CN113584191B - 7种耐药基因多重pcr检测的引物、探针及其试剂盒 - Google Patents
7种耐药基因多重pcr检测的引物、探针及其试剂盒 Download PDFInfo
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Abstract
本发明涉及生物检测技术领域,尤其涉及7种耐药基因多重PCR检测的引物、探针及其试剂盒。本发明可快速、准确及超高灵敏度地对耐碳青霉烯类药物的肠杆菌科细菌中碳青霉烯酶基因KPC、NDM、IMP、VIM、OXA48基因,耐甲氧西林的葡萄球菌mecA基因和耐万古霉素的多重耐药的肠球菌中vanA基因进行联合检测,可实时、快速监测血流感染患者体内的特定耐药基因的变化,及时评估患者状况,为临床医生提供辅助治疗建议。本发明试剂盒亦可用于危重症患者和免疫抑制人群无症状定植者的CRE、MRSA和VRE中7种细菌耐药基因的主动筛查,在科研领域和临床上具有极高的应用价值。
Description
技术领域
本发明涉及生物检测技术领域,尤其涉及7种耐药基因多重PCR检测的引物、探针及其试剂盒。
背景技术
抗菌药物是临床应用最广泛的一类药物,但近年感染性疾病的病死率迅速提升,究其原因在于抗生素滥用,耐药菌带来的用药困难。当前,细菌耐药性的监测/检测重点包括:1、耐碳青霉烯类药物的肠杆菌科细菌(CRE);2、耐甲氧西林的葡萄球菌(MRSA);3、耐万古霉素的多重耐药的肠球菌(VRE)。
碳青霉烯类抗生素对大多数革兰氏阳性及革兰氏阴性病菌感染均有强大的抗菌效果,是临床上抵御严重细菌感染的重要屏障。但是随着碳青霉烯类抗菌药物的大量使用,国内外开始报道对碳青霉烯类抗菌药物不敏感甚至耐药的肠杆菌科细菌,这极大地限制了碳青霉烯类抗菌药物的使用。革兰氏阴性菌对碳青霉烯耐药最主要的机制是产生碳青霉烯酶,按照Ambler分子分类方法,碳青霉烯酶可分为A、B和D三类,A类以blaKPC为主,B类为金属β-内酰胺酶,以blaNDM、blaIMP和blaVIM为主,D类为OXA-48型丝氨酸碳青霉烯酶。我国临床分离的CRE菌株产生碳青霉烯酶以KPC和NDM型为主,少数菌株产生OXA-48、IMP和VIM型碳青霉烯酶。编码碳青霉烯酶基因由质粒介导,细菌一般产单一碳青霉烯酶,少数细菌会产碳青霉烯酶复合酶。CHINET中国细菌耐药监测网对2018年收集自全国39所医院935株CRE菌株的研究结果显示,产KPC、NDM和OXA-48型碳青霉烯酶菌株所占比例分别为51.6%(482/935)、35.7%(334/935)和7.3%(68/935)。头孢他啶/阿维巴坦,对于产KPC-2肠杆菌科细菌感染有良好的效果,但对于金属酶无效。
在过去的10多年里,革兰氏阳性球菌感染所占比例逐渐增加,同时革兰氏阳性球菌对临床常用抗菌药物耐药性日益严重,其中耐甲氧西林葡萄球菌(MRSA)、耐万古霉素肠球菌(VRE)在临床上分离率高,所致感染治疗困难,是当前最受关注的耐药革兰氏阳性球菌。青霉素结合蛋白( Penicillin-binding protein 2a, PBP2a) 是 MRSA 耐药性产生的主要原因, 这种蛋白质对β-内酰胺类抗菌药物的亲和力很低, 而 mecA 基因是调节编码PBP2a 最关键的基因, mecA 基因主要存在于金黄色葡萄球菌染色体 mec 基因盒上。 耐万古霉素肠球菌(VRE)的耐药机制主要是由于肠球菌获得了vanA、vanB、vanC、vanD、vanE等耐药基因后,细胞壁的生物合成途径发生了改变,使其合成的肽聚糖前体与万古霉素等糖肽类抗生素亲和力下降,从而导致肠球菌对万古霉素耐药,通常以vanA、vanB两型较为多见,故最具临床意义。
