CN116479148B - 一种碳青霉烯耐药基因检测试剂盒及其应用 - Google Patents
一种碳青霉烯耐药基因检测试剂盒及其应用 Download PDFInfo
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Abstract
本发明公开了一种碳青霉烯耐药基因检测试剂盒及其应用,属于生物技术领域。本发明的试剂盒具有以下优势:(1)本发明的试剂盒能够在一次检测中实现对NDM、OXA‑48、KPC、VIM、IMP这五大主要的碳青霉烯耐药基因进行检测,同时获得多个耐药基因的检测结果,降低检测成本,节省检测时间;(2)本发明的试剂盒对碳青霉烯耐药基因NDM、OXA‑48、KPC、VIM和IMP的检测的最低可检测浓度为20拷贝/mL,灵敏度高;(3)本发明的试剂盒中的引物探针组合物是通过多重试验设计筛选得到的,具有很高的扩增效率和特异性。
Description
技术领域
本发明属于生物技术领域,具体涉及一种碳青霉烯耐药基因检测试剂盒及其应用。
背景技术
细菌耐药已成为全球公共健康领域的重大挑战,其中尤以碳青霉烯类耐药肠杆菌目细菌(CRE)引起的感染形式最为严峻,碳青霉稀类耐药肠杆菌科是临床医院感染重点监测的病原菌之一。碳青霉烯类抗生素是抗菌谱广、抗菌活性最强的一类β-内酰胺酶抗生素,因其具有对β-内酰胺酶稳定以及毒性低等特点,已成为治疗肺炎克雷伯菌、大肠杆菌和其他肠杆菌科细菌最主要的抗菌药物之一。然而随着碳青霉烯类抗生素的大范围使用,耐药株不断出现,容易引起复发性感染,致使治疗失败和增加病死率。因此开展碳青霉烯耐药基因的检测具有重要意义。
目前实验室检测碳青霉烯酶的方法分为表型检测和基因型检测。表型检测方法包括Carba NP试验、改良碳青霉烯灭活试验、碳青霉烯酶抑制剂增强试验,其中Carba NP试验采用自制试剂,部分试剂有效期短;改良碳青霉烯灭活试验和碳青霉烯酶抑制剂增强试验均需进行过夜培养。基因型检测方法包括酶免疫层析技术和基因检测技术,酶免疫层析技术价格较高。基因检测具有简便、准确、快速、可操作性强等特点。开发一款碳青霉烯耐药基因检测试剂盒,对指导临床医生选择合适的药物,避免加重细菌的耐药和耽误临床诊治具有重要意义。
发明内容
本发明的目的在于克服现有技术的不足,提供一种碳青霉烯耐药基因检测试剂盒及其应用,本发明的检测试剂盒简单高效、灵敏度高。
为实现上述目的,本发明采取的技术方案为:一种用于检测碳青霉烯耐药基因的引物和探针组合物,所述引物和探针组合物包括:用于检测NDM基因的如SEQ ID NO:1~2所示的核苷酸序列的引物和如SEQ ID NO:3所示的核苷酸序列的探针;用于检测OXA-48基因的如SEQ ID NO:4~5所示的核苷酸序列的引物和如SEQ ID NO:6所示的核苷酸序列的探针;用于检测KPC基因的如SEQ ID NO:7~8所示的核苷酸序列的引物和如SEQ ID NO:9所示的核苷酸序列的探针;用于检测VIM基因的如SEQ ID NO:10~11所示的核苷酸序列的引物和如SEQ ID NO:12所示的核苷酸序列的探针;用于检测IMP基因的如SEQ ID NO:13~14所示的核苷酸序列的引物和如SEQ ID NO:15所示的核苷酸序列的探针;如SEQ ID NO:16~17所示的核苷酸序列的内标基因引物和如SEQ ID NO:18所示的核苷酸序列的内标基因探针。
本发明的引物探针组合物能够实现对新德里金属β-内酰胺酶(NDM)、苯唑西林酶(OXA-48)、克雷伯杆菌肺炎碳青霉烯酶(KPC)、金属β-内酰胺酶(VIM)、亚胺培南酶(IMP)这五大主要的碳青霉烯耐药基因同时检测,同时获得多个耐药基因的检测结果,降低检测成本,节省检测时间,可指导临床医生选择合适的药物,避免加重细菌的耐药和耽误临床诊治,有着广阔的应用前景和产业化前景。
本发明还提供一种碳青霉烯耐药基因检测试剂盒,所述试剂盒包括所述引物和探针组合物。
