建立一种实时荧光定量PCR方法检测与鉴定人苍白杆菌及布鲁氏菌  

A real-time fluorescence quantitative PCR method was established for the detection and identification of Ochrobactrum anthropi and Brucella

作　　者：乌伊罕[1] 王利平[1] Wuyihan;Wang Liping(Microbiology Laboratory,Inner Mongolia Autonomous Regional Center for Disease Prevention and Control(Inner Mongolia Academy of Preventive Medicine),Hohhot 010030,China)

机构地区：[1]内蒙古自治区疾病预防控制中心(内蒙古自治区预防医学科学院)微生物实验室,呼和浩特010030

出　　处：《中华地方病学杂志》2024年第5期416-420,共5页Chinese Journal of Endemiology

摘　　要：目的建立一种实时荧光定量PCR方法检测与鉴定人苍白杆菌(Ochrobactrum anthropi,O.anthropi)及布鲁氏菌,并应用于临床样本的检测。方法从美国国立生物技术信息中心(National Center for Biotechnology Information,NCBI)网站GenBank数据库下载苍白杆菌属和与苍白杆菌属相邻近属种菌株的16S核糖体RNA(16S ribosomal RNA,16S rRNA)基因核苷酸序列。使用MEGA 5.1软件构建系统发育树;应用DNAstar(V7.1)软件设计引物及探针,建立检测与鉴定O.anthropi的实时荧光定量PCR方法,验证方法的灵敏性和特异性,并应用该方法检测牧区临床患者血液样本中O.anthropi感染情况。结果共从NCBI网站下载苍白杆菌属17个种37株、与苍白杆菌属相邻近的布鲁氏菌属8个种15株菌株的16S rRNA基因核苷酸序列。16S rRNA基因系统发育树分析发现,O.anthropi与苍白杆菌属O.cytisi、O.lupini、O.tritici种菌株具有高度同源性(相似性均为100.0%),与布鲁氏菌属8个种菌株的相似性为99.1%,与其他13个种苍白杆菌属菌株的相似性为84.5%~98.6%。成功建立了实时荧光定量PCR检测方法(巢氏PCR),检测O.anthropi标准菌株DNA,单一实时荧光定量PCR最低检测限为3.67 fg核酸DNA,循环阈值(cycle threshold,CT)=36.40,巢式PCR检测CT=14.67;且未检出布鲁氏菌属等非苍白杆菌属菌株。对126份临床患者血液样本进行检测,O.anthropi的检出率为17.46%(22/126)。结论建立了实时荧光定量PCR方法检测O.anthropi,该方法具有较高的灵敏性和较强的特异性,可用于O.anthropi与布鲁氏菌的鉴定及临床样本检测。Objective To establish a real-time fluorescence quantitative PCR method for detection and identification of Ochrobactrum anthropi(O.anthropi)and Brucella,and to apply the method in the detection of clinical samples.Methods The gene nucleotide sequences of 16S ribosomal RNA(16S rRNA)of Ochrobactrum spp.and strains which were adjacent to Ochrobactrum spp.were downloaded from the GenBank database of the National Center for Biotechnology Information(NCBI)website in the United States.A phylogenetic tree was constructed by using MEGA 5.1 software.The primers and probes for the real-time fluorescence quantitative PCR assay,which were specific to O.anthropi were designed using DNAstar(V7.1)software,the sensitivity and specificity of the method was verified.And the method was applied to detect O.anthropi infection in blood samples of clinical patients in pastoral areas.Results A total of 37 strains of 16S rRNA gene nucleotide sequences from 17 species of Ochrobactrum spp.and 15 strains from 8 species of Brucella spp.were downloaded from the NCBI website.The phylogenetic tree of 16S rRNA gene revealed a high degree of homology(similarity of 100.0%)between O.anthropi,O.cytisi,O.lupini and O.tritici in Ochrobactrum spp.The similarity was 99.1%between O.anthropi and 8 species of Brucella spp.The similarities was 84.5%-98.6%between O.anthropi and other 13 species of Ochrobactrum spp.The real-time fluorescence quantitative PCR method(nested PCR)was successfully established.For the detection of O.anthropi standard strain DNA,the minimum detection limit of single real-time fluorescence quantitative PCR was 3.67 fg nucleic acid DNA,with a cycle threshold(CT)of 36.40,and a nested PCR detection CT of 14.67,Brucella spp.and other non-Ochrobactrum spp.strains were not detected.Totally 126 blood samples from clinical patients were collected and tested,and the detection rate of O.anthropi was 17.46%(22/126).Conclusion A real-time fluorescence quantitative PCR method has been established to detect O.anthropi,which has high sensitivity an

关 键 词：人苍白杆菌 布鲁氏菌 实时荧光定量PCR 

分 类 号：R516.7[医药卫生—内科学] R446.5[医药卫生—临床医学]