由于常规病原学诊断方法包括标本中细菌培养、分离以及药敏,所需时间长(3-5天),难以及时指导感染性疾病的治疗。随着分子生物学的发展,分子生物学技术在微生物领域的应用,特别是荧光定量PCR和数字PCR检测技术平台的出现后,越来越广泛。数字PCR技术是在qPCR技术上发展起来的第三代PCR技术,理论上可以实现对单个拷贝的目标核酸片段进行扩增检测,是当前灵敏度最高的核酸检测技术。利用多色荧光数字PCR平台,结合不同荧光标记水解探针的应用,可以同时对多个目标片段进行检测。然而如何将数字PCR技术有效的应用于多种耐药基因的检测仍是本领域技术人员亟需克服的难题。
发明内容
有鉴于此, 本发明提供了7种耐药基因多重PCR检测的引物、探针及其试剂盒,可快速、准确及超高灵敏度地检测CRE、MRSA和VRE中7种细菌耐药基因。
为了实现上述发明目的,本发明提供以下技术方案:
7种耐药基因多重PCR检测的引物、探针组,包括:
用于检测KPC基因的SEQ ID NO:1~2所示核苷酸序列的引物和SEQ ID NO:3所示核苷酸序列的探针;
用于检测NDM基因的SEQ ID NO:4~5所示核苷酸序列的引物和SEQ ID NO:6所示核苷酸序列的探针;
用于检测IMP基因的SEQ ID NO:7~8所示核苷酸序列的引物和SEQ ID NO:9所示核苷酸序列的探针;
用于检测VIM基因的SEQ ID NO:10~11所示核苷酸序列的引物和SEQ ID NO:12所示核苷酸序列的探针;
用于检测OXA48基因的SEQ ID NO:13~14所示核苷酸序列的引物和SEQ ID NO:15所示核苷酸序列的探针;
用于检测mecA基因的SEQ ID NO:16~17所示核苷酸序列的引物和SEQ ID NO:18所示核苷酸序列的探针;和
用于检测vanA基因的引SEQ ID NO:19~20所示核苷酸序列的引物和SEQ ID NO:21所示核苷酸序列的探针。
与上述核苷酸序列具有90%以上(例如92%、94%、96%或98%等)同源性且功能相同的核苷酸序列变体,均在本发明的保护范围之内。
本发明引物探针针对耐碳青霉烯类药物的肠杆菌科细菌(CRE)中碳青霉烯酶基因KPC、NDM、IMP、VIM、OXA48基因,耐甲氧西林的葡萄球菌mecA基因和耐万古霉素的多重耐药的肠球菌(VRE)中vanA基因进行联合检测,检测结果准确可靠、灵敏度较高,适用于中国人群的筛查,能有效地起到准确诊断、耐药溯源、耐药控制等作用,从而减少抗生素使用量,降低耐药产生,在科研领域和临床上具有极高的应用价值。
基于以上结果,本发明还提供了所述的引物、探针组在制备CRE、MRSA和VRE中7种耐药基因多重检测的试剂盒中的应用。
本发明还提供了7种耐药基因多重检测的试剂盒,包括本发明所述的引物、探针组。
本发明试剂盒还包括检测试剂和核酸提取试剂。
本发明中,检测试剂用于PCR扩增,核酸提取试剂用于提取待测样本的核酸,本发明对检测试剂和核酸提取试剂的具体组成和来源没有特殊限制,可购自任意厂家。本发明具体实施例中,检测试剂包括无菌水和PCR Mix,包括但不仅限于此,核酸提取试剂为市售天根DP710试剂盒。
本发明还提供了7种耐药基因的多重PCR检测的方法,利用本发明引物探针组或本发明试剂盒进行检测。
本发明检测方法基于多重荧光PCR或多重数字PCR平台进行检测。
“多重数字PCR”是指在同一数字PCR反应体系里加上两对或两对以上引物,同时扩增出多个核酸片段的数字PCR反应。将多重PCR技术与数字PCR技术有机结合而形成的多重数字PCR,根据DNA探针不同荧光度、DNA扩增不同循环数及多种标记荧光同时使用,便可大幅提高数字PCR多重性,多重性可达20-50以上,即同时在一个PCR反应单元内进行20-50个以上的数字PCR反应。
本发明基于多重数字PCR平台的检测方法包括以下步骤:
(1)提取待测样本核酸;
(2)配置数字PCR反应液;
(3)制备液滴芯片;
(4)运行液滴芯片扩增程序后,采用生物芯片阅读仪进行分析后报告输出。
