 2016-12-07 | Ind And Acad Coop Found Myongji Univ | COLONIAL MULTIPLEX PCR PRIMER AND KIT FOR DETECTION OF B-LACTAMATE GENES OF CLASS A, B, C AND D AND METHOD OF USE THEREOF |
| CN105349694A (zh) * | 2015-12-24 | 2016-02-24 | 重庆医科大学 | 检测鲍曼不动杆菌耐药性的引物、试剂盒及其方法 |
| CN105349694B (zh) * | 2015-12-24 | 2020-09-18 | 重庆医科大学 | 检测鲍曼不动杆菌耐药性的引物、试剂盒及其方法 |
| CN106282354A (zh) * | 2016-08-26 | 2017-01-04 | 扬州大学 | 一种鲁氏不动杆菌的检测引物及荧光定量pcr检测方法 |
| CN106282354B (zh) * | 2016-08-26 | 2021-03-12 | 扬州大学 | 一种鲁氏不动杆菌的检测引物及荧光定量pcr检测方法 |
| CN109266658A (zh) * | 2018-10-17 | 2019-01-25 | 昆明理工大学 | 一种鲍曼不动杆菌的特异基因及其引物和应用 |
| CN109628620A (zh) * | 2019-01-22 | 2019-04-16 | 南方医科大学南方医院 | 全序列荧光pcr检测oxa-23家族和oxa-51家族基因型的引物、方法及试剂盒 |
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