[1]陈梅莲,曾见芬,张欣选,等.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[J].现代检验医学杂志,2023,38(02):75-80+159.[doi:10.3969/j.issn.1671-7414.2023.02.014 ]
　CHEN Mei-lian,ZENG Jian-fen,ZHANG Xin-xuan,et al.Establishment and Evaluation of A Rapid Detection Method for 6 Common Clinical Infected Anaerobic Bacteria in Blood Stream by Liquid Phase Chip Technology[J].Journal of Modern Laboratory Medicine,2023,38(02):75-80+159.[doi:10.3969/j.issn.1671-7414.2023.02.014 ]
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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()
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《现代检验医学杂志》[ISSN:/CN:]

卷:
第38卷
期数:
2023年02期
页码:
75-80+159
栏目:
论著
出版日期:
2023-03-15

文章信息/Info

Title:
Establishment and Evaluation of A Rapid Detection Method for 6 Common Clinical Infected Anaerobic Bacteria in Blood Stream by Liquid Phase Chip Technology
文章编号:
1671-7414（2023）02-075-07
作者:
陈梅莲1; 曾见芬1; 张欣选2; 周文娟1; 余洁玲1; 彭兰芬1; 付文金1
（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）
Author(s):
CHEN Mei-lian1;  ZENG Jian-fen1;  ZHANG Xin-xuan2;  ZHOU Wen-juan1;  YU Jie-ling1;  PENG Lan-fen1;  FU Wen-jin1
（1. Department of Clinical Laboratory，Dongguan Houjie Hospital, Guangdong Dongguan 523945, China; 2. Dongguan Nancheng Community Health Service Center, Guangdong Dongguan 523073, China）
关键词:
厌氧菌; 液相芯片; 多重聚合酶链式反应
分类号:
R446.5
DOI:
10.3969/j.issn.1671-7414.2023.02.014
文献标志码:
A
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）＞ 1 000，无非特异性信号；最低检测限可达 103cfu/ml～104cfu/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。
Abstract:
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 >1 000, and there was no non-specific signal. The minimum detectable concentration of the established method could reach to 103cfu/ml ～104 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.

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备注/Memo

备注/Memo:
收稿日期：2022-09-30修回日期：2022-12-16
基金项目：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）。
作者简介：陈梅莲 (1980-)，女，大学本科，副主任技师，研究方向：分子诊断，E-mail: Salala2002@sina.com。
通讯作者：付文金，E-mail：332689892@qq.com。

更新日期/Last Update: 2023-03-15