本发明中,所述待测样本为感染患者血浆样本或无症状定植者肛肠试子样本。
本发明步骤(4)中,所述扩增程序包括:95℃预变性5min;95℃变性15秒,60℃退火30秒,循环40次。
本发明可快速、准确及超高灵敏度地对耐碳青霉烯类药物的肠杆菌科细菌中碳青霉烯酶基因KPC、NDM、IMP、VIM、OXA48基因,耐甲氧西林的葡萄球菌mecA基因和耐万古霉素的多重耐药的肠球菌中vanA基因进行联合检测,具有如下有效效果:
1.本发明采用多色多重的检测方案,将多种目标基因联合检测,单位时间和反应孔内同时获得多个耐药基因的检测结果。
2.本发明引物探针及其试剂盒,可快速、准确及超高灵敏度地检测CRE、MRSA和VRE中7种细菌耐药基因,根据目标耐药基因的定量/半定量检测结果,可实时、快速监测血流感染患者体内的特定耐药基因的变化,及时评估患者状况,为临床医生提供辅助治疗建议。本发明公开的试剂盒亦可用于危重症患者和免疫抑制人群无症状定植者的CRE、MRSA和VRE中7种细菌耐药基因的主动筛查。
具体实施方式
本发明提供了7种耐药基因多重PCR检测的引物、探针及其试剂盒。本领域技术人员可以借鉴本文内容,适当改进工艺参数实现。特别需要指出的是,所有类似的替换和改动对本领域技术人员来说是显而易见的,它们都被视为包括在本发明。本发明的方法及应用已经通过较佳实施例进行了描述,相关人员明显能在不脱离本发明内容、精神和范围内对本文的方法和应用进行改动或适当变更与组合,来实现和应用本发明技术。
本发明采用的试材皆为普通市售品,皆可于市场购得。
下面结合实施例,进一步阐述本发明:
实施例1本发明引物探针
本实施例涉及的引物组合序列如表1所示。
表1引物探针组合序列
目标序列名称 | 引物、探针序列 |
KPC | KPC-F:GGCGCCTAACAAGGATGACA(SEQ ID NO:1);KPC-R:GACGCCCAATCCCTCGAG(SEQ ID NO:2);KPC-P: FAM-GCCGCTGCGGCTAGAC-MGB(SEQID NO:3)。 |
NDM | NDM-F: CTGGATCAAGCAGGAGATCAAC(SEQ ID NO:4);NDM-R:CGCCCATCTTGTCCTGATG(SEQ ID NO:5);NDM-P:VIC-GCCGGTCGCGCTGGC-BHQ1(SEQ ID NO:6)。 |
IMP | IMP-F:GCAAAACTGGTTGTTCCARGTCA(SEQ ID NO:7);IMP-R:AAYCCTTTAACCGCCTGCTCTA(SEQ ID NO:8);IMP-P:VIC-CAAGAGTGATGCGTCTC-BHQ1(SEQ ID NO:9)。 |
VIM | VIM-F:CTCCACGCACTTTCATGACGA(SEQ ID NO:10);VIM-R:CGTACGTTGCCACYCCAGC(SEQ ID NO:11);VIM-P:VIC-CATCAACGCCGCCGAC-BHQ1(SEQ ID NO:12)。 |
OXA-48 | OXA-48-F:CCCAATAGCTTGATCGCCCT(SEQ ID NO:13);OXA-48-R:GTCCATCCCACTTAAAGACTTGGT(SEQ ID NO:14);OXA-48-P:ROX-GATTTGGGCGTGGTTAAGGA-MGB(SEQ ID NO:15)。 |
MECA | MECA-F: AACTACGGTAACATTGATCGCAAC(SEQ ID NO:16);MECA-R: GCATTCCTGGAATAATGACGCTA(SEQ ID NO:17);MECA-P: CY5-TGTTAAAGAAGATGGTATGTGG-BHQ2(SEQ ID NO:18)。 |
