基于液相芯片技术建立快速检测6种临床常见血流感染厌氧菌的方法及评价  

Establishment and Evaluation of A Rapid Detection Method for 6 Common Clinical Infected Anaerobic Bacteria in Blood Stream by Liquid Phase Chip Technology

作　　者：陈梅莲[1] 曾见芬[1] 张欣选 周文娟[1] 余洁玲[1] 彭兰芬[1] 付文金[1] CHEN Mei-lian;ZENG Jian-fen;ZHANG Xin-xuan;ZHOU Wen-juan;YU Jie-ling;PENG Lan-fen;FU Wen-jin(Department of Clinical Laboratory,Dongguan Houjie Hospital,Guangdong Dongguan 523945,China;Dongguan Nancheng Community Health Service Center,Guangdong Dongguan 523073,China)

机构地区：[1]广东省东莞市厚街医院检验科,广东东莞523945 [2]东莞市南城社区卫生服务中心,广东东莞523073

出　　处：《现代检验医学杂志》2023年第2期75-80,159,共7页Journal of Modern Laboratory Medicine

基　　金：东莞市社会科技发展(一般)项目(202050715023529);东莞市社会科技发展(重点)项目(202050715023181)。

摘　　要：目的基于液相芯片技术建立一种可同时快速检测6种临床常见血流感染厌氧菌的方法。方法通过GenBank寻找脆弱拟杆菌、厌氧消化链球菌、具核梭杆菌、中间普雷沃菌、产气荚膜梭菌和痤疮丙酸杆菌的16S rDNA基因序列,经序列比对分析,选择菌内保守菌间特异的区域作靶序列,使用Primer 5.0软件设计特异性引物及探针,建立不对称多重PCR扩增体系,将PCR产物与偶联核酸探针的荧光微球混合物进行杂交,建立一种多重检测方法,并对该方法的特异度、灵敏度和重复性进行评价。收集2020年6月~2022年5月东莞市厚街医院阳性血厌氧培养瓶84例及阴性培养瓶16例,应用建立的方法进行检测,结果与传统培养法进行比较。结果6种厌氧菌靶基因序列经PCR扩增后,电泳成像清晰可见6条目标条带;经反应体系优化,生物素标记与非标记引物浓度比为4∶1时,选择退火温度56℃进行多重扩增,加入5μl PCR产物,52℃温育20 min检测效果最佳;各目标序列荧光强度中位值(median fluorescence intensity,MFI)>1000,无非特异性信号;最低检测限可达103cfu/ml~10^(4)cfu/ml;当菌液浓度为105cfu/ml时,批内及批间的变异系数分别在7.38%和11.10%以下;100例临床样本中6种厌氧菌的检测结果与培养法一致。结论成功建立一种快速、简便、高效的液相芯片方法,可同时检测6种常见血流感染厌氧菌。Objective To establish a method based on liquid chip technology for simultaneous and rapid detecting of 6 common anaerobic bacteria in blood stream infection.Methods The 16S rDNA gene sequences of Bacteroides fragilis,Streptococcus anaerobius,Fusobacterium nucleatum,Prevotella intermedia,Clostridium perfringens and Propionibacterium acnes were searched by GenBank.After sequence alignment analysis,specific regions between conserved bacteria in the bacteria were selected as target sequences.Primer 5.0 software was used to design specific primers and probes.After an asymmetric multiplex PCR amplification,the PCR products hybridized with microsphere mixtures that had been coupled to gene-specific probes,and a multiplex detection system was established.Then the specificity,sensitivity and repeatability of the method were evaluated.From June 2020 to May 2022,84 cases of positive blood anaerobic culture bottles and16 cases of negative culture bottles were collected from Dongguan Houjie Hospital,and the results were compared with the traditional culture methods.Results 6 target bands were clearly visible on electrophoretic imaging.When the concentration ratio of biotin labeled primer to non labeled primer was 4∶1,the best detection effect was obtained by multiple amplification at 56℃,adding 5μl PCR products and incubating at 52℃for 20 minutes.The median fluorescence intensity(MFI)of each target sequence was>1000,and there was no non-specific signal.The minimum detectable concentration of the established method could reach to 103cfu/ml~10^(4) cfu/ml.When the concentration of bacterial solution was 105cfu/ml,the inter-batch and intra-batch CVs were lower than 7.38%and 11.10%,respectively.The detection results of 6 anaerobic bacteria in 100 clinical samples were consistent with the culture method.Conclusion A liquid chip detection method for rapid,simple and efficient detecting of 6 common anaerobic bacteria in blood stream was successfully established.

关 键 词：厌氧菌 液相芯片 多重聚合酶链式反应 

分 类 号：R446.5[医药卫生—诊断学]