作为本发明所述碳青霉烯耐药基因检测试剂盒的优选实施方式,所述试剂盒中用于检测NDM基因、OXA-48基因、KPC基因、VIM基因、IMP基因的引物和探针的浓度均为0.1-0.6μM。
作为本发明所述碳青霉烯耐药基因检测试剂盒的优选实施方式,所述试剂盒中内标基因引物和探针的浓度均为0.05-0.2μM。
作为本发明所述碳青霉烯耐药基因检测试剂盒的优选实施方式,所述试剂盒还包括质控品和PCR Mix。
作为本发明所述碳青霉烯耐药基因检测试剂盒的优选实施方式,所述质控品包括阴性质控品和阳性质控品;所述阴性质控品为灭活的大肠埃希菌;所述阳性质控品为含有目的基因片段灭活的基因工程菌。
作为本发明所述碳青霉烯耐药基因检测试剂盒的优选实施方式,所述目的基因为NDM基因、OXA-48基因、KPC基因、VIM基因和IMP基因。
本发明还提供一种碳青霉烯耐药基因检测方法,包括以下步骤:
(1)提取待测样本核酸;
(2)采用所述的引物和探针组合物进行扩增;
(3)读取各基因Ct值并进行判断。
作为本发明所述方法的优选实施方式,所述待测样本包括粪便样本、直肠拭子、纯菌落样本。
本发明还提供所述的试剂盒在碳青霉烯耐药基因中的应用。
本发明的有益效果:提供一种碳青霉烯耐药基因检测试剂盒,本发明的试剂盒具有以下优势:(1)本发明的试剂盒能够在一次检测中实现对新德里金属β-内酰胺酶(NDM)、苯唑西林酶(OXA-48)、克雷伯杆菌肺炎碳青霉烯酶(KPC)、金属β-内酰胺酶(VIM)、亚胺培南酶(IMP)这五大主要的碳青霉烯耐药基因进行检测,同时获得多个耐药基因的检测结果,降低检测成本,节省检测时间;(2)本发明的试剂盒对碳青霉烯耐药基因NDM、OXA-48、KPC、VIM和IMP的检测的最低可检测浓度为20拷贝/mL,灵敏度高;(3)本发明的试剂盒中的引物探针组合物是通过多重试验设计筛选得到的,具有很高的扩增效率和特异性。
附图说明
图1为本发明碳青霉烯耐药基因检测试剂盒的PCR反应液A检测浓度为200拷贝/mL的标准菌株的结果图;
图2为本发明碳青霉烯耐药基因检测试剂盒的PCR反应液B检测浓度为200拷贝/mL的标准菌株的结果图;
图3为本发明碳青霉烯耐药基因检测试剂盒的PCR反应液A检测浓度为20拷贝/mL的标准菌株的结果图;
图4为本发明碳青霉烯耐药基因检测试剂盒的PCR反应液B检测浓度为20拷贝/mL的标准菌株的结果图;
图5为本发明碳青霉烯耐药基因检测试剂盒的PCR反应液A检测浓度为5拷贝/mL的标准菌株的结果图;
图6为本发明碳青霉烯耐药基因检测试剂盒的PCR反应液B检测浓度为5拷贝/mL的标准菌株的结果图;
图7为本发明碳青霉烯耐药基因检测试剂盒对大肠埃希菌、肺炎克雷伯菌、粪肠球菌、铜绿假单胞菌的检测结果图;
图8为本发明碳青霉烯耐药基因检测试剂盒对阴沟肠杆菌、产酸克雷伯菌、鲍曼不动杆菌、弗氏柠檬酸杆菌的检测结果图;
图9为本发明碳青霉烯耐药基因检测试剂盒对克氏柠檬酸杆菌、奇异变形杆菌、人基因组DNA的检测结果图。
图10为对比例组一的引物探针组合的PCR反应液A检测浓度为200拷贝/mL的标准菌株的结果图。
图11为对比例组一的引物探针组合的PCR反应液B检测浓度为200拷贝/mL的标准菌株的结果图。
图12为对比例1组二的引物探针组合的PCR反应液A检测浓度为200拷贝/mL的标准菌株的结果图。
图13为对比例1组一的引物探针组合的PCR反应液B检测浓度为200拷贝/mL的标准菌株的结果图。
具体实施方式
以下通过实施例形式的具体实施方法,对本发明的上述内容作进一步的详细说明。但不应将此理解为本发明上述主题的范围仅限于以下实施例。实施例中,所使用的实验方法如无特殊说明,均为常规方法,所用的材料、试剂等,如无特殊说明,均可从商业途径得到。
实施例1
本实施例为获得特异性及灵敏度较好的多重引物探针体系组合,共设计了多套引物及探针,并组合进行多重试验,最终优选出特异性好、扩增效率高的引物探针组合如表1所示。
表1
实施例2