VANA | VANA-F:CCGTTCCCGCAGACCTT(SEQ ID NO:19);VANA-R:TTTTTTGCCGTTTCCTGTATCC(SEQ ID NO:20);VANA-P:DYLIGHT755-CAGCAGAGGAGCGAGG-BHQ3(SEQ ID NO:21)。 |
实施例2 本发明检测方法
实验流程如下:
血浆/肛肠试子样本核酸提取→配置数字PCR反应液→液滴芯片生成→扩增→阅读→结果分析与报告输出。
核酸提取实验盒推荐使用领航基因科技(杭州)有限公司生产的血浆游离DNA核酸提取试剂盒进行提取,按照说明书进行核酸提取。
一、靶基因检测方案
1、数字PCR检测流程(配置数字PCR反应液、液滴芯片生成及扩增过程)配制20 μl的液滴PCR检测体系,具体体系配方分别见表2;
表2
组分 | 体积(μl) |
5x Taq Mix | 4 |
Forward Primer (100μM) | 0.06 |
Reverse Primer (100μM) | 0.06 |
Probe (100μM) | 0.04 |
模板 | 5 |
总体积 | 20 |
备注:表2中反应体系为单引物探针对反应体系,同样适用于多重检测体系。
2、将提取的核酸模版加入到体系中并混匀,模板加入量为5 µl,同时配制实验的阳性对照和阴性对照;
3、按照SOP流程将配制好的反应体系加入到液滴芯片加样孔中;将芯片放入样本制备仪,启动仪器生成液滴。
将芯片放入芯片扩增仪中;设置液滴芯片扩增程序,并运行;反应程序如下:95℃预变性5min;95℃变性15秒,60℃退火30秒,循环40次。
二、液滴芯片阅读、结果分析及报告流程
1、扩增结束后,将芯片架拿出放至数字PCR阅读仪的芯片放置台上,打开GenePMS软件,将芯片放置台的温度调至50℃,并设置软件相应参数;
2、芯片放置5分钟后,选择相应的荧光检测通道,开始芯片扫描并分析结果;
3、数据分析与报告输出。
实施例3 本发明试剂盒
1、核酸提取试剂盒(优选为天根DP710试剂盒)
优选市售天根DP710试剂盒按说明进行DNA的提取,具体如下:
分离样本按血浆1:1.5的比例加入裂解液GHH,依次加入蛋白酶K、磁珠和carrierRNA,涡旋振荡混匀后室温孵育20min左右,孵育期间摇晃震荡2次;
将离心管置于磁力架上2min,待磁珠完全吸附时,弃废液;
加750ul缓冲液GDF,上下颠倒混匀30s,使磁珠充分悬浮,将离心管置于磁力架上2min,待磁珠完全吸附时,弃废液;
加750ul漂洗液PWG,上下颠倒混匀30s,使磁珠充分悬浮,将离心管置于磁力架上2min,待磁珠完全吸附时,弃废液,重复该操作一次;
将离心管置于磁力架上,室温晾干5-10min;
加入适量洗脱缓冲液TBC,移液器吹吸使磁珠重新悬浮,56°C孵育5min,期间每2min轻轻晃动使核酸充分洗脱;
将离心管置于磁力架上静置2min,待磁珠完全吸附时,将洗脱液转移至新的离心管中,应用于下一步核酸检测。
2、液滴PCR核酸检测试剂盒
表3
试剂、耗材 | 组分 | 功能 | 来源 |
5 x PCR Mix | PCR所需成分,包括buffer、taq酶,dntp,Mg2+等 | 进行PCR反应 | 本公司通用试剂盒 |
Forward Primer (10μM) | 碱基序列 | 用于PCR反应 | 自主合成,序列见表1 |
Reverse Primer (10μM) | 碱基序列 | 用于PCR反应及逆转录反应引物 | 自主合成,序列见表1 |
Probe (10μM) | 碱基序列和荧光基团标记 | 用于PCR反应,信号扩增指示 | 自主合成,序列见表1 |
模板(DNA样本) | 碱基序列 | 反应体系中的模板 | 上述核酸提取过程中获得 |
实验反应程序:95℃ 10 min(95℃,15 sec, 60℃,30 sec)40 cycles;
实施例4:对比试验
