CN116064859B - 多种病原微生物和耐药基因数字pcr检测的引物探针组及其试剂盒 - Google Patents
多种病原微生物和耐药基因数字pcr检测的引物探针组及其试剂盒 Download PDFInfo
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Abstract
本发明涉及生物检测技术领域,尤其涉及多种病原微生物和耐药基因数字PCR检测的引物探针组及其试剂盒。本发明基于7色荧光通道数字PCR平台,针对多种病原微生物和多种耐药基因的保守区域设计特异性引物、含有不同荧光基团的通用探针和特异性单修饰无荧光基团探针,并结合超多重数字PCR技术实现了每个种类下多个菌种或耐药位点的覆盖。
Description
技术领域
本发明涉及生物检测技术领域,尤其涉及多种病原微生物和耐药基因数字PCR检测的引物探针组及其试剂盒。
背景技术
病原微生物,是指一些侵入人体后,可以导致人体发生感染性疾病,甚至是传染病的微生物。常见的病原微生物包括朊毒体,细菌,支原体,衣原体,立克次体,真菌,螺旋体以及病毒。某些病原微生物反复接触某些化学治疗药物后,其反应性不断减弱,以致最后病原微生物可抵抗该药而不被杀灭或抑制,这就使病原微生物对药物产生耐受性,称为耐药性或抗药性。耐药基因是编码耐药性状的一段核苷酸序列,位于细菌的染色体或者染色体外的质粒上。病原微生物产生耐药性后常使疗效减小或完全失效,直接影响了疾病的治疗效果,因此,针对多种病原微生物核酸和耐药基因鉴定,建立一套系统、高效、快捷的检测方法,可以有效地促进医疗事业的发展。
目前,常用的针对病原微生物核酸和耐药基因的检测方法主要是实时荧光定量PCR(简称qPCR),qPCR可分为荧光染料法(SYBRGreenⅠ法为代表)和荧光探针法(TaqMan法为代表)。SYBRGreenⅠ染料法因具有使用简便,价格便宜等优点而被广泛使用,但其最大的缺点是缺乏特异性,即染料可以与任何dsDNA结合,影响定量结果的准确性。与染料法qPCR不同,TaqMan检测中会增加荧光探针成分,探针通过荧光共振能量转移(FRET)发生作用,具有特异性高、适用多重qPCR的特点。但传统的TaqMan探针技术,通常每个靶标需要一条探针;在一个荧光通道检测多个靶标时,同一荧光标记的探针过多,会导致背景信号过高,造成对比度下降,难以进行信号判读,因而限制了检测靶标数量。
发明内容
有鉴于此,本发明要解决的技术问题在于提供多种病原微生物和耐药基因数字PCR检测的引物探针组及其试剂盒,本发明提供了基于7色荧光通道数字PCR平台,并结合超多重数字PCR技术实现了每个种类下多个菌种或耐药位点的覆盖。
本发明提供了通用探针的结构,其5’端至3’端依次包括:淬灭基团-片段A-荧光基团-片段B-C3。
所述片段A的核苷酸序列如SEQ ID NO:1~7任意一种所示。
所述片段B的核苷酸序列如SEQ ID NO:8~14任意一种所示。
本发明实施例中,所述通用探针的5’端至3’端依次包括:
淬灭基团-SEQ ID NO:1所示的片段A-荧光基团-SEQ ID NO:8所示的片段B-C3;
淬灭基团-SEQ ID NO:2所示的片段A-荧光基团-SEQ ID NO:9所示的片段B-C3;
淬灭基团-SEQ ID NO:3所示的片段A-荧光基团-SEQ ID NO:10所示的片段B-C3;
淬灭基团-SEQ ID NO:4所示的片段A-荧光基团-SEQ ID NO:11所示的片段B-C3;
淬灭基团-SEQ ID NO:5所示的片段A-荧光基团-SEQ ID NO:12所示的片段B-C3;
淬灭基团-SEQ ID NO:6所示的片段A-荧光基团-SEQ ID NO:13所示的片段B-C3;和/或