本实施例的一种碳青霉烯耐药基因检测试剂盒,包括:
(1)PCR反应液A:5mM MgCl2、0.8mM dNTPs、靶标基因引物和探针、内标基因引物对、内标基因探针;所述靶标基因为NDM、OXA-48和KPC;
(2)PCR反应液B:6.5mM MgCl2、1.0mM dNTPs、靶标基因引物对、靶标基因探针、内标基因引物对、内标基因探针;所述靶标基因为VIM和IMP;
(3)PCR预混合溶液:1X buffer、1X热启动Taq酶、1X尿嘧啶糖基化酶;
(4)阴性质控品:灭活的大肠埃希菌;
(5)阳性质控品:含有NDM基因、OXA-48基因、KPC基因、VIM基因和IMP基因片段灭活的基因工程菌。
所述靶标基因引物和探针包括用于检测NDM基因的如SEQ ID NO:1~2所示的核苷酸序列的引物和如SEQ ID NO:3所示的核苷酸序列的探针;用于检测OXA-48基因的如SEQID NO:4~5所示的核苷酸序列的引物和如SEQ ID NO:6所示的核苷酸序列的探针;用于检测KPC基因的如SEQ ID NO:7~8所示的核苷酸序列的引物和如SEQ ID NO:9所示的核苷酸序列的探针;用于检测VIM基因的如SEQ ID NO:10~11所示的核苷酸序列的引物和如SEQID NO:12所示的核苷酸序列的探针;用于检测IMP基因的如SEQ ID NO:13~14所示的核苷酸序列的引物和如SEQ ID NO:15所示的核苷酸序列的探针;所述内标基因引物的核苷酸序列如SEQ ID NO:16~17所示,所述内标基因探针的核苷酸序列如SEQ ID NO:18所示;
所述靶标基因引物和探针的浓度均为0.4μM;所述内标基因引物和探针的浓度均为0.08μM。
实施例3
本发明实施例2的碳青霉烯耐药基因检测试剂盒的检测方法,包括以下步骤:
(1)样本采集:适用样本类型为粪便样本、直肠拭子、纯菌落样本;
(2)核酸提取:采用商品化DNA提取试剂盒,如基于硅胶膜离心柱法的核酸提取试剂或基于磁珠法核酸提取的试剂,并按试剂盒说明书进行操作,最后收集到80μL DNA溶液,直接进行检测,或保存于-80℃。阴性质控品、阳性质控品均需进行提取。
(3)混匀PCR反应液A(检测靶标NDM/OXA-48/KPC/内参基因的引物探针混合物等)和PCR反应液B(检测靶标VIM/IMP/内参基因的引物探针混合物等),根据检测所需总反应数2N,N管中加入10μl PCR反应液A,N管中加入10μl PCR反应液B;
(4)加样:向已分装有试剂的PCR反应管中分别加入阴性质控品,样本DNA溶液,阳性质控品,加样量均为15μL,盖紧管盖,混匀,离心收集溶液置管底;
(5)上机扩增检测:PCR扩增程序及熔解曲线分析程序的设定如表2所示.
表2
(6)结果分析:在满足扩增有效的前提下,各靶标基因阳性Ct判定依据如表3所示。
表3
实施例4
本实施例采用实施例2的碳青霉烯耐药基因检测试剂盒并根据实施例3的检测方法定量检测NDM、OXA-48、KPC、VIM、IMP标准菌株的准确性与灵敏性,具体方法如下:用阴性样本分别将NDM、OXA-48、KPC、VIM、IMP标准菌株稀释为浓度200拷贝/mL、20拷贝/mL、2拷贝/mL,并采用实施例3的具体方法进行检测,每个浓度的样本重复检测20次,检测结果如图1~6所示。
由图1~6可以看出碳青霉烯耐药基因NDM、OXA-48、KPC、VIM、IMP在20拷贝/mL浓度以上的样本均能准确检出,在5拷贝/mL的样本中检出率相对较低为70%-90%,表明本发明试剂盒对病原菌的灵敏度可达到20拷贝/mL。
实施例5
本实施例采用实施例2的碳青霉烯耐药基因检测试剂盒对大肠埃希菌、肺炎克雷伯菌、粪肠球菌、铜绿假单胞菌、阴沟肠杆菌、产酸克雷伯菌、鲍曼不动杆菌、弗氏柠檬酸杆菌、克氏柠檬酸杆菌、奇异变形杆菌进行特异性检测,具体的检测方法为:将大肠埃希菌、肺炎克雷伯菌、粪肠球菌、铜绿假单胞菌、阴沟肠杆菌、产酸克雷伯菌、鲍曼不动杆菌、弗氏柠檬酸杆菌、克氏柠檬酸杆菌、奇异变形杆菌的定量菌液稀释至1×106拷贝/mL,及人基因组DNA作为特异性检测样本按照实施例3的方法进行检测。结果如图7~9所示。
