[1]高正琴.空肠弯曲菌 TaqMan-MGB双重探针实时荧光定量 PCR快速检测[J].现代检验医学杂志,2019,34(06):1-5.[doi:10.3969 ／ j.issn.1671-7414.2019.06.001]
　GAO Zheng-qin.Rapid Detection of Campylobacter Jejuni with TaqMan-MGB Dual Probe Real-time Fluorescence Quantitative PCR[J].Journal of Modern Laboratory Medicine,2019,34(06):1-5.[doi:10.3969 ／ j.issn.1671-7414.2019.06.001]
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空肠弯曲菌 TaqMan-MGB双重探针实时荧光定量 PCR快速检测 ()
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《现代检验医学杂志》[ISSN:/CN:]

卷:
第34卷
期数:
2019年06期
页码:
1-5
栏目:
论著
出版日期:
2019-12-30

文章信息/Info

Title:
Rapid Detection of Campylobacter Jejuni with TaqMan-MGB Dual Probe Real-time Fluorescence Quantitative PCR
文章编号:
1671-7414（2019）06-001-06
作者:
高正琴
（中国食品药品检定研究院，北京　100050）
Author(s):
GAO Zheng-qin
（National Institutes for Food and Drug Control, Beijing 100050,China）
关键词:
空肠弯曲菌; 鞭毛蛋白A; 马尿酸酶; TaqMan-MGB 探针; 实时荧光定量PCR; 快速检测
分类号:
R378, 2；Q503　
DOI:
10.3969 ／ j.issn.1671-7414.2019.06.001
文献标志码:
A
摘要:
目的　开发一种特异、灵敏的TaqMan MGB 双重探针实时荧光定量PCR 方法，用于空肠弯曲菌的快速定量检 测。方法　针对空肠弯曲菌鞭毛蛋白A 和马尿酸酶基因设计特异性引物和探针，建立一种新型TaqMan-MGB 双重探针 实时荧光定量PCR 检测空肠弯曲菌方法，对该方法的定量检测线性范围、特异度、灵敏度、重复性、稳定性进行评价， 应用该方法对临床标本中的空肠弯曲菌进行检测，同时用细菌培养、普通PCR、基因克隆和测序鉴定。结果　建立的 空肠弯曲菌TaqMan MGB 双重探针实时荧光定量PCR 检测方法专属性强，能准确检出空肠弯曲菌，而与其他细菌无交 叉反应，特异度为100%。该技术灵敏度高，能精确定量检测空肠弯曲菌DNA 线性范围达10 个数量级，最低检测限为 4 个菌落形成单位。重复性和稳定性良好，组内和组间相对标准偏差均小于1%。应用该方法成功从78 例临床标本中定 量检出28 例空肠弯曲菌阳性标本，用普通PCR 和基因克隆测序分析确认，细菌培养方法仅获得6 株存活的空肠弯曲菌。 结论　TaqMan MGB 双重探针实时荧光定量PCR 具有快速简便、可靠稳定、特异灵敏的优点，可用于临床标本中空肠 弯曲菌定量检测，值得推广应用。
Abstract:
Objective　To develop a specific and sensitive TaqMan-MGB dual probe real-time fluorescence quantitative PCR assay in order to rapidly detect and quantity Campylobabter jejuni. Methods　Specific primers and probes were designed to conserved regions of flagellin A （flaA） and hippuricase （hipO） genes of Campylobabter jejuni. A novel TaqMan-MGB dual probe real-time fluorescence quantitative PCR assay was developed. The linear range, sensitivity, specificity, reproducibility and stability of the assay were evaluated. The assay was applied to detect Campylobabter jejuni in clinical specimens, and bacterial culture, conventional PCR, gene cloning and sequencing were concurrently performed. Results　It was shown that the explored TaqMan-MGB dual probe real-time fluorescence quantitative PCR assay could accurately detect Campylobabter jejuni with 100% specificity, and had no cross reaction with other bacteria. The technology was demonstrated to be highly sensitive, allowing a precise Campylobabter jejuni DNA quantitation over a range of 10 orders of magnitude, and the limit of detection of the assay was determined to be 4 colony-forming units of Campylobabter jejuni. The reproducibility and stability of this assay was excellent with the relative standard deviation within and between groups of less than 1 percent. The assay was successfully applied to quantifiable detection of Campylobabter jejuni genomic load in 78 clinical specimens, and confirmed using conventional PCR and gene cloning and sequence analysis, in which 28 specimens were positive, while only 6 specimens were positive for Campylobabter jejuni obtained by bacterial culture. Conclusion TaqMan MGB dual probe real-time fluorescence quantitative PCR has the advantages of rapid, simple, reliable, stable, specific and sensitive, which can be used for quantitative detection of Campylobacter jejuni in clinical specimens, and is worthy of popularization and application.

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备注/Memo

备注/Memo:
作者简介：高正琴（1976-），女，博士，副研究员，研究方向：病原生物学与快检新技术研究，E-mail：gaozhengqin@126.com 收稿日期：2019-05-27
更新日期/Last Update: 2019-12-25