淬灭基团-SEQ ID NO:7所示的片段A-荧光基团-SEQ ID NO:14所示的片段B-C3。
本发明中所述通用探针的荧光基团选自FAM、VIC、ROX、CY5、A425、CY5.5或CY7;淬灭基团选自BHQ1、BHQ2或BHQ3。
一些实施例中,所述通用探针的5’端至3’端依次为:
BHQ1-SEQ ID NO:1所示的片段A-dT-FAM-SEQ ID NO:8所示的片段B-C3;
BHQ1-SEQ ID NO:2所示的片段A-dT-VIC-SEQ ID NO:9所示的片段B-C3;
BHQ2-SEQ ID NO:3所示的片段A-dT-ROX-SEQ ID NO:10所示的片段B-C3;
BHQ2-SEQ ID NO:4所示的片段A-dT-CY5-SEQ ID NO:11所示的片段B-C3;
BHQ1-SEQ ID NO:5所示的片段A-dT-A425-SEQ ID NO:12所示的片段B-C3;
BHQ3-SEQ ID NO:6所示的片段A-dT-CY5.5-SEQ ID NO:13所示的片段B-C3;
BHQ3-SEQ ID NO:7所示的片段A-dT-CY7-SEQ ID NO:14所示的片段B-C3。
本发明中,所述通用探针与所述单修饰无荧光探针配合,其中单修饰无荧光探针与扩增产物特异性结合,通用探针与单修饰无荧光探针杂交后,产生荧光信号达到检测目的。
本发明还提供了检测病原微生物和耐药基因的引物探针组,包括本发明所述的通用探针。
本发明提供的引物探针组,还包括特异性引物对,所述特异性引物对靶向病原微生物和耐药基因。
其中,所述病原微生物包括所述病原微生物包括铜绿假单胞菌、大肠杆菌、肺炎克雷伯菌、鲍曼不动杆菌、金黄葡萄球菌、屎肠球菌、粪肠球菌、阴沟肠杆菌、白色念珠菌、光滑念珠菌、近平滑念珠菌、热带念珠菌;
所述耐药基因包括碳青霉烯类耐药-KPC基因、碳青霉烯类耐药-NDM基因、碳青霉烯类耐药-OXA48基因、碳青霉烯类耐药-IMP基因、万古霉素类耐药-VANA基因、万古霉素类耐药-VANB基因、万古霉素类耐药-VANM基因、甲氧西林类耐药-MECA基因或甲氧西林类耐药-MECC基因。
具体地,靶向铜绿假单胞菌的引物对具有如SEQ ID NO:37~38所示的核苷酸序列;
靶向大肠杆菌的引物对具有如SEQ ID NO:39~40所示的核苷酸序列;
靶向肺炎克雷伯菌的引物对具有如SEQ ID NO:41~42所示的核苷酸序列;
靶向鲍曼不动杆菌的引物对具有如SEQ ID NO:43~44所示的核苷酸序列;
靶向金黄葡萄球菌的引物对具有如SEQ ID NO:45~46所示的核苷酸序列;
靶向屎肠球菌的引物对具有如SEQ ID NO:47~48所示的核苷酸序列;
靶向粪肠球菌的引物对具有如SEQ ID NO:49~50所示的核苷酸序列;
靶向阴沟肠杆菌的引物对具有如SEQ ID NO:51~52所示的核苷酸序列;
靶向白色念珠菌的引物对具有如SEQ ID NO:53~54所示的核苷酸序列;
靶向光滑念珠菌的引物对具有如SEQ ID NO:55~56所示的核苷酸序列;
靶向近平滑念珠菌的引物对具有如SEQ ID NO:57~58所示的核苷酸序列;
靶向热带念珠菌的引物对具有如SEQ ID NO:59~60所示的核苷酸序列;
靶向碳青霉烯类耐药-KPC基因的引物对具有如SEQ ID NO:61~62所示的核苷酸序列;
靶向碳青霉烯类耐药-NDM基因的引物对具有如SEQ ID NO:63~64所示的核苷酸序列;
靶向碳青霉烯类耐药-OXA48的引物对具有如SEQ ID NO:65~66所示的核苷酸序列;
靶向碳青霉烯类耐药-IMP基因的引物对具有如SEQ ID NO:67~68所示的核苷酸序列;
靶向万古霉素类耐药-VANA基因的引物对具有如SEQ ID NO:69~70所示的核苷酸序列;