人为往血浆中掺入了50copy/ml的kpc基因作为模拟样本,分别按照本发明试剂盒(实施例1)进行液滴式数字PCR和荧光定量PCR方法检测,结果见表4。
表4
组号 | 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 | 10 | 11 | 12 |
本发明阳性点数 | 11 | 13 | 7 | 10 | 8 | 12 | 8 | 9 | 8 | 12 | 13 | 11 |
空白组阳性点数 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 |
荧光定量PCR Ct值 | 40 | 38 | 39 | 39 | 38 | 40 | 42 | 40 | 39 | 38 | 41 | 42 |
空白组荧光定量PCR Ct值 | 39 | 40 | 42 | 40 | 39 | 38 | 40 | 41 | 38 | 39 | 40 | 42 |
由图4结果可以看出,对于50copy/ml的阳性样本,本发明引物探针结合数字PCR的方法能够准确检测出来,常规的荧光定量无法检测出。
实施例5灵敏度检测实验
取阳性对照品(即含靶基因片段的质粒溶液),测定其浓度并计算拷贝数,按照10倍的浓度梯度稀释,选取5.0~5.0×104拷贝/ml的浓度作为样品,用本发明的试剂盒和检测方法进行检测。
结果表明,本发明试剂盒对病原菌的最低检出限浓度为50个拷贝的基因,即50个拷贝/ml。
结果显示,本发明检测方法可快速、准确及超高灵敏度地检测CRE、MRSA和VRE中7种细菌耐药基因,根据目标耐药基因的定量/半定量检测结果,可实时、快速监测血流感染患者体内的特定耐药基因的变化,及时评估患者状况,为临床医生提供辅助治疗建议。本发明公开的试剂盒亦可用于危重症患者和免疫抑制人群无症状定植者的CRE、MRSA和VRE中7种细菌耐药基因的主动筛查。
以上仅是本发明的优选实施方式,应当指出,对于本技术领域的普通技术人员来说,在不脱离本发明原理的前提下,还可以做出若干改进和润饰,这些改进和润饰也应视为本发明的保护范围。
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Claims (5)
1.7种耐药基因多重PCR检测的引物和探针组,其特征在于,包括:
用于检测KPC基因的SEQ ID NO:1~2所示核苷酸序列的引物和SEQ ID NO:3所示核苷酸序列的探针;
用于检测NDM基因的SEQ ID NO:4~5所示核苷酸序列的引物和SEQ ID NO:6所示核苷酸序列的探针;
用于检测IMP基因的SEQ ID NO:7~8所示核苷酸序列的引物和SEQ ID NO:9所示核苷酸序列的探针;
用于检测VIM基因的SEQ ID NO:10~11所示核苷酸序列的引物和SEQ ID NO:12所示核苷酸序列的探针;
用于检测OXA48基因的SEQ ID NO:13~14所示核苷酸序列的引物和SEQ ID NO:15所示核苷酸序列的探针;
用于检测mecA基因的SEQ ID NO:16~17所示核苷酸序列的引物和SEQ ID NO:18所示核苷酸序列的探针;和
用于检测vanA基因的引SEQ ID NO:19~20所示核苷酸序列的引物和SEQ ID NO:21所示核苷酸序列的探针。
2.权利要求1所述的引物和探针组在制备CRE、MRSA和VRE中7种耐药基因多重检测的试剂盒中的应用。
3.7种耐药基因多重检测的试剂盒,其特征在于,包括权利要求1所述的引物和探针组。
4.根据权利要求3所述的试剂盒,其特征在于,还包括检测试剂和核酸提取试剂。
5.根据权利要求4所述的试剂盒,其特征在于,所述检测试剂包括无菌水、PCR Mix。
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