实时定量PCR检测新生儿全血中肺炎克雷伯菌方法的建立和应用  

Establishment and Application of Taq Man Real-Time PCR for Detection of Klebsiella pneumoniae in Neonatal Blood Sample

作　　者：章晟 庄璐 李秋平 宋婕 张玉佩 朱丽敏 赵丹华 王瑞娟 封志纯 ZHANG Sheng;ZHUANG Lu;LI Qiu-Ping;SONG Jie;ZHANG Yu-Pei;ZHU Li-Min;ZHAO Dan-Hua;WANG Rui-Juan;FENG Zhi-Chun(Affiliated Bayi Children's Hospital, Chinese People's Liberation Army General Hospital,Beijing 100007, China;National Engineering Laboratory for Birth Defects Prevention and Control of Key Technology,Beijing 100007, China;Beijing Key Laboratory of Pediatric Organ Failure,Beijing 100007, China)

机构地区：[1]陆军总医院附属八一儿童医院出生缺陷防控关键技术国家工程实验室儿童器官功能衰竭北京市重点实验室,北京100007

出　　处：《生物技术通讯》2017年第5期665-670,共6页Letters in Biotechnology

基　　金：中国博士后科学基金(2013M542472)

摘　　要：目的:建立实时定量PCR(RQ-PCR)快速检测新生儿血液标本中肺炎克雷伯菌基因组DNA的方法,并进行初步临床应用。方法:选择肺炎克雷伯菌高度保守的二鸟苷酸环化酶基因作为靶基因,设计特异性引物和TaqMan探针,建立RQ-PCR反应体系;采用含靶基因扩增片段的重组质粒建立标准曲线,提取血液中的基因组DNA,对500份新生儿血液标本进行RQ-PCR检测。结果:设计的引物和探针特异性高,检测限为1个拷贝数;500份新生儿血液标本中,RQ-PCR检测为阳性的有18个病例,阳性率为3.6%,明显高于血培养的阳性率2.4%(12个病例),差异具有统计学意义(P<0.01)。结论:RQ-PCR可用于检测新生儿血液标本中的肺炎克雷伯菌,此方法快速、灵敏、特异性强。Objective:To establish a real-time quantitative PCR(RQ-PCR)assay for detecting Klebsiella pneumoniae genomic DNA from neonatal blood sample,and preliminarily apply it in clinical test.Methods:The primers and TaqMan probe were designed targeting the highly conserved diguanylate cyclase gene of K.pneumoniae,and a amplification reaction system was established.The standard curve was built based on the recombinant plasmid DNA containing the amplification of the target gene.The genomic DNA was extracted using QIAamp DNABlood Mini Kit from five hundred neonatal blood sample,followed by RQ-PCR detection.Results:The specificityof primers and probe were high,the detecting limit was1copy.For five hundred neonatal blood sample,the positive rate of RQ-PCR was3.6%(18patients),which was significantly higher than2.4%(12patients)by blood culture.Conclusion:RQ-PCR assays is a rapid,sensitive,and specific method and can be used in the detection ofK.pneumoniae in neonatal blood samples.

关 键 词：实时定量PCR 新生儿 肺炎克雷伯菌 血标本 

分 类 号：Q78[生物学—分子生物学]