靶向万古霉素类耐药-VANB基因的引物对具有如SEQ ID NO:71~72所示的核苷酸序列;
靶向万古霉素类耐药-VANM基因的引物对具有如SEQ ID NO:73~74所示的核苷酸序列;
靶向甲氧西林类耐药-MECA基因的引物对具有如SEQ ID NO:75~76所示的核苷酸序列;
靶向甲氧西林类耐药-MECC基因的引物对具有如SEQ ID NO:77~78所示的核苷酸序列。
本发明提供的引物探针组,还包括单修饰无荧光探针,所述单修饰无荧光探针的核酸序列与所述通用探针中的片段B反向互补,且3’端标记有淬灭基团,其中,所述淬灭基团选自MGB、BHQ2和BHQ3。
具体地,靶向铜绿假单胞菌的单修饰无荧光探针具有如SEQ ID NO:15所示的核苷酸序列,3’端标记有MGB淬灭基团;
靶向大肠杆菌的单修饰无荧光探针具有如SEQ ID NO:16所示的核苷酸序列,3’端标记有MGB淬灭基团;
靶向肺炎克雷伯菌的单修饰无荧光探针具有如SEQ ID NO:17所示的核苷酸序列,3’端标记有MGB淬灭基团;
靶向鲍曼不动杆菌的单修饰无荧光探针具有如SEQ ID NO:18所示的核苷酸序列,3’端标记有MGB淬灭基团;
靶向金黄葡萄球菌的单修饰无荧光探针具有如SEQ ID NO:19所示的核苷酸序列,3’端标记有MGB淬灭基团;
靶向屎肠球菌的单修饰无荧光探针具有如SEQ ID NO:20所示的核苷酸序列,3’端标记有MGB淬灭基团;
靶向粪肠球菌的单修饰无荧光探针具有如SEQ ID NO:21所示的核苷酸序列,3’端标记有MGB淬灭基团;
靶向阴沟肠杆菌的单修饰无荧光探针具有如SEQ ID NO:22所示的核苷酸序列,3’端标记有MGB淬灭基团;
靶向白色念珠菌的单修饰无荧光探针具有如SEQ ID NO:23所示的核苷酸序列,3’端标记有MGB淬灭基团;
靶向光滑念珠菌的单修饰无荧光探针具有如SEQ ID NO:24所示的核苷酸序列,3’端标记有MGB淬灭基团;
靶向近平滑念珠菌的单修饰无荧光探针具有如SEQ ID NO:25所示的核苷酸序列,3’端标记有MGB淬灭基团;
靶向热带念珠菌的单修饰无荧光探针具有如SEQ ID NO:26所示的核苷酸序列,3’端标记有MGB淬灭基团;
靶向碳青霉烯类耐药-KPC基因的单修饰无荧光探针具有如SEQ ID NO:27所示的核苷酸序列,3’端标记有MGB淬灭基团;
靶向碳青霉烯类耐药-NDM基因的单修饰无荧光探针具有如SEQ ID NO:28所示的核苷酸序列,3’端标记有BHQ2淬灭基团;
靶向碳青霉烯类耐药-OXA48的单修饰无荧光探针具有如SEQ ID NO:29所示的核苷酸序列,3’端标记有MGB淬灭基团;
靶向碳青霉烯类耐药-IMP基因的单修饰无荧光探针具有如SEQ ID NO:30所示的核苷酸序列,3’端标记有MGB淬灭基团;
靶向万古霉素类耐药-VANA基因的单修饰无荧光探针具有如SEQ ID NO:31所示的核苷酸序列,3’端标记有BHQ1淬灭基团;
靶向万古霉素类耐药-VANB基因的单修饰无荧光探针具有如SEQ ID NO:32所示的核苷酸序列,3’端标记有BHQ1淬灭基团;
靶向万古霉素类耐药-VANM基因的单修饰无荧光探针具有如SEQ ID NO:33所示的核苷酸序列,3’端标记有BHQ1淬灭基团;
靶向甲氧西林类耐药-MECA基因的单修饰无荧光探针具有如SEQ ID NO:34所示的核苷酸序列,3’端标记有BHQ2淬灭基团;
靶向甲氧西林类耐药-MECC基因的单修饰无荧光探针具有如SEQ ID NO:35所示的核苷酸序列,3’端标记有MGB淬灭基团。
本发明还提供了所述通用探针或所述引物探针组在在制备多种病原微生物和耐药基因的数字PCR检测试剂中的应用。