由图7~9可知,采用本发明的试剂盒检测大肠埃希菌、肺炎克雷伯菌、粪肠球菌、铜绿假单胞菌、阴沟肠杆菌、产酸克雷伯菌、鲍曼不动杆菌、弗氏柠檬酸杆菌、克氏柠檬酸杆菌、奇异变形杆菌以及人基因组DNA,检测结果均为阴性,表明本试剂盒特异性良好。
对比例1
本对比例为采用实施例1相同的引物探针设计方法设计得到的引物探针组合,具体引物序列如表4所示。由于碱基互补配对原则,引物和(或)探针之间会形成二聚体,多种靶标联合检测时,引物和探针众多,引物和引物、探针和探针或者引物和探针之间容易发生二聚体,要保证设计的保守性(保守性对检测的准确性至关重要),又要考虑不同引物探针之间的相互干扰,需要精心对引物探针进行设计优化并反复测试验证。因此,本发明还设计了其余的一些引物探针组成了不同的检测体系1、2(对比例1组合1、2)。根据实施例3的检测方法采用上述两组引物探针组合分别检测NDM、OXA-48、KPC、VIM、IMP标准菌株的准确性,其中标准备菌株的浓度为10000拷贝/mL,扩增结果如图10~13所示。从图中可以看出两组引物探针组合检测只出现部分靶标的扩增曲线,且扩增效果差,而其他靶标基因甚至无典型扩增曲线。
表4
最后应当说明的是,以上实施例仅用以说明本发明的技术方案而非对本发明保护范围的限制,尽管参照较佳实施例对本发明作了详细说明,本领域的普通技术人员应当理解,可以对本发明的技术方案进行修改或者等同替换,而不脱离本发明技术方案的实质和范围。
Claims (9)
1.一种用于检测碳青霉烯耐药基因的引物和探针组合物,其特征在于,所述引物和探针组合物包括:用于检测NDM基因的如SEQ ID NO:1~2所示的引物和如SEQ ID NO:3所示的探针;用于检测OXA-48基因的如SEQ ID NO:4~5所示的引物和如SEQ ID NO:6所示的探针;用于检测KPC基因的如SEQ ID NO:7~8所示的引物和如SEQ ID NO:9所示的探针;用于检测VIM基因的如SEQ ID NO:10~11所示的引物和如SEQ ID NO:12所示的探针;用于检测IMP基因的如SEQ ID NO:13~14所示的引物和如SEQ ID NO:15所示的探针;如SEQ ID NO:16~17所示的内标基因引物和如SEQ ID NO:18所示的内标基因探针。
2.一种碳青霉烯耐药基因检测试剂盒,其特征在于,所述试剂盒包括如权利要求1所述引物和探针组合物。
3.根据权利要求2所述的试剂盒,其特征在于,所述试剂盒中用于检测NDM基因、OXA-48基因、KPC基因、VIM基因、IMP基因的引物和探针的浓度均为0.1-0.6μM。
4.根据权利要求2所述的试剂盒,其特征在于,所述试剂盒中内标基因引物和探针的浓度均为0.05-0.2μM。
5.根据权利要求2所述的试剂盒,其特征在于,所述试剂盒还包括质控品和PCR Mix。
6.根据权利要求5所述的试剂盒,其特征在于,所述质控品包括阴性质控品和阳性质控品;所述阴性质控品为灭活的大肠埃希菌;所述阳性质控品为含有目的基因片段灭活的基因工程菌;所述目的基因为NDM基因、OXA-48基因、KPC基因、VIM基因和IMP基因。
7.一种碳青霉烯耐药基因检测方法,其特征在于,包括以下步骤:
(1)提取待测样本核酸;
(2)采用权利要求1所述的引物和探针组合物进行扩增;
(3)读取各基因Ct值并进行判断;
所述方法为非疾病的诊断或治疗目的的方法。
8.根据权利要求7所述的方法,其特征在于,所述待测样本包括粪便样本、直肠拭子、纯菌落样本。
9.根据权利要求2~6任一项所述的试剂盒在检测碳青霉烯耐药基因中的应用,其特征在于,所述应用为非疾病的诊断或治疗目的的应用。
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