本发明还提供了一种检测多种病原微生物和耐药基因的试剂盒,包括本发明所述的通用探针或引物探针组。
本发明提供的试剂盒中,还包括内标基因的引物和探针组合,具体序列为:IC-UP7:ctgcacgaagctctttttcccgcgacggatctacgtcacagcg(SEQ ID NO:36);IC-P:
cgcgacggatctacgtcacagcg(SEQ ID NO:102);IC-F:gcttcttgtggagctcgacaa(SEQID NO:79);IC-R:ccgtcagcaacttcgttttca(SEQ ID NO:80)。
本发明还提供了多种病原微生物和耐药基因多重数字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)配置多重数字PCR扩增体系;
(3)运行扩增程序:95℃5min【95℃15s,60℃1min】*40;
(4)输出结果。
利用本发明引物探针可实现12种病原微生物包括铜绿假单胞菌、大肠杆菌、肺炎克雷伯菌、鲍曼不动杆菌、金黄葡萄球菌、屎肠球菌、粪肠球菌、阴沟肠杆菌、白色念珠菌、光滑念珠菌、近平滑念珠菌、热带念珠菌,和9种耐药基因包括碳青霉烯类耐药-KPC基因、碳青霉烯类耐药-NDM基因、碳青霉烯类耐药-OXA48基因、碳青霉烯类耐药-IMP基因、万古霉素类耐药-VANA基因、万古霉素类耐药-VANB基因、万古霉素类耐药-VANM基因、甲氧西林类耐药-MECA基因、甲氧西林类耐药-MECC基因的联合检测。
本发明针对12种病原微生物、9种耐药基因的保守区域,共特异性设计了42条引物、7条含有不同荧光基团的通用探针和21条特异性的单修饰无荧光基团探针,通用探针和单修饰探针配合产生荧光信号,可以检测样品中是否含有铜绿假单胞菌、大肠杆菌、肺炎克雷伯菌、鲍曼不动杆菌、金黄葡萄球菌、屎肠球菌、粪肠球菌、阴沟肠杆菌、白色念珠菌、光滑念珠菌、近平滑念珠菌、热带念珠菌,以及是否含有碳青霉烯类耐药-KPC基因、碳青霉烯类耐药-NDM基因、碳青霉烯类耐药-OXA48基因、碳青霉烯类耐药-IMP基因、万古霉素类耐药-VANA基因、万古霉素类耐药-VANB基因、万古霉素类耐药-VANM基因、甲氧西林类耐药-MECA基因、甲氧西林类耐药-MECC基因位点。
本发明采用常规双修饰探针的单重扩增体系以及通用探针和单修饰探针配合的多重扩增体系,两种体系的检测结果一致,保证了针对靶标基因设计含有不同荧光基团的通用探针,并结合特异性单修饰无荧光基团探针进行多重数字PCR的可行性。
具体实施方式
本发明提供了多种病原微生物和耐药基因数字PCR检测的引物探针组及其试剂盒,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本发明的引物探针组设计
本实施例涉及的引物探针组序列如表1所示。
其中:UP1-UP7表示7条不同颜色的通用探针。F表示上游引物,R表示下游引物,P表示探针。例如TL-F表示铜绿假单胞菌的上游引物,TL-R表示铜绿假单胞菌的下游引物,TL-P表示铜绿假单胞菌常规的双修饰探针,TL-UP1表示铜绿假单胞菌特异性的单修饰无荧光基团的探针;
表1
实施例2数字PCR扩增体系
一、单重扩增体系如表2所示
表2
二、多重扩增体系如表3所示
表3
三、扩增程序
95℃5min【95℃15s,60℃1min】*40
四、结果判断
1、单重扩增体系检测结果如表4所示
表4
2、多重扩增体系检测结果如表5所示
表5
结果显示,铜绿假单胞菌、大肠杆菌、肺炎克雷伯菌、鲍曼不动杆菌仅在FAM和CY7有检测值,其他通道均无检测值。
金黄葡萄球菌、屎肠球菌、粪肠球菌、阴沟肠杆菌仅在VIC和CY7有检测值,其他通道均无检测值。
白色念珠菌、光滑念珠菌、近平滑念珠菌、热带念珠菌仅在ROX和CY7有检测值,其他通道均无检测值。
碳青霉烯类耐药基因仅在CY5和CY7有检测值,其他通道均无检测值。
万古霉素类耐药基因仅在A425和CY7有检测值,其他通道均无检测值。
甲氧西林类耐药基因仅在CY5.5和CY7有检测值,其他通道均无检测值。
综合上述,本发明提供的引物和探针能够准确对模拟样本进行检测,检测结果与预期完全相符,说明本检测体系的准确性正常;本发明中检测多种病原微生物以及耐药基因的体系具有特异性;两种检测体系的检测结果基本一致,说明本发明的多重检测体系具有稳定性。
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。
Claims (3)
1.检测病原微生物和耐药基因的引物探针组,其特征在于,由通用探针、特异性引物对和单修饰无荧光探针组成;
所述病原微生物为铜绿假单胞菌、大肠杆菌、肺炎克雷伯菌、鲍曼不动杆菌、金黄葡萄球菌、屎肠球菌、粪肠球菌、阴沟肠杆菌、白色念珠菌、光滑念珠菌、近平滑念珠菌或热带念珠菌;
所述耐药基因为碳青霉烯类耐药-KPC基因、碳青霉烯类耐药-NDM基因、碳青霉烯类耐药-OXA48基因、碳青霉烯类耐药-IMP基因、万古霉素类耐药-VANA基因、万古霉素类耐药-VANB基因、万古霉素类耐药-VANM基因、甲氧西林类耐药-MECA基因或甲氧西林类耐药-MECC基因;
所述通用探针为:
淬灭基团- SEQ ID NO:1所示的片段A -荧光基团- SEQ ID NO:8所示的片段B-C3、
淬灭基团- SEQ ID NO:2所示的片段A -荧光基团- SEQ ID NO:9所示的片段B-C3、淬灭基团- SEQ ID NO:3所示的片段A -荧光基团- SEQ ID NO:10所示的片段B-C3、
淬灭基团- SEQ ID NO:4所示的片段A -荧光基团- SEQ ID NO:11所示的片段B-C3、
淬灭基团- SEQ ID NO:5所示的片段A -荧光基团- SEQ ID NO:12所示的片段B-C3、
淬灭基团- SEQ ID NO:6所示的片段A -荧光基团- SEQ ID NO:13所示的片段B-C3、和
淬灭基团- SEQ ID NO:7所示的片段A -荧光基团- SEQ ID NO:14所示的片段B-C3;
所述通用探针中,所述荧光基团选自FAM、VIC、ROX、CY5、A425、CY5.5或CY7,所述淬灭基团选自BHQ1、BHQ2或BHQ3;
所述特异性引物对为:
靶向铜绿假单胞菌的引物对的核酸序列如SEQ ID NO:37~38所示;
靶向大肠杆菌的引物对的核酸序列如SEQ ID NO:39~40所示;
靶向肺炎克雷伯菌的引物对的核酸序列如SEQ ID NO:41~42所示;
靶向鲍曼不动杆菌的引物对的核酸序列如SEQ ID NO:43~44所示;
靶向金黄葡萄球菌的引物对的核酸序列如SEQ ID NO:45~46所示;
靶向屎肠球菌的引物对的核酸序列如SEQ ID NO:47~48所示;
靶向粪肠球菌的引物对的核酸序列如SEQ ID NO:49~50所示;
靶向阴沟肠杆菌的引物对的核酸序列如SEQ ID NO:51~52所示;
靶向白色念珠菌的引物对的核酸序列如SEQ ID NO:53~54所示;
靶向光滑念珠菌的引物对的核酸序列如SEQ ID NO:55~56所示;
靶向近平滑念珠菌的引物对的核酸序列如SEQ ID NO:57~58所示;
靶向热带念珠菌的引物对的核酸序列如SEQ ID NO:59~60所示;
靶向碳青霉烯类耐药-KPC基因的引物对的核酸序列如SEQ ID NO:61~62所示;
靶向碳青霉烯类耐药-NDM基因的引物对的核酸序列如SEQ ID NO:63~64所示;
靶向碳青霉烯类耐药-OXA48的引物对的核酸序列如SEQ ID NO:65~66所示;
靶向碳青霉烯类耐药-IMP基因的引物对的核酸序列如SEQ ID NO:67~68所示;
靶向万古霉素类耐药-VANA基因的引物对的核酸序列如SEQ ID NO:69~70所示;
靶向万古霉素类耐药-VANB基因的引物对的核酸序列如SEQ ID NO:71~72所示;
靶向万古霉素类耐药-VANM基因的引物对的核酸序列如SEQ ID NO:73~74所示;
靶向甲氧西林类耐药-MECA基因的引物对的核酸序列如SEQ ID NO:75~76所示;
和靶向甲氧西林类耐药-MECC基因的引物对的核酸序列如SEQ ID NO:77~78所示;
所述单修饰无荧光探针为:
靶向铜绿假单胞菌的单修饰无荧光探针的核酸序列如SEQ ID NO:15所示,3’端标记有MGB淬灭基团;
靶向大肠杆菌的单修饰无荧光探针的核酸序列如SEQ ID NO:16所示,3’端标记有MGB淬灭基团;
靶向肺炎克雷伯菌的单修饰无荧光探针的核酸序列如SEQ ID NO:17所示,3’端标记有MGB淬灭基团;
靶向鲍曼不动杆菌的单修饰无荧光探针的核酸序列如SEQ ID NO:18所示,3’端标记有MGB淬灭基团;
靶向金黄葡萄球菌的单修饰无荧光探针的核酸序列如SEQ ID NO:19所示,3’端标记有MGB淬灭基团;
靶向屎肠球菌的单修饰无荧光探针的核酸序列如SEQ ID NO:20所示,3’端标记有MGB淬灭基团;
靶向粪肠球菌的单修饰无荧光探针的核酸序列如SEQ ID NO:21所示,3’端标记有MGB淬灭基团;
靶向阴沟肠杆菌的单修饰无荧光探针的核酸序列如SEQ ID NO:22所示,3’端标记有MGB淬灭基团;
靶向白色念珠菌的单修饰无荧光探针的核酸序列如SEQ ID NO:23所示,3’端标记有MGB淬灭基团;
靶向光滑念珠菌的单修饰无荧光探针的核酸序列如SEQ ID NO:24所示,3’端标记有MGB淬灭基团;
靶向近平滑念珠菌的单修饰无荧光探针的核酸序列如SEQ ID NO:25所示,3’端标记有MGB淬灭基团;
靶向热带念珠菌的单修饰无荧光探针的核酸序列如SEQ ID NO:26所示,3’端标记有MGB淬灭基团;
靶向碳青霉烯类耐药-KPC基因的单修饰无荧光探针的核酸序列如SEQ ID NO:27所示,3’端标记有MGB淬灭基团;
靶向碳青霉烯类耐药-NDM基因的单修饰无荧光探针的核酸序列如SEQ ID NO:28所示,3’端标记有BHQ2淬灭基团;
靶向碳青霉烯类耐药-OXA48的单修饰无荧光探针的核酸序列如SEQ ID NO:29所示,3’端标记有MGB淬灭基团;
靶向碳青霉烯类耐药-IMP基因的单修饰无荧光探针的核酸序列如SEQ ID NO:30所示,3’端标记有MGB淬灭基团;
靶向万古霉素类耐药-VANA基因的单修饰无荧光探针的核酸序列如SEQ ID NO:31所示,3’端标记有BHQ1淬灭基团;
靶向万古霉素类耐药-VANB基因的单修饰无荧光探针的核酸序列如SEQ ID NO:32所示,3’端标记有BHQ1淬灭基团;
靶向万古霉素类耐药-VANM基因的单修饰无荧光探针的核酸序列如SEQ ID NO:33所示,3’端标记有BHQ1淬灭基团;
靶向甲氧西林类耐药-MECA基因的单修饰无荧光探针的核酸序列如SEQ ID NO:34所示,3’端标记有BHQ2淬灭基团;
和靶向甲氧西林类耐药-MECC基因的单修饰无荧光探针的核酸序列如SEQ ID NO:35所示,3’端标记有MGB淬灭基团。
2.权利要求1所述的引物探针组在制备多种病原微生物和耐药基因的数字PCR检测试剂中的应用。
3.试剂盒,其特征在于,其包括权利要求1所述的引物探针组以及PCR反应试剂。
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